U.S. patent application number 12/670739 was filed with the patent office on 2010-09-09 for stochastic confinement to detect, manipulate, and utilize molecules and organisms.
Invention is credited to James Q. Boedicker, Cory Gerdts, Toan Huynh, Rustem F. Ismagilov, Christian Kastrup, Hyun Jung Kim, Matthew K. Runyon, Feng Shen.
Application Number | 20100227767 12/670739 |
Document ID | / |
Family ID | 39882696 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100227767 |
Kind Code |
A1 |
Boedicker; James Q. ; et
al. |
September 9, 2010 |
STOCHASTIC CONFINEMENT TO DETECT, MANIPULATE, AND UTILIZE MOLECULES
AND ORGANISMS
Abstract
Methods of detecting organisms e.g. bacteria using stochastic
confinement effects with microfluidic technologies involving plugs
are provided. Signal amplification methods for the detection of
molecules are also disclosed.
Inventors: |
Boedicker; James Q.;
(Chicago, IL) ; Ismagilov; Rustem F.; (Chicago,
IL) ; Kastrup; Christian; (Cambridge, MA) ;
Gerdts; Cory; (Romeoville, IL) ; Huynh; Toan;
(Chicago, IL) ; Kim; Hyun Jung; (Chicago, IL)
; Runyon; Matthew K.; (Chicago, IL) ; Shen;
Feng; (Chicago, IL) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
39882696 |
Appl. No.: |
12/670739 |
Filed: |
July 28, 2008 |
PCT Filed: |
July 28, 2008 |
PCT NO: |
PCT/US08/71374 |
371 Date: |
January 26, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60962426 |
Jul 26, 2007 |
|
|
|
61052490 |
May 12, 2008 |
|
|
|
Current U.S.
Class: |
506/7 ; 435/29;
435/32 |
Current CPC
Class: |
C12N 11/04 20130101;
C12M 35/08 20130101; C12M 23/34 20130101; C12M 25/01 20130101; G01N
33/54313 20130101; G01N 33/542 20130101; C12Q 1/18 20130101; C12N
1/20 20130101; C12N 1/38 20130101; C12P 39/00 20130101; C12M 1/14
20130101 |
Class at
Publication: |
506/7 ; 435/29;
435/32 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C12Q 1/02 20060101 C12Q001/02; C12Q 1/18 20060101
C12Q001/18 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This work was supported in part by Grant No. 0526693 awarded
by the National Science Foundation (NSF) CRC and under grant
numbers EB01903, GM075827, and GM074961 awarded by the National
Institutes of Health (NIH). The government has certain rights in
the invention.
Claims
1. A method of detecting bacteria in a sample, comprising: creating
an array of plugs by introducing a first plug fluid into a flow of
carrier fluid in a microchannel; wherein the majority of plugs in
the array do not contain a bacterium; wherein the first plug fluid
is substantially immiscible with the carrier fluid and comprises a
concentration of the sample diluted such that at most 2 bacteria
are present in any plug; and analyzing the array for the presence
of bacteria.
2. The method of claim 1, wherein the array is analyzed for a
detectable signal produced by the bacteria, wherein the detectable
signal is a substance produced by the bacteria or is produced when
the bacteria consumes a substance in the plug.
3. The method of claim 2, wherein the substance is selected from
the group consisting of oxygen, carbon, a protein produced by the
bacteria, a molecule produced by a bacterial enzymatic reaction,
and a redox/potential sensitive indicator.
4-5. (canceled)
6. The method of claim 1, wherein the sample is from human, soil or
marine.
7. (canceled)
8. The method of claim 1, wherein the bacteria are at a higher
concentration in the plugs than in the sample.
9. The method of claim 1, wherein the plugs contain different
species of bacteria.
10. The method of claim 1, further comprising introducing a plug
fluid comprising media capable of supporting bacterial growth into
the plug.
11. The method of claim 10, wherein the detectable signal is
produced by growth of the bacteria.
12. The method of claim 1, wherein the plugs comprise a substance
capable of inducing virulence in the bacteria.
13. The method of claim 1, wherein the at least two plug comprise a
substance capable of lysing the bacteria.
14. (canceled)
15. The method of claim 1, further comprising assaying the bacteria
detected for a biological activity.
16. The method of claim 1, further comprising identifying the
bacteria.
17. The method of claim 1, further comprising splitting a plug that
has been determined to contain bacteria into multiple plugs, each
containing at least one of the bacteria.
18. The method of claim 1, further comprising conducting a
polymerase chain reaction on the contents of the at least two plugs
prior to analyzing the at least two plugs for the detectable
signal.
19. A method of detecting bacteria comprising: flowing at least two
plugs in a carrier fluid through a microchannel; wherein each plug
comprises a plug fluid substantially immiscible with the carrier
fluid; wherein a first plug comprises a means for detecting a first
species of bacteria; wherein a second plug comprises a means for
detecting a second species of bacteria different from the first
species of bacteria; introducing a sample optionally comprising
bacteria into the first and second plugs, wherein the bacteria
produce a detectable signal; and analyzing the plugs for the
detectable signal.
20-28. (canceled)
29. A method of screening for antibiotic activity comprising:
flowing at least two plugs in a carrier fluid through a
microchannel; wherein each plug comprises a plug fluid immiscible
with the carrier fluid and media capable of supporting bacterial
growth; wherein the first plug comprises a first antibiotic
candidate; wherein the second plug comprises a second antibiotic
candidate; introducing a sample which comprises bacteria into the
first and second plugs; and detecting the presence of bacterial
growth.
30-44. (canceled)
45. A method of detecting bacteria in an aqueous sample,
comprising: dividing a sample into a plurality of subsamples in a
microfluidic device, where each subsample is separated from another
by a fluorinated liquid, each subsample has a volume that is a
nanoliter or less, the majority of the subsamples do not contain a
bacterium, and each subsample has at most 2 bacteria; and analyzing
the volumes for the presence of bacteria.
Description
RELATED APPLICATIONS
[0001] The present patent document claims the benefit of the filing
date under 35 U.S.C. .sctn.119(e) of Provisional U.S. Patent
Application Ser. Nos. 60/962,426, filed Jul. 26, 2007, and
61/052,490 filed May 12, 2008, which are hereby incorporated by
reference.
BACKGROUND
[0003] Bacterial infections are a major health problem, leading to
more than 130,000 deaths from sepsis annually in the United States
alone. (G. S. Martin, D. M. Mannino, S. Eaton and M. Moss, N. Engl.
J. Med., 2003, 348, 1546-1554) These deaths are often the result of
nosocomial, or hospital acquired, infections and frequently involve
drug resistant strains of bacteria. (B. M. Farr, Curr. Opin.
Infect. Dis., 2004, 17, 317-322; G. J. Moran, A. Krishnadasan, R.
J. Gorwitz, G. E. Fosheim, L. K. McDougal, R. B. Carey and D. A.
Talan, N. Engl. J. Med., 2006, 355, 666-674) In addition,
bacteremia, the presence of bacteria in the blood, is one of the
major causes of sepsis and generally requires a minimum of a day or
more to diagnose, increasing the chances of patient mortality. (S.
D. Carrigan, G. Scott and M. Tabrizian, Clin. Chem., 2004, 50,
1301-1314) Patient mortality rates further increase when
inappropriate antimicrobial treatment is administered, which is
estimated to occur in 23-30% of cases. (S. D. Carrigan, G. Scott
and M. Tabrizian, Clin. Chem., 2004, 50, 1301-1314)
[0004] Shortening the time necessary to detect and identify an
effective antibiotic regimen to treat bacterial infections could
significantly decrease the mortality rate and reduce the cost of
treating patients with sepsis and other aggressive bacterial
infections. (H. B. Nguyen, E. P. Rivers, F. M. Abrahamian, G. J.
Moran, E. Abraham, S. Trzeciak, D. T. Huang, T. Osborn, D. Stevens
and D. A. Talan, Ann. Emerg. Med., 2006, 48, 28-54) However,
attempts to reduce the assay time of traditional diagnosis and
characterization techniques are impeded by the necessity to
incubate bacterial specimens for hours to days to increase the cell
density of the sample to detectible levels. To overcome this
challenge, new PCR-based detection methods enable diagnosis in the
one to four hour time frame. (S. Poppert, A. Essig, B. Stoehr, A.
Steingruber, B. Wirths, S. Juretschko, U. Reischl and N.
Wellinghausen, J. Clin. Microbiol., 2005, 43, 3390-3397; K. P.
Hunfeld, Int. J. Med. Microbiol., 2007, 297, 32-32.) However, these
methods only provide a genetic profile of the infecting bacterial
species and lack the ability to directly test the bacteria's
function, such as susceptibility to particular antibiotics.
Although some types of antibiotic resistance have genetic markers,
such as the mecA gene for instance, (K. Murakami, W. Minamide, K.
Wada, E. Nakamura, H. Teraoka and S. Watanabe, J. Clin. Microbiol.,
1991, 29, 2240-2244) genetic markers have not been identified for
all antibiotic resistant strains of bacteria. Therefore, antibiotic
susceptibility is more accurately determined by a functional assay,
especially for bacterial strains with unknown resistance
mechanisms.
[0005] There are many people (10.sup.5-10.sup.6) affected every
year, and there is no method of detection that works. Early
diagnosis of the presence and type of bacteria in a patient's blood
stream would help prevent the death of millions of people dying
from sepsis. Currently, blood is drawn from a patient and cultures
are done to grow the bacteria. However it usually takes days to
weeks to grow enough bacteria to detect them, and by the time they
grow in the culture, they also grow in the patient, and the patient
becomes very sick.
[0006] The broth in the blood culture bottle is the first step in
creating an environment in which bacteria will grow. It contains
all the nutrients that bacteria need to grow. If the physician
expects anaerobic bacteria to grow, oxygen will be kept out of the
blood culture bottle; if aerobes are expected, oxygen will be
allowed in the bottle.
[0007] The bottles are placed in an incubator and kept at body
temperature. They are watched daily for signs of growth, including
cloudiness or a color change in the broth, gas bubbles, or clumps
of bacteria. When there is evidence of growth, the laboratory does
a gram stain and a subculture. To do the gram stain, a drop of
blood is removed from the bottle and placed on a microscope slide.
The blood is allowed to dry and then is stained with purple and red
stains and examined under the microscope. If bacteria are seen, the
color of stain they picked up (purple or red), their shape (such as
round or rectangular), and their size provide valuable clues as to
what type of microorganism they are and what antibiotics might work
best. To do the subculture, a drop of blood is placed on a culture
plate, spread over the surface, and placed in an incubator.
[0008] If there is no immediate visible evidence of growth in the
bottles, the laboratory looks for bacteria by doing gram stains and
subcultures. These steps are repeated daily for the first several
days and periodically after that.
[0009] When bacteria grow, the laboratory identifies it using
biochemical tests and the Gram stain. Sensitivity testing, also
called antibiotic susceptibility testing is performed as well. The
bacteria are tested against many different antibiotics to see which
antibiotics can effectively kill it.
[0010] All information is passed on to the physician as soon as it
is known. An early report, known as a preliminary report, is
usually available after one day. This report will tell if any
bacteria have been found yet, and if so, the results of the gram
stain. The next preliminary report may include a description of the
bacteria growing on the subculture. The laboratory notifies the
physician immediately when an organism is found and as soon as
sensitivity tests are complete. Sensitivity tests may be complete
before the bacteria are completely identified. The final report may
not be available for five to seven days. If bacteria are found, the
report will include its complete identification and a list of the
antibiotics to which the bacteria is sensitive.
[0011] What is needed is a faster and better method of detecting
organisms, including improving the accuracy and decreasing the
man-hours associated with standard blood culturing, and shortening
the time necessary to detect and identify an effective antibiotic
regimen to treat bacterial infections.
BRIEF SUMMARY
[0012] In one embodiment, a method of detecting an organism is
provided. The method comprises flowing at least two plugs in a
carrier fluid through a microchannel; introducing a sample
optionally comprising the organism into the first and second plugs;
and analyzing the at least two plugs for the detectable signal.
Each plug comprises a plug fluid that is substantially immiscible
with the carrier fluid and the organism produces a detectable
signal.
[0013] In a second embodiment, a method of detecting bacteria in a
patient is provided. The method comprises flowing at least two
plugs in a carrier fluid through a microchannel; introducing a
patient sample optionally comprising bacteria into the at least two
plugs; and analyzing the at least two plugs for the detectable
signal. Each plug comprises a plug fluid that is substantially
immiscible with the carrier fluid and the bacteria produce a
detectable signal.
[0014] In a third embodiment, a method of detecting bacteria is
provided. The method comprises flowing at least two plugs in a
carrier fluid through a microchannel; introducing a sample
optionally comprising bacteria into the first and second plugs,
wherein the bacteria produce a detectable signal; and analyzing the
plugs for the detectable signal. Each plug comprises a plug fluid
substantially immiscible with the carrier fluid. The first plug
comprises a means for detecting a first species of bacteria and the
second plug comprises a means for detecting a second species of
bacteria different from the first species of bacteria.
[0015] In a fourth embodiment, a method of detecting bacteria is
provided comprising flowing at least two plugs in a carrier fluid
through a microchannel; introducing a sample optionally comprising
bacteria into the first and second plugs; and detecting the
presence of bacteria bound beads. Each plug comprises a plug fluid
substantially immiscible with the carrier fluid. The first plug
comprises a first antibody bound bead, the first antibody bound
bead comprising a first bead and a first antibody that binds to a
first bacteria. The second plug comprises a second antibody bound
bead, the second antibody bound bead comprising a second bead and a
second antibody that binds to a second bacteria different than the
first bacteria. The bacteria bind to the antibody bound beads to
form bacteria bound beads.
[0016] In a fifth embodiment, a method of screening for antibiotic
activity is provided. The method comprises flowing at least two
plugs in a carrier fluid through a microchannel. Each plug
comprises a plug fluid substantially immiscible with the carrier
fluid and an antibody bound bead, the antibody bound bead
comprising a bead and an antibody that binds bacteria; introducing
a sample which comprises bacteria into the first and second plugs;
and detecting the presence of either antibody bound beads or
bacteria bound beads. The first plug comprises a first antibiotic
candidate and the second plug comprises a second antibiotic
candidate. The bacteria bind to the antibody bound beads to form
bacteria bound beads.
[0017] In a sixth embodiment, a method of screening for antibiotic
activity is provided comprising flowing at least two plugs in a
carrier fluid through a microchannel; introducing a sample which
comprises bacteria into the first and second plugs; detecting the
presence of either antibody bound beads or bacteria bound beads.
Each plug comprises a plug fluid substantially immiscible with the
carrier fluid and an antibody bound bead, the antibody bound bead
comprising a bead and an antibody that binds bacteria. The first
plug comprises a first antibiotic candidate and the second plug
comprises a second antibiotic candidate. The bacteria bind to the
antibody bound beads to form bacteria bound beads.
[0018] In a seventh embodiment, a method of screening for
antibiotic activity is provided comprising flowing at least two
plugs in a carrier fluid through a microchannel; introducing a
sample which comprises bacteria into the first and second plugs;
and detecting the presence of bacterial growth. Each plug comprises
a plug fluid immiscible with the carrier fluid and media capable of
supporting bacterial growth. The first plug comprises a first
antibiotic candidate and the second plug comprises a second
antibiotic candidate.
[0019] In an eighth embodiment, a method of detecting molecules is
provided comprising providing a first set of molecules that
constitute an autocatalytic loop, capable of amplification of one
of the components; providing a second set of molecules that
modulate the autocatalytic loop when the autocatalytic loop reacts
with the target molecules; and analyzing for the presence of the
target molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates a method of detecting bacteria using
stochastic confinement of bacteria into plugs reduces detection
time. (a) A schematic drawing illustrating the increase in cell
density resulting from the stochastic confinement of an individual
bacterium in a nanoliter-sized plug. (b) A schematic drawing
illustrates the experimental procedure to compare the detection of
bacteria incubated in nanoliter-sized plugs and bacteria incubated
in a milliliter-scale culture. (c) An illustrative graph of
decreased detection time vs. the log of the plug volume. (d) An
illustrative graph of detection times vs. cell density for bacteria
incubated in plugs (circles) and bacteria incubated in 96 well
plates (crosses) with similar initial cell densities.
[0021] FIG. 2. illustrates a method for screening many antibiotics
against a bacterial sample using a combination stochastic
confinement with microfluidic hybrid and/or cartridge methods. (a)
A schematic drawing illustrating the formation of plugs of
bacteria, viability indicator, and antibiotic from a preformed
array of plugs of different antibiotics. (b) An illustrative graph
of fluorescence intensity of the control plugs with no antibiotic
(+, blank1, positive control) and vancomycin (A, VCM, negative
control). (c) An illustrative bar graph shows the results of the
antibiotic screen against the Methicillin Resistant S. aureus
(MRSA), indicating that this strain of MRSA was resistant to four
antibiotics, but sensitive to two. (d) An illustrative chart shows
the agreement between the susceptibility profiles (S, sensitive and
R, resistant) of MRSA determined by the plug-based microfluidic
screen and the control susceptibility screen using Mueller Hinton
plates.
[0022] FIG. 3 illustrates detecting active and inactive particles.
(a) a solution of target (large circles) and non-target (small
circles) particles; (b) active particle decorated with antibodies
or small peptides; (c) decorated target particle separated from
non-target particles and concentrated by stochastic confinement;
(d) rapid detection and optical readout.
[0023] FIG. 4 illustrates stochastic confinement of particles by
(a) encapsulation in droplets, (b) placement in pores of a
membrane, and (c) confinement on materials with restricted
transport.
[0024] FIG. 5 is an illustrative method for analyte detection. (a)
particles stochastically confined into plugs which undergo
two-stage amplification on chip to give a macroscale readout; (b)
particles confined on membranes which translate their activity to a
signal seen by the naked eye on an upper layer; (c) particles
trapped in a gel with amplification cascades incorporated localize
the output signal over the active particle.
[0025] FIG. 6 is an illustrative method used to identify the
minimal inhibitory concentration (MIC) of cefoxitin (CFX) for
Methicillin Sensitive S. aureus (MSSA) and Methacillin Resistant S.
aureus (MRSA). (a) a schematic drawing illustrates formation of
plugs of bacteria, viability indicator, and an antibiotic at
varying concentrations; (b and c) Using 24 mg/L CFX as the
baseline, graphs show the average change in intensity of plugs
greater than (solid) and less than (striped) 3 times the baseline
for MRSA (b) and MSSA (c).
[0026] FIG. 7 is an illustrative combination of stochastic
confinement with the plug-based microfluidic assay used to
determine susceptibility of bacteria to an antibiotic in a natural
matrix, blood plasma. (a) a schematic drawing illustrating
formation of plugs of bacteria, viability indicator, antibiotic,
and plasma/LB mixture; (b and c) Images and linescans of four
representative plugs made from a 1:1 blood plasma/LB sample
inoculated with MRSA without (left) and with (right) the addition
of AMP; (d and e) Images and linescans of four representative plugs
made from a 1:1 blood plasma/LB sample inoculated with MSSA without
(left) and with (right) the addition of AMP
[0027] FIG. 8 is an illustrative schematic description of a test
strip with amplification system.
[0028] FIG. 9 is an illustrative schematic drawing of a test strip
with both detection region and control "timer" region.
[0029] FIG. 10 is an illustrative chemical amplification process
involving a one-step positive feedback.
[0030] FIG. 11 is an illustrative chemical amplification process
involving a two-step positive feedback.
[0031] FIG. 12 is an illustrative chemical amplification process
with a cascade of two positive feedback loops.
[0032] FIG. 13 is an illustrative two-step amplification cascade
involving blood coagulation enzymes.
[0033] FIG. 14 is a graph of time to get response versus amount of
input obtained by simulation.
[0034] FIG. 15 is a graph of blood clotting time varied with size
of patch of tissue factor.
[0035] FIG. 16 is an illustrative example of combining
amplification cascades with stochastic confinement.
[0036] FIG. 17 is an illustrative example of selective detection of
particles by using stochastic confinement.
DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED
EMBODIMENTS
Definitions
[0037] The term "organism" refers to any organisms or
microorganism, including bacteria, yeast, fungi, viruses, protists
(protozoan, micro-algae), archaebacteria, and eukaryotes. The term
"organism" refers to living matter and viruses comprising nucleic
acid that can be detected and identified by the methods of the
invention. Organisms include, but are not limited to, bacteria,
archaea, prokaryotes, eukaryotes, viruses, protozoa, mycoplasma,
fungi, and nematodes. Different organisms can be different strains,
different varieties, different species, different genera, different
families, different orders, different classes, different phyla,
and/or different kingdoms.
[0038] Organisms may be isolated from environmental sources
including soil extracts, marine sediments, freshwater sediments,
hot springs, ice shelves, extraterrestrial samples, crevices of
rocks, clouds, attached to particulates from aqueous environments,
involved in symbiotic relationships with multicellular organisms.
Examples of such organisms include, but are not limited to
Streptomyces species and uncharacterized/unknown species from
natural sources.
[0039] Organisms included genetically engineered organisms.
[0040] Further examples of organisms include bacterial pathogens
such as: Aeromonas hydrophila and other species (spp.); Bacillus
anthracis; Bacillus cereus; Botulinum neurotoxin producing species
of Clostridium; Brucella abortus; Brucella melitensis; Brucella
suis; Burkholderia mallei (formally Pseudomonas mallei);
Burkholderia pseudomallei (formerly Pseudomonas pseudomallei);
Campylobacter jejuni; Chlamydia psittaci; Clostridium botulinum;
Clostridium botulinum; Clostridium perfringens; Coccidioides
immitis; Coccidioides posadasii; Cowdria ruminantium (Heartwater);
Coxiella burnetii; Enterovirulent Escherichia coli group (EEC
Group) such as Escherichia coli-enterotoxigenic (ETEC), Escherichia
coli-enteropathogenic (EPEC), Escherichia coli --O157:H7
enterohemorrhagic (EHEC), and Escherichia coli-enteroinvasive
(EIEC); Ehrlichia spp. such as Ehrlichia chaffeensis; Francisella
tularensis; Legionella pneumophilia; Liberobacter africanus;
Liberobacter asiaticus; Listeria monocytogenes; miscellaneous
enterics such as Klebsiella, Enterobacter, Proteus, Citrobacter,
Aerobacter, Providencia, and Serratia; Mycobacterium bovis;
Mycobacterium tuberculosis; Mycoplasma capricolum; Mycoplasma
mycoides ssp mycoides; Peronosclerospora philippinensis; Phakopsora
pachyrhizi; Plesiomonas shigelloides; Ralstonia solanacearum race
3, biovar 2; Rickettsia prowazekii; Rickettsia rickettsii;
Salmonella spp.; Schlerophthora rayssiae varzeae; Shigella spp.;
Staphylococcus aureus; Streptococcus; Synchytrium endobioticum;
Vibrio cholerae non-01; Vibrio cholerae O1; Vibrio parahaemolyticus
and other Vibrios; Vibrio vulnificus; Xanthomonas oryzae; Xylella
fastidiosa (citrus variegated chlorosis strain); Yersinia
enterocolitica and Yersinia pseudotuberculosis; and Yersinia
pestis.
[0041] Further examples of organisms include viruses such as:
African horse sickness virus; African swine fever virus; Akabane
virus; Avian influenza virus (highly pathogenic); Bhanja virus;
Blue tongue virus (Exotic); Camel pox virus; Cercopithecine
herpesvirus 1; Chikungunya virus; Classical swine fever virus;
Coronavirus (SARS); Crimean-Congo hemorrhagic fever virus; Dengue
viruses; Dugbe virus; Ebola viruses; Encephalitic viruses such as
Eastern equine encephalitis virus, Japanese encephalitis virus,
Murray Valley encephalitis, and Venezuelan equine encephalitis
virus; Equine morbillivirus; Flexal virus; Foot and mouth disease
virus; Germiston virus; Goat pox virus; Hantaan or other Hanta
viruses; Hendra virus; Issyk-kul virus; Koutango virus; Lassa fever
virus; Louping ill virus; Lumpy skin disease virus; Lymphocytic
choriomeningitis virus; Malignant catarrhal fever virus (Exotic);
Marburg virus; Mayaro virus; Menangle virus; Monkeypox virus;
Mucambo virus; Newcastle disease virus (VVND); Nipah Virus; Norwalk
virus group; Oropouche virus; Orungo virus; Peste Des Petits
Ruminants virus; Piry virus; Plum Pox Potyvirus; Poliovirus; Potato
virus; Powassan virus; Rift Valley fever virus; Rinderpest virus;
Rotavirus; Semliki Forest virus; Sheep pox virus; South American
hemorrhagic fever viruses such as Flexal, Guanarito, Junin,
Machupo, and Sabia; Spondweni virus; Swine vesicular disease virus;
Tick-borne encephalitis complex (flavi) viruses such as Central
European tick-borne encephalitis, Far Eastern tick-borne
encephalitis, Russian spring and summer encephalitis, Kyasanur
forest disease, and Omsk hemorrhagic fever; Variola major virus
(Smallpox virus); Variola minor virus (Alastrim); Vesicular
stomatitis virus (Exotic); Wesselbron virus; West Nile virus;
Yellow fever virus; and South American hemorrhagic fever viruses
such as Junin, Machupo, Sabia, Flexal, and Guanarito.
[0042] Further examples of organisms include parasitic protozoa and
worms, such as: Acanthamoeba and other free-living amoebae;
Anisakis sp. and other related worms Ascaris lumbricoides and
Trichuris trichiura; Cryptosporidium parvum; Cyclospora
cayetanensis; Diphyllobothrium spp.; Entamoeba histolytica;
Eustrongylides sp.; Giardia lamblia; Nanophyetus spp.; Shistosoma
spp.; Toxoplasma gondii; Filarial nematodes and Trichinella.
Further examples of analytes include allergens such as plant pollen
and wheat gluten.
[0043] Further examples of organisms include fungi such as:
Aspergillus spp.; Blastomyces dermatitidis; Candida; Coccidioides
immitis; Coccidioides posadasii; Cryptococcus neoformans;
Histoplasma capsulatum; Maize rust; Rice blast; Rice brown spot
disease; Rye blast; Sporothrix schenckii; and wheat fungus.
[0044] Further examples of organisms include worms such as C.
Elegans and pathogenic worms nematodes.
[0045] "Particle" as used herein refers to an organism, molecule,
cell, a viral particle, spore, and the like.
[0046] "Patient sample" refers to a sample obtained from a patient
or person and includes blood, feces, urine, saliva or other bodily
fluid, preferably blood. Food samples may also be analyzed.
[0047] "Sample" refers to any sample potentially comprising an
organism. Environments for finding organisms include, but are not
limited to geothermal and hydrothermal fields, acidic soils,
sulfotara and boiling mud pots, pools, hot-springs and geysers
where the enzymes are neutral to alkaline, marine actinomycetes,
metazoan, endo and ectosymbionts, tropical soil, temperate soil,
arid soil, compost piles, manure piles, marine sediments,
freshwater sediments, water concentrates, hypersaline and
super-cooled sea ice, arctic tundra, Sargosso sea, open ocean
pelagic, marine snow, microbial mats (such as whale falls, springs
and hydrothermal vents), insect and nematode gut microbial
communities, plant endophytes, epiphytic water samples, industrial
sites and ex situ enrichments. Additionally, a sample may be
isolated from eukaryotes, prokaryotes, myxobacteria (epothilone),
air, water, sediment, soil or rock, a plant sample, a food sample,
a gut sample, a salivary sample, a blood sample, a sweat sample, a
urine sample, a spinal fluid sample, a tissue sample, a vaginal
swab, a stool sample, an amniotic fluid sample and/or a buccal
mouthwash sample.
[0048] Microfluidics is an attractive platform for rapid
single-cell functional analysis. (M. Y. He, J. S. Edgar, G. D. M.
Jeffries, R. M. Lorenz, J. P. Shelby and D. T. Chiu, Anal. Chem.,
2005, 77, 1539-1544; A. Grodrian, J. Metze, T. Henkel, K. Martin,
M. Roth and J. M. Kohler, Biosens. Bioelectron., 2004, 19,
1421-1428; D. B. Weibel, W. R. DiLuzio and G. M. Whitesides, Nat.
Rev. Microbiol., 2007, 5, 209-218; Y. Marcy, T. Ishoey, R. S.
Lasken, T. B. Stockwell, B. P. Walenz, A. L. Halpern, K. Y. Beeson,
S. M. D. Goldberg and S. R. Quake, PLoS Genet., 2007, 3, 1702-1708;
J. El-Ali, S. Gaudet, A. Gunther, P. K. Sorger and K. F. Jensen,
Anal. Chem., 2005, 77, 3629-3636; A. Huebner, M. Srisa-Art, D.
Holt, C. Abell, F. Hollfelder, A. J. Demello and J. B. Edel, Chem.
Commun., 2007, 1218-1220; H. M. Yu, C. M. Alexander and D. J.
Beebe, Lab Chip, 2007, 7, 726-730; C. J. Ingham, A. Sprenkels, J.
Bomer, D. Molenaar, A. van den Berg, J. Vlieg and W. M. de Vos,
Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 18217-18222; R. D.
Whitaker and D. R. Walt, Anal. Chem., 2007, 79, 9045-9053.) Plugs,
for example, droplets of aqueous solution surrounded by a
fluorinated carrier fluid, provide a simple platform for
manipulating samples with no dispersion or losses to interfaces.
(H. Song, D. L. Chen and R. F. Ismagilov, Angew. Chem.-Int. Edit.,
2006, 45, 7336-7356; H. Song, J. D. Tice and R. F. Ismagilov,
Angew. Chem.-Int. Edit., 2003, 42, 768-772.) Microfluidic
plug-based assays provide the ability to reduce detection time by
confining bacterium into nanoliter-sized plugs. This confinement,
referred to as "stochastic confinement" decreases detection time by
confining the sample into plugs that either have a single
bacterium, or are empty. This approach increases the effective
concentration of the bacterium, and allows released molecules to
accumulate in the plug. Such stochastic trapping is commonly used
for single-cell analysis in microfluidic devices, (M. Y. He, J. S.
Edgar, G. D. M. Jeffries, R. M. Lorenz, J. P. Shelby and D. T.
Chiu, Anal. Chem., 2005, 77, 1539-1544; Y. Marcy, T. Ishoey, R. S.
Lasken, T. B. Stockwell, B. P. Walenz, A. L. Halpern, K. Y. Beeson,
S. M. D. Goldberg and S. R. Quake, PLoS Genet., 2007, 3, 1702-1708;
A. Huebner, M. Srisa-Art, D. Holt, C. Abell, F. Hollfelder, A. J.
Demello and J. B. Edel, Chem. Commun., 2007, 1218-1220; S.
Takeuchi, W. R. DiLuzio, D. B. Weibel and G. M. Whitesides, Nano
Lett., 2005, 5, 1819-1823; P. Boccazzi, A. Zanzotto, N. Szita, S.
Bhattacharya, K. F. Jensen and A. J. Sinskey, App. Microbio.
Biotech., 2005, 68, 518-532; V. V. Abhyankar and D. J. Beebe, Anal.
Chem., 2007, 79, 4066-4073) and similar techniques have been used
for single molecule and single enzyme work. (H. H. Gorris, D. M.
Rissin and D. R. Walt, Proc. Natl. Acad. Sci. U.S.A., 2007, 104,
17680-17685; A. Aharoni, G. Amitai, K. Bernath, S. Magdassi and D.
S. Tawfik, Chem. Biol., 2005, 12, 1281-1289; O. J. Miller, K.
Bernath, J. J. Agresti, G. Amitai, B. T. Kelly, E. Mastrobattista,
V. Taly, S. Magdassi, D. S. Tawfik and A. D. Griffiths, Nat.
Methods, 2006, 3, 561-570; J. Huang and S. L. Schreiber, Proc.
Natl. Acad. Sci. U.S.A., 1997, 94, 13396-13401; D. T. Chiu, C. F.
Wilson, F. Ryttsen, A. Stromberg, C. Farre, A. Karlsson, S.
Nordholm, A. Gaggar, B. P. Modi, A. Moscho, R. A. Garza-Lopez, O.
Orwar and R. N. Zare, Science, 1999, 283, 1892-1895; J. Yu, J.
Xiao, X. J. Ren, K. Q. Lao and X. S. Xie, Science, 2006, 311,
1600-1603.) Microfluidics also enables simultaneous execution of
numerous assays of bacterial function from a single bacterial
sample in the same experiment, which is especially useful for rapid
antibiotic susceptibility screening. Previously, gel microdroplets
had been utilized for susceptibility screening. (Y. Akselband, C.
Cabral, D. S. Shapiro and P. McGrath, J. Microbiol. Methods, 2005,
62, 181-197; C. Ryan, B. T. Nguyen and S. J. Sullivan, J. Clin.
Microbiol., 1995, 33, 1720-1726) However, this method did not take
advantage of the stochastic confinement effects in plugs or
high-throughput screening methods of current microfluidic
technologies. Confinement effects as described herein are increased
if the gel droplets are surrounded by a barrier substantially
impermeable to released products (for example, a fluorous liquid or
a non-porous solid). An immiscible fluid surrounding the droplet
will form a barrier to prevent or reduce loss of released products
from a cell, enabling the released products to accumulate more
rapidly and reach higher concentrations in the droplet Microfluidic
technology offers two advantages over traditional bacterial
detection and drug screening methods: 1) stochastic confinement of
single cells from dilute samples concentrates the bacteria,
eliminates the need for pre-incubation, and reduces detection time;
2) each assay can be performed by using an individual bacterium,
enabling hundreds of assays to be performed using a single, low
density bacterial sample without pre-incubation. This technology
will reduce the time needed to diagnose bacterial infections and
enable patient-specific antibiotic regimens. This technology also
has the advantage of separating objects into individual and
separate volumes of fluid by forming plugs. A further advantage is
that by using techniques such as the hybrid method to perform high
throughput screening of multiple reagents and conditions using only
a small volume sample.
[0049] Examples of microfluidic technology including descriptions
of uses, applications and techniques for plug-based methods of
analysis, manipulation of plugs, hybrid plug merging, cartridge
formation, use and handling, and holding component and loading
component handling, use and formation, use of markers and fluid
handling include U.S. Pat. No. 7,029,091; U.S. Published Patent
Applications 2005/0087122 A1, 2005/0019792 A1, 2007/0172954 A1,
2007/0195127 A1, 2007/0052781 A1, 2006/0003439 A1, 2006/0094119 A1,
2006/0078893 A1; 2006/0078888 A1, 2007/0184489 A1, 2007/0092914 A1,
2005/0221339 A1, 2007/0003442 A1, 2006/0163385 A1, 2005/0172476 A1,
2008/0003142 A1, 2008/0014589 A1; and WIPO published international
applications WO 07/081,386 A2, WO 07/081,387 A1, WO 07/133,710 A2,
WO 07/081,385 A2, WO 08/063,227 A2, WO 07089541 A2, WO 07/030,501
A2, WO 06/096571 A2. These references are incorporated by reference
in their entirety.
[0050] Approaches that may be used for bacterial detection include:
1) the use of cartridges (cartridges pre-loaded with reagents for
different detection methods/growth media/antibiotics may be used to
detect and identify bacteria); 2) the hybrid method to test many
antibiotics/substrates/detection growth conditions or culturing
conditions at different concentrations; and 3)
screening/sorting.
[0051] In addition to concentrating the sample by stochastic
confinement, other microfluidic on-chip approaches may be used to
preconcentrate a sample before detection. One method is to flow the
initial sample through a device which contains a structure (such as
a filter) that would collect all of the objects (cells, particles,
molecules or proteins bound to particles) needed to be detected.
The structure traps the objects (through size exclusion such as a
filter or through specific chemical or physical interactions with
the objects) but enables the aqueous fluid in which the objects are
suspended to pass through the structure. Once all of the objects
have been collected in the structure, the objects may be
resuspended by another aqueous flow such that the volume of aqueous
fluid used to resuspend the objects is less than the volume in
which the objects were originally suspended. The resuspended
objects, now at a higher concentration, may then be loaded into
plugs or droplets for further concentration due to stochastic
confinement.
[0052] Other concentration techniques may be used such as
centrifugation, attaching magnetic beads to the particles of
interest, or having surfaces which selectively bind to the target
and then release the target at a later time. For example, see S.
Song and A Singh, Analytical and Bioanalytical Chemistry, Volume
384, Number 1/January, 2006, 41-43, and P. Gridzinski, J Yang, R H
Liu, M D Ward, Biomedical Microdevices, Volume 5, Number
4/December, 2003, pg. 303-310.
[0053] The experiments may be done by slowly flowing plugs into a
tube, incubating them as they flow (with heating/cooling for PCR if
need be along the way), flowing them by the detector, and then
dumping them. This may address the "storage of 1,000,000 plugs"
problem.
[0054] The 1,000,000 plugs problem with stochastic confinement
refers to the fact that to concentrate a sample 1,000,000 fold,
999,999 empty plugs must be formed for every 1,000,000 fold
concentrated plug. One approach to this problem is do some initial
screening using a less sophisticated method (such as flow cytometry
or optical scanning) to sort plugs into occupied/unoccupied groups.
These simple tests would not provide a detailed characterization of
the object in the plug, but would rapidly determine whether or not
the plug is occupied by an object. Then the advantage of stochastic
confinement can still be realized by running more tests to rapidly
characterize the occupied plugs. It is also possible to run an
initial screen that does take advantage of stochastic confinement,
by first running a general test for the presence of the object (a
simple fluorescent viability assay for bacteria) and then sorting
the plugs into occupied/unoccupied before adding additional sets of
reagents to test for further characterization of the object. It is
also possible to take a blood sample (for example 10 mL), make it
into a bulk emulsion, apply the procedure above, and then sort the
plugs or analyze the plugs using flow cytometry and the like.
[0055] A possible method for encapsulating large numbers of cells
is similar to sequential merging of reagents to plugs flowing in a
1D microfluidic channel (straight channel). However, the cells are
introduced into a 2D channel (width of channel much larger than
width of cell), and the cells flow through regions in which various
reagents are applied to the cells. The 2D channel should be
approximately similar in height to the cells such that the cells
flowing through the device form a monolayer. For detection, plugs
may or may not be encoded. If the plugs are not encoded, plugs may
be analyzed for those that respond for more complex tests like
susceptibility tests. Plugs may be encoded by position, or by an
internal marker (for example a fluorescent marker of different
colors) if not encoded. The same tests may be done by performing
multiple non-encoded tests in parallel.
Blood Cultures
[0056] There are many variables involved in performing a blood
culture. Before a person's blood is drawn, the physician must make
several decisions based on knowledge of infections and the person's
clinical condition and medical history.
[0057] Several groups of microorganisms, including bacteria,
viruses, mold, and yeast, can cause blood infections. The bacteria
group can be further broken down into aerobes and anaerobes. Most
aerobes do not need oxygen to live. They can grow with oxygen
(aerobic microbes) or without oxygen (anaerobic microbes).
[0058] Based on the clinical condition of the patient, the
physician determines what group of microorganisms is likely to be
causing the infection and then orders one or more specific types of
blood culture, including aerobic, anaerobic, viral, or fungal (for
yeasts and molds). Each specific type of culture is handled
differently by the laboratory. Most blood cultures test for both
aerobic and anaerobic microbes. Fungal, viral, and mycobacterial
blood cultures can also be done, but are less common.
[0059] The physician must also decide how many blood cultures
should be done. One culture is rarely enough, but two to three are
usually adequate. Four cultures are occasionally required. Some
factors influencing this decision are the specific microorganisms
the physician expects to find based on the person's symptoms or
previous culture results, and whether or not the person has had
recent antibiotic therapy.
[0060] The time at which the cultures are to be drawn is another
decision made by the physician. During most blood infections
(called intermittent bacteremia) microorganisms enter the blood at
various time intervals. Blood drawn randomly may miss the
microorganisms. Since microorganisms enter the blood 30-90 minutes
before the person's fever spikes, collecting the culture just after
the fever spike offers the best likelihood of finding the
microorganism. The second and third cultures may be collected at
the same time, but from different places on the person, or spaced
at 30-minute or one-hour intervals, as the physician chooses.
During continuous bacteremia, such as infective endocarditis,
microorganisms are always in the blood and the timing of culture
collection is less important. Blood cultures should always be
collected before antibiotic treatment has begun.
[0061] Bacteria are the most common microorganisms found in blood
infections. Laboratory analysis of a bacterial blood culture
differs slightly from that of a fungal culture and significantly
from that of a viral culture.
[0062] Blood is drawn from a person and put directly into a blood
culture bottle containing a nutritional broth. After the laboratory
receives the blood culture bottle, several processes must be
completed: providing an environment for the bacteria to grow;
detecting the growth when it occurs; identifying the bacteria that
grow; testing the bacteria against certain antibiotics to determine
which antibiotic will be effective.
[0063] Given that a typical 5 mL blood sample from a patient with
bacteremia contains a cell density of 100 CFU/mL, (L. G. Reimer, M.
L. Wilson and M. P. Weinstein, Clin. Microbiol. Rev., 1997, 10,
444-7) the methods of the present invention are capable of
performing dozens of functional tests on such a sample.
Patient-specific characterization of bacterial species not only
allows more rapid and effective treatment, but also enables
in-depth characterization of bacterial infections at the population
level. Such detailed characterization can aid in tracking and
identifying new resistance patterns in bacterial pathogens. (S. K.
Fridkin, J. R. Edwards, F. C. Tenover, R. P. Gaynes and J. E.
McGowan, Clin. Infect. Dis., 2001, 33, 324-329; and R. T. Horvat,
N. E. Klutman, M. K. Lacy, D. Grauer and M. Wilson, J. Clin.
Microbiol., 2003, 41, 4611-4616) The principles of these methods,
stochastic single-cell confinement and multiple functional assays
without sample pre-incubation, can also be applied to other areas,
including performing functional tests on field samples, detecting
contamination of food or water, separating and testing samples with
mixtures of species, measuring functional heterogeneity in
bacterial populations, and monitoring industrial bioprocesses.
[0064] Other patient samples that may be collected include feces,
urine or saliva. The latter would be useful for assessing oral
health. Tears may be collected for diagnosing eye infections.
Interstitial fluid (also referred to as tissue fluid or
intercellular fluid) may be collected and use to diagnose
infections in the pleural space around the lungs.
[0065] Examples of bacterial infections, include, but are not
limited to those listed in Table 1.
TABLE-US-00001 TABLE 1 Types of Bacterial Infection Type of
Infection Description Examples Inapparent No detectable clinical
Asymptomatic (subclinical) symptoms of infection gonorrhea in women
and men Dormant (latent) Carrier state Typhoid carrier Accidental
Zoonosis or Anthrax, cryptococcal environmanetal or infection, and
inadvertent exposures laboratory exposure, respectively
Opportunistic Infection caused by Serrati or Candida normal flora
or infection of the transient bacteria genitourinary tract when
normal host defenses are compromised Primary Clinically apparent
Shigella dysentery (e.g. invasion and multiplication of microbes in
body tissues, causing local tissue injury) Secondary Microbial
invasion Bacterial pneumonia subsequent to primary following viral
lung infection infection Mixed Two or more Anaerobic abscess (E.
coli microbes infecting the and Bacteroides same tissue fragilis)
Acute Rapid onset (hours or Diptheria days); brief duration (days
or weeks) Chronic Prolonged duration Mycobacterial (months or
years) diseases (tus and leprosy) Localized Confined to a small
Staphylococcal boil area or to an organ Generalized Disseminated to
many Gram-negative body regions bacteria (gonococcernia) Pyogenic
Pus-forming Staphylococcal and streptococcal infection Retrograde
Microbes ascending E. coli urinary tract in a duct or tube
infection against the flow of secretions or excretions Fulminant
Infections that occur Airborne Yersinia suddenly and pestis
(pneumonic intensely plague)
[0066] Bacterial Detection
[0067] To monitor the presence and metabolically active bacteria in
plugs, a fluorescent viability indicator alamarBlue.RTM. was added
to the cultures. The active ingredient of alamarBlue is the
fluorescent redox indicator resazurin. (J. O'Brien and F. Pognan,
Toxicology, 2001, 164, 132-132.) Resazurin is reduced by electron
receptors used in cellular metabolic activity, such as NADH and
FADH, to produce the fluorescent molecule resofurin. Therefore,
fluorescence intensity in a plug is correlated with the presence
and metabolic activity of a cell, in this case, a bacterium.
Because resazurin indicates cell viability, resazurin-based assays
have been used previously in antibiotic testing. (S. G. Franzblau,
R. S. Witzig, J. C. McLaughlin, P. Torres, G. Madico, A. Hernandez,
M. T. Degnan, M. B. Cook, V. K. Quenzer, R. M. Ferguson and R. H.
Gilman, J. Clin. Microbiol., 1998, 36, 362-366; A. Martin, M.
Camacho, F. Portaels and J. C. Palomino, Antimicrob. Agents
Chemother., 2003, 47, 3616-3619; K. T. Mountzouros and A. P.
Howell, J. Clin. Microbiol., 2000, 38, 2878-2884; C. N. Baker and
F. C. Tenover, J. Clin. Microbiol., 1996, 34, 2654-2659.) Resazurin
may be used to detect both the presence of a live bacterium and the
response of bacteria to drugs, such as antibiotics. Stochastic
confinement decreases detection time because in a plug that has the
bacterium, the bacterium is at an effectively higher concentration
than in the starting solution, and the signal-to-noise required for
detection is reached sooner since the product of reduction of
resazurin accumulates in the plug more rapidly.
[0068] To demonstrate the ability of stochastic confinement to
reduce detection time, a single sample of Staphylococcus aureus (S.
aureus) containing the fluorescent viability indicator was split.
Half of the culture was used to generate plugs of nanoliter volume,
and the other half remained as a milliliter-scale culture. Both the
nanoliter plugs and the milliliter-scale culture were incubated for
2.8 h at 37.degree. C. After incubation, the milliliter-scale
culture was used to form plugs. This experimental procedure is
illustrated in FIG. 1b. Line scans indicate that confining the
bacteria at the beginning of incubation (t=0), led to a few
occupied plugs with a high fluorescence intensity and many empty
plugs with low fluorescence intensity (solid line). All plugs made
from the milliliter-scale culture had an intermediate intensity
(dotted line). Confining bacteria into plugs of nanoliter volume
reduced the time required to detect a change in fluorescence
intensity of the viability indicator. Bacteria confined to and
incubated in nanoliter-sized plugs showed a greater change in
fluorescence intensity after 2.8 h than the bacteria incubated in
the "unconfined" milliliter-scale culture (FIG. 1c). Line scans of
the plugs of bacteria that were incubated in plugs showed many
empty plugs with low fluorescence intensity and a few occupied
plugs with high fluorescence intensity (FIG. 1b, top). However,
lines scans of plugs of bacteria that were incubated in the
milliliter-scale culture have a lower, uniform fluorescence
intensity (FIG. 1b, bottom). Therefore, bacteria confined to
nanoliter-sizes plugs may be detected earlier than bacteria in a
milliliter-scale culture.
[0069] In plugs containing single bacterium, the detection time was
proportional to plug volume. Detection time was defined as the time
at which the increase in fluorescence intensity reached a maximum.
When single bacterium were confined in plugs ranging from 1 mL to
1500 mL in volume, detection time increased with the log of plug
volume (FIG. 1c), implying that bacteria were dividing
exponentially inside the plugs. This result is similar to previous
estimates that detection time decreases by about 1.5 h for every
order of magnitude increase in cell density. (P. Kaltsas, S. Want
and J. Cohen, Clin. Microbiol. Infect., 2005, 11, 109-114) The
detection times measured for bacteria incubated in plugs were
similar to detection times measured for bacteria incubated in a 96
well plate from cultures with similar initial cell densities (FIG.
1d). This result implies that incubation in plugs had no adverse
effects on growth of bacteria.
[0070] Detecting low concentrations of species (down to single
molecules and single bacteria) is a challenge in food, medical, and
security industries. Plugs may allow one to concentrate such
samples and perform analysis. For example, a sample containing
small amounts of DNA of interest in the presence of an excess of
other DNA may be amplified. Amplification may be detected if plugs
are made small enough that some plugs contain single DNA molecules
of interest, and other plugs contain no DNA molecules of interest.
This separation into plugs effectively creates plugs with higher
DNA of interest concentration than in the original sample.
Amplification of DNA in those plugs, for example by PCR, may lead
to higher signal than amplification of the original sample. In
addition, localization of bacteria in plugs by a similar method may
create a high local concentration of bacteria (1 per very small
plug), making them easier to detect. For some bacteria that use
quorum sensing, this may be a method to activate and detect them.
Such bacteria may be inactive/non-pathogenic and difficult to
detect at low concentrations due to lack of activity, but at a high
concentration of bacteria, the concentration of a signaling
molecule increases, activating the bacteria. If a single bacterium
is localized in a plug, the signaling molecule produced by a
bacterium cannot diffuse away and its concentration will rapidly
increase, triggering activation of the bacterium, making it
possible for detection. In addition, plugs may be used to localize
cells and bacteria by creating gels or matrixes inside plugs.
Bacteria and other species (particles and molecules) may be
collected and concentrated into plugs by putting air through a plug
fluid such as water, and then using that plug fluid to generate
plugs. For example, by making smaller plugs from the initial plug,
some of the newly formed smaller plugs will contain sample while
other plugs will not contain the sample, but only buffer, for
example. This results in concentrated sample containing plugs
because some of the plugs do not contain any of the sample.
[0071] This method is not limited to liquid samples. Microorganisms
and other particulate matter can be detected in gaseous samples,
such as samples of air taken at airports or along train routes.
There are numerous methods for collecting airborne particles in
water, for example, as described in U.S. Pat. Nos. 7,201,878,
7,243,560, and 5,855,652 all incorporated by reference herein in
their entirety. After collecting the airborne particulate matter in
water, these samples can then be added directly to the fluorinated
oil carrier fluid to form plugs.
[0072] PCR techniques are disclosed in the following published US
patent applications and International patent applications: US
2008/0166793 A1, WO 08/069,884 A2, US 2005/0019792 A1, WO
07/081,386 A2, WO 07/081,387 A1, WO 07/133,710 A2, WO 07/081,385
A2, WO 08/063,227 A2, US 2007/0195127 A1, WO 07/089,541 A2, WO
07030501 A2, US 2007/0052781 A1, WO 06096571 A2, US 2006/0078893
A1, US 2006/0078888 A1, US 2007/0184489 A1, US 2007/0092914 A1, US
2005/0221339 A1, US 2007/0003442 A1, US 2006/0163385 A1, US
2005/0172476 A1, US 2008/0003142 A1, and US 2008/0014589 A1, all of
which are incorporated by reference herein in their entirety.
[0073] Amplifications of nucleic acids have been performed via
polymerase chain reactions (PCR). The key concept of PCR comprises
genetic template (primer), thermostable DNA polymerase, and the
circuit for regulating temperature. By combining these components
in a miniaturized microfluidic device capable of implementing
cartridge and/or hybrid methods, a high concentration of
interesting DNA fragments from a very small amount of genetic
sample via trivial PCR methods may be collected. It has been
confirmed that PDMS is a heat-stable material, indicating it is a
suitable material for the PCR process.
[0074] With respect to the cartridge, the plug-based microfluidic
platform may be used to setup a huge number of reaction centers in
nano- or pico-liter volume scale, extending into the femtoliter and
microliter scales. With respect to the hybrid method, various
conditions of reactions and samples may be incorporated, split, and
merged in a microfluidic device.
[0075] For example, a sample comprising 1% of DNA of interest in
the presence of 99% of background DNA would need to be amplified
enough to harvest and use. However, amplification of 1% of DNA by a
factor of 100 only increases the total amount of DNA by a factor of
2, resulting in detection difficulties. By making small enough
plugs to comprise a single molecule of DNA of interest, and
amplifying each plug by means of conventional PCR techniques in a
microfluidic device, highly amplified PCR products of target 1% of
DNA in a plug may be obtained. Assume that the probability of
appearance of target DNA as a single molecule in a plug is one in
every 10.sup.th plug, then the ratio of target DNA/total DNA is
1:10 in such plugs. 1000-Fold amplifications of target DNA molecule
in those plugs, affords a 100:10 ratio of target to background DNA,
resulting in a 10 fold increase of the total amount of DNA.
Conclusively, the target DNA from the very low concentration may
easily be detected under the presence of large background signals.
In the microfluidic device, a platform cartridge incorporating a
carrier fluid channel, a sampling channel, and one or two PCR
reagent channels may be made. By regulating each fluid, individual
plugs containing a single molecule of target DNA or different
molecules of DNA from the sample may be generated. Along the
channel containing plugs, heating regions are placed in the channel
region by repeating heating and cooling process for the
denaturation and renaturation of DNA samples.
[0076] Stochastic confinement has applications in the isolation and
screening of rare particles or cells from a sample. When samples
are stochastically confined, the result is a set of isolated
volumes of fluid, most with either no particles or 1 particle. In
this way, rare particles are segregated from ubiquitous particles.
The separation of rare particles enables the direct assay of the
function and detection of rare particles without interference from
other particles in the system.
[0077] Even if a rare particle has high activity, since it is at
low concentration in the bulk sample it may not be detectable due
to dilution of the signal and low signal to noise ratio as a result
of low background reaction from the ubiquitous particles. Once rare
particles are isolated and concentrated through stochastic
confinement, other plug based microfluidic technologies can be used
to screen the rare particle.
[0078] For example, if the original sample contains 100 rare
particles, since stochastic confinement enables the separation and
screening of individual particles, this enables the rare particle
to be screened against up to 100 different conditions. After
confining all of the particles into droplets, those droplets may be
merged with thousands of different conditions. If the droplets are
randomly merged with screening conditions, then up to 100 different
conditions will be screened against the rare particles. If the rare
particle is allowed to divide, then the plug containing the rare
particle can be split into several smaller plugs and each plug
assayed or screened independently, for example, using the hybrid
and/or cartridge method. Examples include combining the plug
containing many rare particles with plugs of various reagents and
conditions in order to determine the function and optimal
conditions for the rare particle. As an additional example, several
stochastically confined organisms may be allowed to grow inside an
array of plugs. After the array is split into four daughter arrays,
each daughter array may be interrogated by a different technique or
reagent, while preserving the identity of plugs and their
relationship in the daughter array (for example, 37.sup.th plug in
the first array corresponds to the 37.sup.th plug in the second,
third, and forth array). The results may be combined to provide
information on the response of corresponding daughter plugs to each
of the techniques. In addition, some of the daughter arrays may be
retained as a reference culture. When the results from the other
three arrays are known, the reference culture arrays may be used
for further manipulation, characterization, assaying, and isolation
of organisms.
[0079] Isolation of rare particles through stochastic confinement
may also be combined with the hybrid and/or cartridge method for
screening growth/virulence activation/assay conditions against the
rare cells. For example, if a sample contains 10,000 cells, 100 of
which belong to a rare cell type, stochastic confinement may be
used to isolate the 100 rare cells into plugs containing only a
single rare cell and no other types of cells. Then the plugs
containing the rare cell types may be used in hybrid and/or
cartridge screening by the following methods.
[0080] The plugs generated from the stochastic confinement may be
combined with screening conditions using the hybrid and/or
cartridge method. Plugs containing the rare cells are randomly
distributed throughout all of the plugs generated (many of which do
not contain a rare cell). If many plugs (100's, 1000's) are merged
with a single screening condition, it is likely that at least one
of the plugs for that condition will contain a rare cell. In this
way, multiple growth/assay/virulence activation conditions may be
screened against the rare cell type. A separate test may be needed
to separate plugs containing rare cells from the other plugs
generated by confinement. It may be best to sort the plugs into
rare cell/common cell/empty after merging with the hybrid and/or
cartridge screen to reduce detection time. Instead of sorting for
rare cells first (which may take time) and then merging with
screening conditions, the screening and determining the presence of
rare cell may be done simultaneously.
[0081] Alternatively, it may be desirable to first separate out
plugs containing rare cells from plugs containing common cells or
no cells. This may be done using antibodies, binding assays,
testing for function specific to the rare cell combined with
automated sorting mechanisms (optical, magnetic, FACS). Once the
rare cell type plugs have been isolated, a hybrid and/or cartridge
screen can be used to screen for growth/assay/virulence activating
conditions or to run a multitude of functional and genetic tests on
the rare cell type.
[0082] Another application of stochastic confinement is to
accurately count populations of cells. Since confinement isolates 1
cell per plug and may be used to perform tests to identify the type
of cell in each plug, confinement may be used to determine the
density (number of cells of type X per volume of sample) or the
ratio (100 cells of type X for every 1 of type Y) of cells in a
sample. This may also be used to find ratios of phenotypes of cell
populations that are genetically identical (25% of Staphylococcus
cells are resistant to oxacillin or 30% of cells will induce
virulence in response to host protein X).
[0083] Screening for growth conditions is an important application
because an estimated 99% of all microbes cannot be cultured by
standard techniques. Unculturability of these organisms may be due
to: 1) nutrient levels of media are too high; 2) requirement of
specific ion concentrations; and 3) requirement for additional
factors (unusual compounds not found in most media). Confinement
has two effects: 1) it reduces competition from other microbes,
giving rare and slow growing cell types time to reproduce; and 2)
it can be combined with the hybrid and/or cartridge method to
screen for media additives and the concentration of the additive to
find new growth conditions for a previously uncultured microbe or a
microbe which is difficult to grow or slow growing under current
conditions. In addition, one may also use control of surface
chemistry provided by plugs to enhance growth of organisms.
Compounds that modulate surface chemistries may be incorporated
into the hybrid screen in addition to or along with compounds
modulating growth conditions.
[0084] In addition, an organism might be releasing products such as
quorum sensing molecules and will not initiate growth until a
threshold concentration of released products have accumulated.
Confinement will put microbial cells at higher initial cell density
and enable cells to grow and optionally activate genes associated
with high cell density. Some organisms may be able to grow in
culture, which were previously believed to be not culturable by
standard techniques due to their slow growth or growth to low
densities. These organisms may still undergo a sufficient number of
divisions when stochastically confined, and therefore allow further
detection by less-sensitive techniques that require multiple copies
of the organism to be present. When plugs are used to create
stochastic confinement, growth of the organism allows further
manipulation and analysis that cannot be done on a single plug (for
example requiring mutually incompatible methods) by splitting the
plugs, injecting reagents into them and monitoring results. These
steps may be performed sequentially, where the results of the first
experiment guides the design of the second experiment, or in
parallel. Examples of mutually incompatible methods include
reagents that produce similar signals (such as fluorescence in the
same range of wavelengths), or methods that require different
conditions (such as different solvents or pH values), or require
different states of the organisms (such as a functional test that
requires an alive organism, and a staining protocols that kills the
organism).
[0085] Implementation of this type of screening may be done in many
ways. For example, a known organism may be screened in the presence
of many media and culturing conditions (varying ion concentrations,
known autoinducers, amount of confinement, temperature, pH, protein
additives, reaction oxygen species, stress inducers; changing
carbon source and concentration of carbon source; changing nitrogen
source and concentration of nitrogen source; changing availability
of various trace metals (Mn, Mo, Cu, Pt, etc.), adding drugs known
to interfere with specific cellular activities; adding transport
and ion channel inhibitors, small molecules involved in cellular
communication, virulence activators, etc.). After using the hybrid
method to screen through many conditions, functional tests or other
assays may be performed in plugs to determine if compounds have
been generated with properties of interest (such as drug targets,
antibiotic compounds, ion channel inhibitors, virulence activation,
virulence inhibition, degradation of various compounds, binding
affinity, etc.).
[0086] One idea to investigate molecules released by microorganisms
is called OSMAC (one strain many compounds)), as described in Big
Effects from Small Changes: Possible Ways to Explore Nature's
Chemical Diversity by Helge Bjorn Bode, Barbara Bethe, Regina Hofs,
Axel Zeeck, Chem Bio Chem Volume 3 Issue 7, Pages 619-627. Small
changes in culturing conditions (for example, media composition,
aeration, culture vessel, addition of enzyme inhibitors)
drastically change the metabolites that are released from a cell.
The molecules released may aid in detection of the organism, or may
have functional uses such as antibiotics.
[0087] Therefore, even though strain B. subtilis can be cultured
and a lot is known about its genome, useful compounds that it is
capable of releasing may be missed simply because the organism has
never been grown under specific conditions such as, for example,
certain concentration of phosphate ions, addition of protease
inhibitor, and addition of 10 uM autoinducer 2. Therefore, using
hybrid and/or cartridge like approaches, a larger range of
metabolites, released compounds, and drug leads may be probed
simply by running high throughput screens of various media
conditions/additives. Even changes of a single component in the
media (for example, phosphate from 20 mM to 1 uM) may activate the
production and release of a previously unknown metabolite or
compound.
[0088] Release of compounds is highly dependent on culturing
conditions. It is known that phosphate levels, temperature,
nutrient availability all influence the production and release of
various metabolites (J. E. Gonzalez-Pastor, E. C. Hobbs, R. Losick,
Science, 2003, 301, 510). Hybrid and/or cartridge methods developed
previously may enable the screening of media conditions and
concentrations of additives both for communities, common species,
and rare species of microbes.
[0089] Implementation of this type of screening may be done by
taking a known organism and screening many media and culturing
conditions (ion concentrations, known autoinducers, amount of
confinement, temperature, pH, protein additives, reaction oxygen
species, stress inducers, carbon sources, concentration of carbon
source, nitrogen sources, concentration of nitrogen source,
availability of various trace elements and their chemical form (Mn,
Mo, Cu, Pt, V, B etc. and corresponding ions), and/or by adding
drugs known to interfere with specific cellular activities, adding
transport and ion channel inhibitors, and/or small molecules
involved in cellular communication, virulence activators, and the
like.). After using hybrid and/or cartridge method to screen
through many conditions, functional tests or other assays may be
performed in plugs to determine if compounds have been generated
with properties of interest (such as drug targets, antibiotic
compounds, ion channel inhibitors, virulence activation, virulence
inhibition, degradation of various compounds, binding affinity,
etc.). Alternatively, this method may also be used with rare cells
isolated from natural samples such as soil, aquatic environments
including sea water and marine sediments and surfaces, an animal's
digestive tract, environmentally contaminated sites including soil,
water or air, sludge used in environmental remediation, etc. Cells
which are unculturable (no known conditions cause them to
divide/reproduce outside of natural environment) may also be used.
If small volumes are used, activity may be detected even from a
single cell without division.
[0090] It is known that some strains of microbes will not initiate
growth or some cellular functions unless cell density is above a
minimal threshold. It is also possible that although the process is
occurring, the rate at low cell densities may be so slow that it
would take weeks or months to observe growth or the function.
Therefore, by placing single cells in very confined spaces with
small volumes it is likely that they will initiate high density
processes and that many processes will have increased rates. This
is especially important in the case of rare cells, since the sample
may only control 1 or a few copies of the rare cell. Small volume
confinement makes it possible to achieve high cell densities of
rare cell types.
[0091] Stochastic confinement may be used to isolate rare organisms
from various sources, including: soil extract from various types of
soil environments and soil layers (including the surface layer,
subsoil, substratum), ice shelves, marine and freshwater sediments,
naturally occurring biofilms, hot springs, hydrothermal vents,
extraterrestrial samples, crevices of rocks, attached to
particulates from aqueous environments, growing on or inside of
manmade structures, clouds, gastrointestinal tract, and found
forming a symbiotic relationship inside of a host organism.
Specifically, stochastic confinement may be used to isolate rare
cells from soil extract. Once rare cells have been obtained, the
plugs containing the cells may be incubated overnight to allow for
growth or secretion of molecules. The plugs containing the rare
cells may then be used as an input into the hybrid and/or cartridge
method. For example, to find rare cells or their secreted molecules
that may stimulate production of antibiotics and other compounds by
Streptomyces species, each plug containing the rare cell may then,
for example, be merged with 1000's of plugs containing cells of
Streptomyces species. After plugs containing streptomyces have been
merged with rare cell supernatant and incubated, screening for
antibiotic production is performed. In this way, compounds in the
supernatant of the rare cell, or rare cells directly, may induce
the production of new antibiotic compounds.
[0092] In another example, stochastic confinement may be used to
isolate cells from ocean sediments. Once plugs containing cells
have been collected, the hybrid and/or cartridge method may be used
to screen various media conditions such as phosphate concentration
(from 0 to 100 .mu.M), autoinducer 2 concentration (from 0 to 100
.mu.M), and glucose concentration (from 0 to 10 mM). The cells are
then incubated in the new media conditions. Various
functional/genetic tests are performed in the plugs to determine
which media conditions yield growth and or production of compound
with desired properties
[0093] Alternatively, cells which are unculturable (no known
conditions cause them to divide/reproduce outside of natural
environment) may be used. If small volumes are used, one may be
able to detect activity even from a single cell without
division.
[0094] Plug based methods may be used to collect the lysate or cell
free supernatant from various types of cells and merge these
solutions with other cells to elicit metabolite/compound
production. Lysate/supernatant may be diluted during the screen
(concentration screen using hybrid method). In this way,
uncharacterized/unknown compounds/combinations of compounds may be
screened to elicit production of useful compounds.
[0095] It should be noted that using stochastic confinement to
enumerate particles including organisms and cells would also be
useful for counting the occurrence of rare cell types in a sample.
Stochastic confinement has applications in isolation of rare
particles from samples with many other ubiquitous particles besides
a few particles of interest. When samples are stochastically
confined, the result is a set of isolated volumes of fluid, most
with either no particles or one particle. Rare particles are
therefore separated from ubiquitous particles. The separation of
rare particles enables the direct assay of the rare particles
without interference from other particles in the system. Even if a
rare particle has high activity, since it is at low concentration
in the bulk sample it may not be detectable due to dilution of the
signal and low signal to noise or low signal to background
(potentially low background reaction from the ubiquitous
particles). Once rare particles are isolated and concentrated
through stochastic confinement, other plug based microfluidic
technologies may be used to screen the rare particle. If the rare
particle is allowed to divide, then the plug containing the rare
particle can be split into several smaller plugs and assayed or
screened using the hybrid and/or cartridge method (combining the
plug containing many rare particles with plugs of various reagents
and conditions in order to determine the function and optimal
conditions for the rare particle). In addition, if the original
sample contains 10 rare particles, since stochastic confinement
enables the separation and screening of individual particles, this
enables the rare particle to be screened against up to 10 different
conditions. When rare particles are bacteria, care must be taken
with bacteria to avoid forming biofilms during incubation which
might grow and adhere to the wall, interfering with enumerations.
Thus it is preferable to use inert surfaces in plugs.
[0096] Two important purposes when dealing with the particles of
interest are detection and harvest, both of which are enabled or
greatly enhanced by stochastic confinement when the particles of
interest are rare particles mixed with many other ubiquitous
particles. Without stochastic confinement, the background is too
high compared to the signal for detection, and the probability of
isolating the particles of interest is too low for harvest.
[0097] Occasions when detection is needed include, but are not
limited to, when the particles are rare cells such as cancer cells
in general, cancer stem cells, or fetal cells in maternal blood, or
when the particles are bacteria or viruses causing some diseases,
or when the particles are some toxic materials released by some
industrial procedure, or particles are results of some military,
civil, or natural event that needs detection.
[0098] On the other hand, if the particles of interest have some
special functions that are useful, isolating and possibly
multiplying them is important in efficiently utilizing these
special functions. For example, stem cells isolated and multiplied
from adults may be used for treatment, bypassing the need for
embryonic stem cells. Natural products used in medicine may be
produced by isolating and multiplying natural cells that produce
them. Bacteria with novel functions such as cleaning up hydrocarbon
waste, degrading other environmental pollutants including
halogenated compounds, converting biomass into more easily
utilizable fuels such as ethanol or butanol or methane, oxidizing
methane, or fixing nitrogen may be isolated, multiplied and used
for appropriate purposes.
[0099] Therefore, after the particles are separated and the plugs
containing the particles of interest are distinguished from others
by a primary assay, one or multiple further assays may be done to
detect particles of interest and/or one or multiple types of
particles detected from the primary assay may be used alone or in
combinations for appropriate functions.
[0100] The basis for detection of the particles include but are not
limited to: 1) surface properties including functional groups on
the surface of particles and signaling molecules on cells
(antigens, receptors, sugar groups, lipids, etc); 2) materials
inside the particles (chemicals enclosed in materials, DNA, RNA in
general, microRNA, signaling molecules in general, proteins, and
the like)--these materials may require further processing to the
particles to be exposed and used for detection; and 3) chemical
exchange with the environment (production and/or consumption of
chemicals by material, uptake and/or secretion of molecules such as
food, waste, signaling molecules in general, ions, novel molecules,
etc. by cells). These chemical exchanges may occur naturally or
with human intervention for example by stimulation with
reagents.
Specific applications
[0101] Detection of diseases by examination of fetal materials in
maternal blood
[0102] Prenatal diagnosis of genetic diseases plays an important
role in pregnancy, at least in informing the parents about the
possibilities. Women of 35 years of age or more have high risk of
abnormalities. However, since there are also many more pregnancies
in the "low-risk" 26 year-old group, most (about 70%) abnormalities
occur in this group. (Daniilidis, A.; Kouzi-Koliakou, K., Fetal
cells in maternal circulation--potentials for prenatal control.
Journal of Biological Research-Thessaloniki 2006, 6, 119-130.)
[0103] Most effective current methods are invasive, where samples
are taken directly from the fetus. These procedures have a risk of
miscarriage (1-2%). (Daniilidis, A.; Kouzi-Koliakou, K., Fetal
cells in maternal circulation--potentials for prenatal control.
Journal of Biological Research-Thessaloniki 2006, 6, 119-130.)
Thus, these invasion methods are usually applied to those in the
high-risk group only. Because of the risk of miscarriage and
because of the high collective occurrence of abnormalities in the
"low-risk" group that are usually not screened, a non-invasive and
reliable method to detect or predict genetic disorders is in high
demand.
[0104] One promising possibility is using fetal materials in
maternal blood. If the cells and free DNA in the mother's blood may
be used effectively to detect genetic disorders, the risk of
miscarriage by invasive procedure is diminished and virtually any
expecting mother may have a blood draw to check for possible
genetic disorders. However, the biggest challenge is the small
number of fetal cells (1-6 cells/mL (Daniilidis, A.;
Kouzi-Koliakou, K., Fetal cells in maternal circulation--potentials
for prenatal control. Journal of Biological Research-Thessaloniki
2006, 6, 119-130.)) and DNA in maternal blood.
[0105] Previously developed methods to isolate such cells involve
enrichment methods such as density gradient centrifugation and
selective lysis, and sorting methods such as fluorescence-activated
cell sorting (FACS) and magnetic-activated cell sorting (MACS).
Methods to detect genetic disorders include (PCR) and fluorescent
in situ hybridization (FISH).
[0106] FACS and MACS depend on tagging the fetal cells with
fluorescent or paramagnetic antibodies and using fluorescence
intensity as a signal to separate the cells with flow cytometry or
using magnets to separate the cells. These techniques rely on
specific antibodies. In a recent study in which blood samples
spiked with fetal nucleated red blood cells were used to check the
sorting procedure which used density-gradient centrifugation, MACS,
and selective lysis, only 37% were recovered. (Ponnusamy, S.;
Mohammed, N.; Ho, S. S. Y.; Zhang, H. M.; Chan, Y. H.; Ng, Y. W.;
Su, L. L.; Mahyuddin, A. P.; Venkat, A.; Chan, J.; Rauff, M.;
Biswas, A.; Choolani, M., In vivo model to determine fetal-cell
enrichment efficiency of novel noninvasive prenatal diagnosis
methods. Prenatal Diagnosis 2008, 28, (6), 494-502.)
[0107] Prenatal diagnosis of fetal physiology, non-genetic
diseases, or genetic disorders by fetal cells and possible use of
fetal cells.
[0108] Stochastic confinement and autocatalytic kinetics with
threshold, as discussed above and in the amplification section, may
allow one to detect reliably plugs containing fetal cells in
maternal blood and separate them out to use techniques in further
detection or application. The two important advantages of this
system versus current techniques are:
[0109] 1) Because of the threshold kinetics, the result in each
plug is binary or pseudo-binary. In other words, there is a large
contrast between plugs containing fetal cells and other plugs
(which contain other cells or no cells). Therefore, the signal used
to mechanically sort the cells is clear and error in this step is
avoided. For example, in conventional FACS background fluorescence
and photobleaching may make the results deviate from being
ideal.
[0110] 2) Stochastic confinement allows for reliable detection even
if the specificity of an antibody label is not ideal. As long as
binding to the fetal cells (such as fetal nucleated red blood cell)
is at least two orders of magnitude stronger than undesired binding
to non-fetal cells, the kinetic threshold may be adjusted to lie in
between the two concentrations of antibodies in plugs. The
adjustment may be carried out by choosing an appropriate
amplification method and tuning the concentration of such method
(see section about amplification). There is a need to determine
properties of fetal cells (such as having a particular disease or
not). Using antibodies that selectively bind to fetal cells of
interest, stochastic confinement may be used to detect such
properties. Even though the contrast provided by the specificity of
the antibodies may not be much, stochastic confinement and/or an
amplification method may greatly enhance this contrast.
[0111] The general steps of this method include:
[0112] obtaining about 10-20 mL of blood sample from the expecting
mother (the volume is chosen because even in such cases when
amplification methods may detect single cells, a 1 mL sample of
blood still has a significant probability to not contain the cells
of interest when the concentration of the fetal cells is 1-6
cells/mL);
[0113] coarse enrichment by density gradient centrifugation or
other methods (optional);
[0114] stochastic confinement into plugs;
[0115] primary detection (such as by using antibodies (such as
monoclonal antibody against H315 for trophoblasts, (Daniilidis, A.;
Kouzi-Koliakou, K., Fetal cells in maternal circulation--potentials
for prenatal control. Journal of Biological Research-Thessaloniki
2006, 6, 119-130) monoclonal antibody against transferring receptor
for erythroblass, (Bianchi, D. W.; Flint, A. F.; Pizzimenti, M. F.;
Knoll, J. H. M.; Latt, S. A., Isolation of Fetal DNA from Nucleated
Erythrocytes in Maternal Blood. Proceedings of the National Academy
of Sciences of the United States of America 1990, 87, (9),
3279-3283.) etc.));
[0116] optionally if an amplification method is needed to enhance
the contrast: merging with plugs containing chemicals or materials
needed in the amplification method (if these chemical and materials
are added with the antibody this step is not needed);
[0117] using the plugs containing fetal cells of interest for
further assays (such as FISH or PCR to look for genetic disorders,
or any other possible methods to detect certain properties of
interest of fetal cells) if necessary; and/or
[0118] using the plugs containing fetal cells of interest for
applications if there are functions associated with such fetal
cells in need, with or without multiplying these cells.
[0119] Fetal materials (including DNA) in maternal blood and
markers of disorder after stochastic confinement may also be
detected with methods described in the section entitled
"amplification."
[0120] Detection
[0121] The following articles, describing methods for concentrating
cells and/or chemicals by making small volume plugs with low
numbers of items to no items being incorporated into the plugs,
with specific applications involving PCR, are incorporated by
reference herein: Anal Chem. 2003 Sep. 1; 75(17):4591-8.
Integrating polymerase chain reaction, valving, and electrophoresis
in a plastic device for bacterial detection. Koh C G, Tan W, Zhao M
Q, Ricco A J, Fan Z H; Lab Chip. 2005 April; 5(4):416-20. Epub 2005
Jan. 28. Parallel nanoliter detection of cancer markers using
polymer microchips. Gulliksen A, Solli L A, Drese K S, Sorensen O,
Karlsen F, Rogne H, Hovig E, Sirevag R.; Ann N Y Acad. Sci. 2007
March; 1098:375-88. Development of a microfluidic device for
detection of pathogens in oral samples using upconverting phosphor
technology (UPT). Abrams W R, Barber C A, McCann K, Tong G, Chen Z,
Mauk M G, Wang J, Volkov A, Bourdelle P, Corstjens P L, Zuiderwijk
M, Kardos K, Li S, Tanke H J, Sam Niedbala R, Malamud D, Bau H;
Sensors, 2004. Proceedings of IEEE 24-27 Oct. 2004
Page(s):1191-1194 vol. 3. A microchip-based DNA purification and
real-time PCR biosensor for bacterial detection. Cady, N.C.;
Stelick, S.; Kunnavakkam, M. V.; Yuxin Liu; Batt, C. A.; Science.
2006 Dec. 1; 314(5804):1464-7. Microfluidic Digital PCR Enables
Multigene Analysis of Individual Environmental Bacteria. Elizabeth
A. Ottesen, Jong Wook Hong, Stephen R. Quake, Jared R. Leadbetter;
Electrophoresis 2006, 27, 3753-3763. Automated screening using
microfluidic chip-based PCR and product detection to assess risk of
BK virus-associated nephropathy in renal transplant recipients.
Govind V. Kaigala, Ryan J. Huskins, Jutta Preiksaitis, Xiao-Li
Pang, Linda M. Pilarski, Christopher J. Backhouse; Journal of
Microbiological Methods 62 (2005) 317-326. An insulator-based
(electrodeless) dielectrophoretic concentrator for microbes in
water. Blanca H. Lapizco-Encinas, Rafael V. Davalos, Blake A.
Simmons, Eric B. Cummings, Yolanda Fintschenko; Anal. Chem. 2004,
76, 6908-6914. Electrokinetic Bioprocessor for Concentrating Cells
and Molecules. Pak Kin Wong, Che-Yang Chen, Tza-Huei Wang, and
Chih-Ming Ho; Lab Chip, 2002, 2, 179-187. High sensitivity PCR
assay in plastic micro reactors. Jianing Yang, Yingjie Liu, Cory B.
Rauch, Randall L. Stevens, Robin H. Liu, Ralf Lenigk and Piotr
Grodzinski; Anal. Chem. 2005, 77, 1330-1337. High-Throughput
Nanoliter Sample Introduction Microfluidic Chip-Based Flow
Injection Analysis System with Gravity-Driven Flows. Wen-Bin Du,
Qun Fang, Qiao-Hong He, and Zhao-Lun Fang; Science Vol 315 5 Jan.
2007, 81-84. Counting Low-Copy Number Proteins in a Single Cell. Bo
Huang, Hongkai Wu, Devaki Bhaya, Arthur Grossman, Sebastien
Granier, Brian K. Kobilka, Richard N. Zare; Nature Biotechnology
Vol 22 (4), April 2004. A nanoliter-scale nucleic acid processor
with parallel architecture. Hong J W, Studer V, Hang G, Anderson W
F, and Quake S R; Electrophoresis 2002, 23, 1531-1536. A nanoliter
rotary device for polymerase chain reaction. Jian Liu, Markus
Enzelberger, and Stephen Quake; Biosensors and Bioelectronics 20
(2005) 1482-1490. Microchamber array based DNA quantification and
specific sequence detection from a single copy via PCR in nanoliter
volumes. Yasutaka Matsubara, Kagan Kerman, Masaaki Kobayashi,
Shouhei Yamamura, Yasutaka Morita, Eiichi Tamiya; US Patent
Application 2005/0019792 A1, "Microfluidic device and methods of
using same"; and Nature Methods 3, 541-543 (2006) "Overview:
methods and applications for droplet compartmentalization of
biology" John H Leamon, Darren R Link, Michael Egholm &
Jonathan M Rothberg.
[0122] Plugs offer advantages over cuvettes. For example, with
plugs, the optical path may be made very thin, so if a bacterium is
labeled (for example, with fluorescent antibodies) it is easy to
detect. Alternatively, the same effect may be obtained by squeezing
a blood sample between two coverslips separated by a 20 .mu.m gap,
which may be created by placing a thin metal (gold or Pt) wire
between the cover slips. Thus, the cover slips may be covered with
something to which bacteria stick, and factors that makes them grow
biofilms or multiply aggressively. Probes may be added for
detecting these activities or colonies.
[0123] Another advantage is diffusion control in a plug versus a
coverslip or cuvette. For example, in a cuvette, a material
produced by bacteria can diffuse away and become diluted. In a
plug, the material produced by bacteria builds up to a high enough
concentration that it is easy to detect. The material produced or
the multiplying bacteria may be detected. Once the bacteria start
growing, they grow until nutrients in the plug are depleted.
Monitoring the decrease in a nutrient attributable to the presence
of the bacteria provides evidence of the bacteria. For example, the
decrease in O.sub.2 attributable to the presence of the bacteria
may be done using agglutination beads.
[0124] Oxygen or other gases or mixtures of gases may need to be
provided to encourage growth. Gases may be introduced by various
methods including by dissolving them in the fluorocarbon carrier
fluid. Care may be taken to avoid evaporation of oil/media
particularly for incubation at 37.degree. C. The plugs may be
sealed in glass or placed in a Teflon capillary that is permeable
to gases. The capillary is then placed in a vial of media that is
the same (isotonic but not comprising indicators, cells and other
components which will not partition across tubing). In short, the
oxygen concentration of blood in any sepsis assay may need to be
controlled in a way other than what is typically done with blood
tests.
[0125] Many bacteria and bacterial components can directly activate
individual coagulation factors. However, direct initiation of the
coagulation cascade and the formation of a propagating clot is not
typically observed when humans are infected. These bacterial
components usually activate low levels of coagulation factors, but
this activation does not result in the amplification and positive
feedback necessary to form a clot that can grow and propagate. For
example, Staphylococcus aureus (S. aureus) produces coagulase, a
protein that binds prothrombin stoichiometrically and leads to
cleavage of fibrinogen to fibrin. However, this conversion simply
precipitates fibrin and does not result in production of thrombin,
feedback, or amplification of the coagulation cascade. Escherichia
coli (E. coli) that express the protein Curli are also known to
activate coagulation factors, such as factor XII. This process was
shown to cause slower initiation of coagulation due to depletion of
factor XII. Bacteria do initiate coagulation in some organisms,
such as horseshoe crabs, but this mechanism of controlling
infection is believed to have been lost during evolution of
vertebrates.
[0126] Crab blood is known to clot rapidly on contact with
bacteria. Plugs with grown bacteria may be merged with plugs that
contain crab blood. Clotting occurs rapidly when the crab blood
contacts the bacteria, and secondary indicators such as fluorescent
indicators or dyes for "crab thrombin" may be used to detect
bacteria. Other clotting systems are disclosed in Kastrup et al.
Acc Chem. Res. "Using chemistry and microfluidics to understand the
spatial dynamics of complex biological networks." 2008 April;
41(4):549-58.
[0127] Bacteria export materials out of the cell, for various
reasons including to attack the host, to digest food, to fight, to
signal, etc. These chemical messengers become more concentrated
inside a plug than inside the original sample of blood. If the
chemical messenger is an enzyme, a fluorogenic substrate for the
enzyme would indicate the presence of the bacteria. Human blood has
esterases, etc, that may interfere, but the use of enzymes specific
to bacteria would allow detection of the bacteria. In fact, if one
had a panel of 30 substrates, one may set up 30 tubes with 1000
plugs each, and looks for specific substrates lighting up (if there
is substrate for one bacterium), or one may look for patterns of
substrates lighting up if there is a more complicated relationship
(each bacterium has a pattern of substrates associated with
it).
[0128] Another type of molecule exported by bacteria are signaling
molecules. If a single bacterium produces activators, these
activators can accumulate in the plug, turning a single bacterium
ON into the attack mode, and making it easier to detect.
[0129] Many types of cells react to high cell density by activating
behaviors and specific genes through the process of quorum sensing.
For instance, the human opportunistic pathogen Pseudomonas
aeruginosa releases signaling molecules (homo serine lactones or
other signals known as autoinducers) which accumulate in the space
surrounding the cells and enable the cells to measure their local
cell density. Many pathogens (such as Pseudomonas aeruginosa)
activate virulence behaviors in response to the activation of
quorum sensing. Because detection of cells in a virulent state is
of interest, stochastic confinement would place cells at a high
enough density that they should activate virulence mechanisms which
may then be detected. In this way, stochastic confinement can
detect cells with the potential for virulence, even if they
currently have not yet activated virulence in the patient.
Additionally, virulence activation often involves the upregulation
of enzymes involved in infection, such as lipases, coagulases, and
proteases that may be used as detection targets, specifically as
detection targets of virulent species. The virulence enzymes and
other released molecules may be used to activate detection
mechanisms.
[0130] Another possibility is to lyse the cells in the media to
detect something that is only produced by bacteria.
[0131] PCR may also be applied to plugs. PCR may be overwhelmed by
the background DNA of human cells. In the case of plugs, if plugs
are made small enough there would be only one cell per plug (human
or bacterial) which eliminate background issues (if
bacterial-specific primers are used background from other cells
would not interfere). Binding of bacteria to human cells, or
bacteria hiding inside of human cells would not be problematic due
to the minimal background. Plugs may also be spiked to stimulate
bacteria to make them easier to detect.
[0132] If bacteria can be detected in people at low concentration
(before infection becomes a real threat), these methods may be used
for general clinical practice to screen high risk patients
including babies and the elderly for bacteria. Viral infections may
also be monitored by this method by using vesicles that a virus
would try to enter, and detecting the entry by fluorogenic
substrates, or by the destruction of vesicles (detected, for
example, by a Ca/Fluo4 system).
[0133] Methods of detecting bacteria using microfluidic based
techniques comprise: 1) screening a sample against various reagents
which results in a detectable signal in the presence of bacteria;
and 2) screening these samples in parallel or in series against
different drugs to determine which drug is best suited for killing
the bacteria in the sample. Part 1) of this technique may include
screening a sample against preloaded, plug-based cartridges that
comprise bacteria-specific reagents. If the sample comprises
bacteria that are specific for a reagent in one of the plugs a
signal will be detected in that plug. Examples of the detection
methods include, but are not limited to: i) magnetic based
detection, ii) optical detection, and iii) oxygen detection. Part
2) of this technique may include preloading plug-based cartridges
with various antibiotics that are known to kill certain types of
bacteria. WO 05-056826 A1 which discloses methods is incorporated
by reference herein in its entirety.
[0134] In some aspects of the present invention, the method of
detecting bacteria comprises confinement of a bacterium into a
small volume plug, wherein the confinement induces virulence
activation of the bacteria through a quorum sensing type mechanism.
The activation of virulence in the bacteria may induce the
upregulation of various virulence factors such as proteins,
enzymes, and small molecules. The upregulation and release of these
virulence factors may be used as a detection target for the
presence of virulent bacteria, may be used to detect a bacterium
with the potential to activate virulence mechanisms, or may be used
as targets to detect a specific species or type of bacteria. The
upregulation and release of these virulence factors such as
lipases, proteases, and coagulases may be incorporated as the
initial step in an enzymatic detection cascade.
[0135] In some aspects of the method of detection of bacteria, the
plug comprises a substance capable of lysing the bacteria. Lysis
may be accomplished by standard detergent-based bacterial cell
lysis. For example, frozen and thawed cell pellets are incubated
with lysis buffer that is supplemented with lysozyme, which help
disrupt cell walls (Lysozyme hydrolyzes .beta.(1.fwdarw.4) linkages
between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in
peptidoglycan and between N-acetyl-D-glucosamine residues in
chitodextrin). Gram-negative bacteria may be hydrolyzed in the
presence of EDTA that chelates metal ions in the outer bacterial
membrane. Cells are incubated with lysis buffer for about 30 min,
on ice. If the target is a nucleic acid, proteinase should be
supplemented, whereas if the target is a protein, nucleases should
be supplemented. To separate cell debris and insoluble protein
(e.g., inclusion bodies), the sample is centrifuged (14,000 g, 30
min, 4.degree. C.) and the supernatant collected. The supernatant
comprises the soluble protein fraction, which can be further
purified or directly analyzed, for example by SDS-PAGE.
[0136] Methods for Detecting Bacteria
[0137] In some aspects, the method for detecting bacteria comprises
detecting bacteria in various samples using microfluidic based
techniques, and screening bacteria using a hybrid and/or
cartridge-based method to detect microorganisms by detecting their
binding to beads.
[0138] Binding bacteria to magnetic beads may be accomplished by
using specific (antibodies, chemical link between bacteria and
bead) or non-specific (charge of bacteria, general molecule
expressed on outside of bacteria) methods. Bound and unbound
magnetic beads may be separated by microfluidic sorting techniques
which rely on differences in diffusion based on size or changes of
magnetic potential due to bound bacteria. Bound and unbound
magnetic beads may also be separated using a magnetic field because
migration in the field will be reduced for bound beads with
increased drag. This method may be used to isolate specific types
of bacteria from a sample or to remove all bacteria from other
parts of the sample matrix. Liquid containing bacteria bound to
beads may then be used to make plugs. Various techniques may be
used to detect the presence of a magnetic bead in a plug, including
measuring the electric current induced by a moving magnetic
particle. Another detection method incorporates a hard-drive head
to detect magnetic particles. Another detection method takes a
relative measurement of the magnetic beads to measure relative
orientation rate in the field. Another detection method relies on
bacteria binding to multiple beads and the detection method being
able to distinguish between single unbound beads and groups of
bound beads (for example by changes in the amplitude of the spike
in the detector).
[0139] Magnetic beads may also be used to detect proteins or other
molecules by either chemically linking the target to the bead, or
by first attaching an antibody to the target. The antibody then
recognizes and attaches to the magnetic bead.
[0140] Optical detection schemes may also be used. Beads may have
an optical signal, and a detection method which differentiates
single unbound beads vs. groups of beads may be used to detect the
target.
[0141] If the beads-based method provides means to count the number
of detected objects, then bead detection schemes may be used to
monitor the proliferation of bound objects after exposure to a
compound, such as an antibiotic. The bead method may be used to
isolate the bacteria from the sample and then introduce the
antibiotic to the bacteria. Addition of more beads and enumeration
of bound beads after incubation with the antibiotic may be used to
determine whether or not the antibiotic inhibited proliferation of
the bound bacteria.
[0142] Other detection schemes may be used to detect the bacteria
themselves, such as oxygen or carbon dioxide detectors. Oxygen
detection schemes include formation of Prussian blue as a function
of oxygen presence, and fluorescence based oxygen sensors.
[0143] The sample may be detected attached to a bead, free in
solution, or after deposition on the wall of the channel. Instead
of a bead, bacteria may be agglutinated.
[0144] In some aspects, an array of pre-formed nanoliter sized
droplets, or plugs is generated. Each plug comprises one or more
beads. In each plug, all beads are substantially similar. Each bead
has a specific binding affinity, provided by an antibody for
example, for a microorganism or a subset of microorganisms, for
example, bacteria, viruses, or fungi. The binding affinity can also
be for a specific small particle, such as a pollen grain or a
spore. The beads themselves are detectable, for example a magnetic
bead or a fluorescent bead. Ideally, the binding event is also
detectable.
[0145] If the binding event of one or more beads with a bacterium
is directly detectable, the assay may be performed by the steps of
injecting the sample into each of the plugs, incubating to allow
the detection, and performing the detection. Direct detection may
be accomplished by taking advantage of the difference in the
magnitude and frequency of the signals produced by many unbound
beads inside of a plug vs a microorganism that carries with it the
same number of beads bound. Many unbound beads would generate
several smaller signals, while an organism with the same number of
beads bound would generate a single signal of higher amplitude.
[0146] If the binding event is not detectable, then the detection
can be performed by separating bound beads from unbound beads, and
detecting bound beads. The separation may be performed by a range
of methods including a diffusive filter, a Brownian ratchet, or in
the case of magnetic beads, a magnetic filter that applies a
magnetic field to bias motion of the beads. Separation may require
separating the plug fluid from the carrier fluid, flowing through
the filter, and re-forming the plugs by adding carrier fluid to the
plug fluid that passed the filter and contains predominantly bound
beads. Detection is performed by a range of methods, including
scanning the detector over the array of plugs, or flowing the array
of plugs past the detector. Magnetic detection may be performed by
detecting currents generated by moving magnetic beads, and may also
incorporate technologies used in hard disk drives.
[0147] Spacer and index plugs may be used in the original array of
plugs as disclosed in WO 08-079274 A1, the entirety of which is
hereby incorporated by reference. Index plugs may contain markers
detectable by the same method used to detect binding of bacteria to
beads.
[0148] This method is discussed in the context of microorganisms
and particles, but it is applicable to the detection of molecules
and other objects by using a larger bead carrying antibodies
against the molecule, and a set of detectable beads carrying
another antibody against the same molecule. In the absence of the
molecule, the detectable beads do not bind larger beads and remain
dispersed. In the presence of the molecule, the detectable beads
bind to the molecules and therefore to the larger beads, and become
detectable by the methods described here.
[0149] To perform an antibiotic screen, plugs may contain
antibiotics, and the plugs injected with the sample may be allowed
to incubate to permit growth of microorganisms. Antibiotics are
recognized and are substances which inhibit the growth of or kill
microorganisms. Examples of antibiotics include, but are not
limited to, chlorotetracycline, bacitracin, nystatin, streptomycin,
polymicin, gramicidin, oxytetracyclin, chloramphenicol, rifampicin,
cefsulodin, cefotiam, mefoxin, penicillin, tetracycline,
chloramphenicol, minocycline, doxycycline, vancomycin, bacitracin,
kanamycin, neomycin, gentamycin, erythromycin, cephalosporins,
geldanamycin, and analogs thereof. Examples of cephalosporins
include cephalothin, cephapirin, cefazolin, cephalexin, cephradine,
cefadroxil, cefamandole, cefoxitin, cefaclor, cefuroxime,
cefonicid, ceforanide, cefotaxime, moxalactam, ceftizoxime,
ceftriaxone, and cefoperazone. Additional examples of antibiotics
that may be used are in US 2007/0093894 A1, hereby incorporated by
reference in its entirety. Detection of differences in growth and
microbial populations in the absence and presence of each
antibiotic would provide information on antibiotic susceptibility.
First the bacteria in the sample are counted. Then, the bacteria
sample is merged into the cartridge containing plugs of different
growth media and different antibodies along with some as "blank"
media and "blank" antibiotics plugs. Recount is preformed by
merging with magnetic beads to see which one had bacteria
reproducing.
[0150] In some aspects, the detectable signal is produced by the
growth of the bacteria. Optical detection comprising optical
methods for detection may use fluorescent nanoparticles instead of
magnetic ones. One may also use control of surface chemistry
provided by plugs to enhance growth of organisms. One method
comprises merging the sample with a cartridge, the cartridge may
optionally have growth tablets; monitoring bacterial growth by a
change in oxygen concentration; and recording the readout. The
readout may be but is not limited to a change in volume of oxygen
bubbles the device, a change in optical signal due to the presence
of an oxygen sensitive substrate (the substrate may be incorporated
in many ways including in solution, on beads, or immobilized on a
film that coats the device), or calorimetric reactions for
detecting oxygen.
[0151] Bacteria detection by using agglutination may also be
employed. This method comprises merging sample with plugs that
comprise substance that induces agglutination in the presence of
bacteria including for example, antibody labeled beads; forming
agglutination screens on a large scale for bacteria detection;
filling preformed plugs with beads covered with antibodies specific
for different bacteria; and monitoring which plugs result in clumps
of beads indicating presence of bacteria. Monitoring may be done by
eye or some other detection technique. Secondary agglutination may
also be used.
[0152] In addition, multiple assays may be performed in one device,
for example by splitting a sample into a plurality of samples using
techniques described above for splitting plugs.
[0153] One strategy is to change the hydrophobicity of the channel.
Aqueous plugs with fluorinated oil as the carrier fluid will not
wet hydrophobic channel walls. Therefore, to have a plug stick to
the channel wall it is necessary to create a region of the channel
in which the surface chemistry of the channel is hydrophilic.
[0154] A hydrophilic patch may be useful in 2 ways: i) a
hydrophilic patch may capture a plug and hold it in place; and ii)
a hydrophilic patch may temporarily come into contact with a plug
as it is flowing by. This brief contact may result in a small
portion of the plug fluid being deposited on the hydrophilic region
of the channel wall. Capturing a plug (i) or depositing a portion
of a plug onto the channel wall (i) may enable the assays to be run
on the plug fluid.
[0155] Methods which require a surface such as ELISA, or an oxygen
sensor incorporated into the surface, etc. may then be performed at
the hydrophilic patch of channel wall. There are some papers which
suggest that measurements performed on surfaces are more sensitive
than measurements in bulk fluid. More generally, the concept of
controlling channel wall surface chemistry can be implemented in
the trafficking and measurement of plug fluids.
[0156] Another strategy for manipulating plugs is to control
pressure drops in the channels by channel constriction. From
Hagen-Poiseuille's law, if a channel gets narrower, the pressure
drop quickly increases. If the pressure drop in one branch of a
microfluidic network becomes narrow, plugs will not be able squeeze
through the constriction because the capillary pressure required
for the plug to squeeze through will be higher than the pressure
drop over the device. In this way, channel constriction may be used
to collect plugs and stop them from moving.
[0157] By designing a device with specified pressure drops in each
channel in a network of branching channels, it may be possible to
automatically sort the large number of plugs needed for some
stochastic confinement applications. For example, a main channel
carrying plugs splits into 10 different smaller channels. If each
channel has a different diameter/length (i.e. pressure drops for
each channel are different) this would create a bias in the loading
of plugs. A system may be designed such that the 1st 100 plugs
would be loaded into channel 1, then the pressure drop in channel 1
becomes greater than channel 2 due to the presence of many plugs,
therefore the next 100 plugs would flow into channel 2, etc. This
type of bulk sorting of plugs can be achieved simply by designing
the channels with specific pressure drops and does not rely on
turning on and off pumps, opening and closing valves, or other
"active" plug sorting mechanisms.
[0158] Certain embodiments of the invention may be used to detect
sepsis in 3 to 4 hours from a blood sample from a patient, and in
other embodiments detection times may be reduced to 20 minutes or
less. In a 5 to 10 mL blood sample from an infected patient, there
might be 100 to 1,000 bacteria. Allowing the sample to culture
overnight can increase those numbers by 10 to 100-fold. It would
also be useful to know patterns of antibiotic resistance, and
current methods are very tedious.
[0159] Possible means for detecting bacteria in a sample include
stains and dyes, as used in flow cytometry, and which are well
known in the art. Alternatively, one may look for a uniform change
in color, appearance, scattering or optical density across a plug.
almarBLUE.TM. (resazurin) is a fluorescent redox sensitive dye that
can be used to detect living cells.
[0160] Certain embodiments of the invention may be used to detect
different strains of bacteria, including Pseudomonas,
Staphylococcus, E. Coli, etc.
[0161] When testing whole blood for bacteria one can use known
methods to get rid of white blood cells, which would also be
metabolically active, before testing for the presence of bacteria.
For example, there are lysing agents well known in the art that
selectively lyse eukaryotic cells.
[0162] A severely infected patient can have 10.sup.6 bacteria
(CFU)/mL in the blood. There are known methods for detecting
10.sup.2 to 10.sup.3 bacteria/mL, but only with culturing
overnight. For example, the PCR-based LightCycler.TM. can detect
10.sup.3, but only can only detect bacteria with known,
predetermined, target gene sequences, and it does not give any
functional information, for example concerning antibiotic
resistance, and it is relatively slow, taking 6 to 8 hours. Other
methods, using 96 well plates have demonstrated the detection of
10.sup.6 bacteria in a 200 .mu.l sample in 2 to 3 hours, 10.sup.5
bacteria in approximately two hours, and 10.sup.3 bacteria in 8 to
9 hours. However, it is desirable to be able to detect low
concentrations of clinically relevant bacteria in 3 to 4 hours or
less.
[0163] Another means of detecting and typing cells is PCR
amplification of 16S-23S rRNA., as described in Vliegen, I., et
al., "Rapid identification of bacteria by real-time amplification
and sequencing of the 16S rRNA gene" Journal of Microbiological
Methods 66 (2006) 156-164, and patent application WO 96/119585,
hereby incorporated by reference in its entirety. This can be
accelerated by using a rapid microchip PCR method described
recently that uses infrared light to achieve a 12 minute PCR
reaction. See "On-chip pressure injection for integration of
infrared-mediated DNA amplification with electrophoretic
separation" Christopher J. Easley, a James M. Karlinseya and James
P. Landers, Lab Chip, 2006, 6, 601-610.
[0164] Fluorogenic media, which change color in the presence of
specific bacteria, can also be used to detect cells. Chromogenic
media include, for example, Difco mEl agar, Merck/EMD
Chromocult.TM. Coliform Agars, Chromocult.TM. Enterococci
Agar/Broth, or Fluorocult.RTM. LMX Broth, BL ml agar, IDEXX
Colilert, CPI ColiTag and Merck/EMD ReadyCult.RTM.. Typical enzyme
substrates linked to chromogens or fluorogens include ONPG, CPRG,
and MUG. These are also available in ready-to-use format, e.g. BBL
ml agar and `convenience` packs, e.g. IDEXX Colilert, CPI ColiTag
and Merck/EMD ReadyCult.RTM..
[0165] Bacteria can also be detected using simple growth and
density measurements of plugs. Such measurements may aid detection
and characterization of specific antibiotics that block the ability
of the bacteria to grow, after combining plugs with specific
antibiotics.
[0166] Microchannel PCR is described in U.S. Pat. No. 6,990,290,
rapid bacterial PCR is described in U.S. Pat. No. 6,673,578, and a
sepsis detection chip is described in US 2005-130185 A1, all
incorporated by reference herein in their entirety.
[0167] Confining bacteria in small spaces might influence their
phenotype, and gene regulation. For example, one bacterium inside
of a small volume may respond as if it is in a culture with high
cell density because communication molecules that it secretes, such
as homoserine lactones, activate quorum sensing. This can be used
to an advantage of this to decrease detection times or limits,
and/or trigger virulence.
[0168] Staining
[0169] Typically to monitor bacterial growth, blood sample bottles
are placed in an incubator and kept at body temperature. They are
watched daily for signs of growth, including cloudiness or a color
change in the broth, gas bubbles, or clumps of bacteria. When there
is evidence of growth, the laboratory does a gram stain and a
subculture. To do the gram stain, a drop of blood is removed from
the bottle and placed on a microscope slide. The blood is allowed
to dry and then is stained with purple and red stains and examined
under the microscope. If bacteria are seen, the color of stain they
picked up (purple or red), their shape (such as round or
rectangular), and their size provide valuable clues as to what type
of microorganism they are and what antibiotics might work best. To
do the subculture, a drop of blood is placed on a culture plate,
spread over the surface, and placed in an incubator.
[0170] Aggregation of a signal can be used to detect the presence
of a single organism in a plug. With reference to FIG. 3, a single
bacterium can simultaneously bind to many antibodies. If each of
these antibodies has a signal, then the signal would be localized
and therefore become detectable. For instance if each antibody was
tagged with a fluorescent marker, then although single fluorescent
markers may not be detected, the co-localized signal from many
markers would be above the threshold for detection. Similarly, if
each antibody was tagged with an enzyme, then many antibodies close
together would create a high local concentration of the enzyme.
Many enzymatic cascades (such as initiation of blood coagulation)
require a threshold local concentration of enzyme. In this way,
single antibodies would not be detected, but a local cluster of
antibodies all bound to the same bacteria would create a detectable
local concentration of enzymes. In a more specific example, if the
bacteria sample was in blood or in a solution that contained the
coagulation cascade, a detection method may involve antibodies
which bind to the bacteria but also are tagged with the
metalloprotease InhA (expressed by Bacillus species which induce
blood clotting). A bacterium that is recognized by the antibody
will bind to many antibodies and create a cluster of antibodies and
therefore a localized cluster of InhA. The coagulation cascade will
respond to the local cluster of InhA and initiate coagulation.
[0171] An additional effect of aggregation of a signal in the
decrease in the background signal. For instance, if there are 100
molecules of signal in a 1 mL sample, then before clustering the
background is 100 molecules/mL. After clustering 80 of the
molecules together followed by stochastic confinement (assume in a
1 femtoliter space), the signal is now 80 molecules/fL and the
background is now 20 molecules/mL because 80 molecules of signal
have been removed from the bulk solution. In this way, aggregation
of a signal increases the signal to noise ratio.
[0172] Another advantage of stochastic confinement is to segregate
the signal/analyte from background material. For example, in the
detection of bacterial strain "A" in a sample that contained
numerous contaminant strain "C", assays in the bulk may experience
interference from the presence of strain "C". This interference may
be in the form of "C" influencing the behavior of "A", or "C"
influencing the components of the assay itself. Interference
problems may be increased if the sample contains an abundance of
"C" and few "A". By forming plugs which separate species "A" from
"C", the assay may be run successfully for the presence of "A" as
some plugs will contain only strain "A" and none of strain "C". As
long as the threshold is tuned properly, the amplification cascade
can selectively respond to only the active, target particles even
in the presence of a large excess of interfering particles.
[0173] In addition to stochastic confinement using plug-based
microfluidics, there are other methods that may be used to achieve
confinement of individual organisms or molecules as shown in FIG.
4. One such method is to generate an array of microwells.
Microwells can be defined as a small compartment with volumes of
nanoliters or less. The well is open on the top and the walls are
impermeable to either the particles to be detected, molecules used
in the detection system, or both. Once the wells are loaded, a top
barrier may be placed over the wells in order to completely confine
the particle(s) and molecules in the well. Even without complete
confinement, a well with a single open wall will still experience
some confinement effects due to decreased flux of particles and
molecules into and out of the well. Another confinement method is
to trap the particles in the matrix of a gel or polymer.
Confinement effects will be achieved by reducing the diffusion of
both the particles and the molecules used in the detection scheme.
In this way, high local concentrations of molecules used for
detection will accumulate around the particle. Adding an
impermeable boundary to the bottom and/or top of the matrix (such
as glass, plastic, etc.) will further increase confinement effects
due to decreased flux of the accumulating signal away from the
particle.
[0174] Stochastic confinement of individual bacteria into plugs of
nanoliter volume or smaller volumes reduces detection time.
[0175] To reduce the time required to detect bacteria in a sample,
a microfluidic device was designed to confine single bacterium into
plugs of nanoliter volume. In principle, when generating plugs with
a small volume from a solution with a low concentration of
bacteria, much of the volume of the initial solution forms plugs
that contain no bacteria. There are a few occupied plugs, each
occupied by single bacterium. As a result, the concentration of
bacteria in the occupied plugs is greater than the concentration of
bacteria in the initial solution. For example, if plugs of
nanoliter volume were made from a culture with an initial bacterial
concentration of 10.sup.5 CFU/mL, one in ten plugs would receive a
single bacterium, as illustrated in FIG. 1a. The concentration of
cells in these occupied plugs would be one bacterium per nanoliter
or 10.sup.6 CFU/mL. In other words, 10.sup.5 CFU/mL corresponds on
average to 0.1 bacterium per 1 mL, and confining this solution into
nanoliter plugs creates plugs with 0 bacteria per nanoliter plug
and with 1 bacterium per nanoliter plug. CFU/mL refers to the
colony forming units (CFU), a measure of live bacteria, per
milliliter. Confinement effects are increased as the volume of
plugs is decreased, therefore when confinement effects could be
achieved in nanoliter plugs, picoliter plugs or femtoliter plugs
would have increased confinement effects.
[0176] Targeting virulence factor for screening antibacterial drugs
is a potential way to develop novel drugs. To escape
cross-resistance of current drugs, targeting to virulence factors
involved in human pathogenesis is considered essential. It has also
been suggested that using surrogate infection model systems to
screen novel drugs is a key issue for detecting and investigating
the virulence factor.
[0177] Bacterial virulence may also be used to identify new
therapeutics. Targeting virulence helps to preserve the many
symbioses between the microorganism and host that contribute to
human health. Targeting bacterial adhesion and toxin production and
function are good approaches to developing the antivirulence drugs
which will prevent the assembly of adhesive machinery or toxin
expression or secretion. Quorum-sensing systems, two-component
response systems, and biofilm formation can additional targets for
virulence factors control.
[0178] The following methods are the ways in which pathogens cause
disease in humans: adhesion, colonization, invasion, immune
response inhibitors, and toxins. Since pathogenic bacteria have
different methods of inducing virulence, conditions for
inducing/monitoring/evaluating virulence are useful targets for
drug discovery.
[0179] A representative example of inducing virulence is the
generation of reactive oxygen species (ROS). Inflammatory cells
have defense mechanisms against invading microorganisms and are
known to exert their antimicrobial actions by releasing reactive
oxygen species (ROS), proteolytic enzymes and other toxic
metabolites. While these ROS and other toxic compounds damage
pathogens and host cells together, antioxidant defense system such
as superoxide dismutase (SOD), catalase, glutathione, and
glutathione peroxidase also affect the host cells specifically.
[0180] Standard Method Vs Stochastic Confinement
[0181] The standard procedure for a patient presenting a bacterial
infection in the blood, is to take a blood sample from the patient,
the blood sample having a cell density of 100 CFU/mL. If the
patient has an infection of methicillin resistant Staphylococcus
aureus, and traditional culture based methods are used, detection
of the Staphylococcus aureus takes about 12 hours. At that point
the susceptibility of the pathogen to antibiotics would not yet be
known; antibiotic testing would require another 6 hours.
[0182] If the blood sample is stochastically confined into 0.1 mL
plugs, detection takes less than 4 hours. Because stochastic
confinement has the ability to screen individual bacterium, up to
100 different antibiotic conditions may be screened from a 1 mL
blood sample without a preincubation step. Detection time for
bacteria with S. aureus decreases by 1.5 hours for every order of
magnitude increase in cell density at time zero (i.e. 100 CFU/mL
takes 12 hours, 1000 CFU/mL take 9.5 hours). Therefore with
confinement in nL plugs, concentration can be increased from 100
CFU/mL to 10.sup.7 CFU/mL which decreases detection time by 7.5
hours. (P. Kaltsas, S. Want and J. Cohen, Clin. Microbiol. Infect.,
2005, 11, 109-114.) In 23-30% of all cases, an inappropriate
antibiotic is initially administered (S. D. Carrigan, G. Scott and
M. Tabrizian, Clin. Chem., 2004, 50, 1301-1314.)
[0183] In the standard method, the infection is identified after 12
hours and then it takes 6 additional hours to screen antibiotics.
Many patients would not receive appropriate antibiotic treatment
for over 18 hours, which means that the infection has worsened and
the chance of mortality is greatly increased.
[0184] In the stochastic confinement method, infection and
antibiotic sensitivity of infection is known after 4 hours or less.
Antibiotic treatment can begin much sooner. Other advantages of
stochastic confinement are the ability to run multiple tests on the
sample without preincubation, therefore the clinician can have an
in depth characterization of the pathogen (antibiotic sensitivity,
serotype, strain, genetic information, other functional tests like
propensity for virulence) within a few hours. Since confinement
involves testing individual cells, heterogeneity in the
activity/phenotype within the population of cells may be tested.
For instance, it may be detected that 1% of bacteria in the sample
are resistant to oxacillin even though 99% of the cells are
sensitive. A traditional method might not detect this difference.
Consider starting with a sample at 100 CFU/mL in which only 1% of
cells are resistant. Detection time of resistant cells would be for
a density of 1 CFU/mL. In the standard method, protocols for
running the test might not be long enough (an additional 3 hours or
more) to even detect this type of resistance in the standard
method. In addition, subpopulations of resistant cells typically
grow more slowly, increasing the chances of not detecting the cells
in traditional tests.
[0185] Stochastic confinement is useful for large scale monitoring
of resistant strains in the population and offers many benefits.
Bacterial infections in the hospital setting do not routinely
undergo in depth characterization of the infecting strain.
Stochastic confinement provides a cheap and efficient method of
characterizing pathogens so that hospital bacterial infections can
be characterized routinely in the hope of better controlling them.
In addition, functional tests are a better way to characterize
pathogens than genetic tests because the genetic marker for a new
resistance mechanism can be found immediately. Therefore there is
less of a time delay between identification a new resistance
mechanism and the discovery of a genetic marker useful for the
diagnosis of the resistant strain. Stochastic confinement also
enables increased tracking of resistance patterns leading to early
recognition of resistant strains and enables health care agencies
to track the spread of resistant strains.
[0186] Functional detection is possible as well wherein target or
host cells and potentially infectious organisms are added into
plugs and monitored for infection. Human/mammalian, bird, live
stock, plants/crops can all be used as the target or host cells for
these kinds of infectivity assays. The same techniques can be used
to look for ways of reducing infectivity of microbes in any of
these systems. The term virulence is used to mean direct
infectivity, or killing remotely, any kind of pathogenicity, or
negatively affecting the host cell in other ways or sporulating of
bacterial spores, or activation of viral particles, or transition
of bacterial cells from dormant to active form (relevant to
tuberculosis). "Particle" refers to a cell, a viral particle, a
spore, and the like.
[0187] The same ideas can be applied to general screening of cells
and their activity such as switching from one state to another
which may depend on the concentration of soluble or surface-bound
factors, or presence of other cells. Examples include stem-cell
differentiation and cancer cell activation.
[0188] The hybrid approach is described in Liang Li, Debarshi
Mustafi, Qiang Fu, Valentina Tereshko, Delai L. Chen, Joshua D.
Tice, and Rustem F. Ismagilov, "Nanoliter microfluidic hybrid
method for simultaneous screening and optimization validated with
crystallization of membrane proteins", PNAS 2006 103: 19243-19248
as well as in publications incorporated by reference above.
[0189] An example of chemical systems capable of amplification is
described in Kastrup et al. PNAS 2006 Oct. 24; 103(43):15747-52 as
well as in publications incorporated by reference above. This
simple chemical model system, built by using a modular approach,
can be used to predict the spatiotemporal dynamics of complex
chemical networks. Microfluidics is used to create in vitro
environments that expose both the complex network and the model
system to surfaces patterned with patches presenting stimuli. Such
chemical model systems, implemented with microfluidics, may be used
to predict spatiotemporal dynamics of complex biochemical
networks.
[0190] Reducing virulence is a good strategy to fight microbial
infections. Targeting virulence factors for screening antibacterial
drugs is a potential way to develop novel drugs. To escape
cross-resistance of current drugs, targeting to virulence factors
involved in human pathogenesis is considered essential. To find
compounds that reduce virulence, conditions need to be presented
that induce virulence so that the virulence and the effects of
drugs on virulence can be monitored. There are a number of ways
that pathogens cause disease in humans including adhesion,
colonization, invasion, immune response inhibition, and toxins.
Since pathogenic bacteria have different ways of inducing
virulence, conditions of inducing, monitoring, and evaluating
virulence should be selected as the target.
[0191] Conditions that lead to virulence are often not known.
Components may include the presence of host's cellular signals,
accumulation of secreted microbial factors, presence of
surface-bound cellular components and signals, presence of host
cells, presence of molecular species associated the host's
environment, presence of molecular species present on surfaces of
host cells, reactive oxygen species, or reactive nitrogen
species.
[0192] One representative example of inducing virulence is the
generation of reactive oxygen species (ROS). Inflammatory cells
show defense mechanism against invading microorganisms and are
known to exert their antimicrobial actions by releasing reactive
oxygen species (ROS), proteolytic enzymes and other toxic
metabolites. While these ROS and other toxic compounds damage
pathogens and host cells together, antioxidant defense system such
as superoxide dismutase (SOD), catalase, glutathione, and
glutathione peroxidase protect the host cells. Bacteria may detect
the presence of ROS to control their virulence.
[0193] The bacterial detection method may be used to determine
conditions that induce virulence using the hybrid method. Any of
the factors that induce virulence may be introduced into plugs
containing microbial cells and their concentration varied. In
addition, surface chemistries may be used to incorporate molecules
present on host's cell surfaces. These surface chemistries may also
be screened in the context of the hybrid and/or cartridge method. A
combination of several cartridges, each containing a different set
of reagents (e.g. solution-based and surface-based) may be used to
screen combinations of reagents at different concentrations.
Activation of virulence may be observed as a function of solution
and/or surface conditions, leading to determination of optimal
conditions and/or conditions that are most physiologically
relevant. These methods may be used in combination with
confinement.
[0194] An array of host cells may be introduced using the hybrid
and/or cartridge method to determine the kinds or types of cells
that a microorganism may be virulent against. The hybrid and/or
cartridge approach may be used to screen a wide range of
microorganisms against a particular host to determine which
microorganisms may be virulent against the host.
[0195] Confinement, alone or in combination with other factors, may
be used to induce virulence. For example, confinement may lead to
accumulation of secreted microbial factors turning on virulence.
Virulence induced under confinement may be more physiologically
relevant.
[0196] Using the methods described herein, microbial cells or
particles isolated from a patient may be tested for their ability
to induce virulence. Hybrid and/or cartridge methods may be
especially attractive for such determination because they allow
variation in the concentration of the inducing factors.
[0197] Co-confinement of microbial cells and host cells
(human/mammalian or bird/live stock/plants/crops hosts) may further
improve induction of virulence, as both microbial and host factors
may accumulate within the confined volume. Such co-confinement may
be created, for example, by either forming plugs from at least two
streams (one containing a suspension of microbes and the other a
suspension of target cells), or by creating plugs containing one
type of cells or particles (e.g., microbial cells or viral
particles), and injecting them with a suspension containing the
other type of particle (e.g., target cells).
[0198] Changes in virulence may be monitored using a variety of
methods including assaying for the secretion of surface expression
of molecules/proteins/enzymes associated with virulence including
lipases, virulence factors, iron siderophores, and the like. The
assay may be tailored for the specific type of virulence
mechanisms. For example, for adhesion, the ability of cells to
stick to a host cell and detecting secretion of molecules known to
promote adhesion to other cells is important. In colonization and
invasion, disruption of cell membranes and promotion of endocytosis
is important and thus, injection systems to inject protein/genetic
material into another cell (for example via the type III secretion
system) may be useful. For immune response inhibitors, molecules
that bind to antibodies, formation of capsules around the cell,
induction of fibrin formation to surround the bacterial cell and
prevention of recognition by the host are important. For toxins,
molecules are released that may be detected using fluorescence
assays, agglutination assays, light producing reactions, color
change reactions and the like. Also, production of certain factors
associated with virulence (e.g. the lethal factor associated with
B. anthracis virulence) may be detected, or functional tests of the
effects on the target cell may be used.
[0199] In terms of ROS, P. aeruginosa induction of oxidative stress
in the host cells may be assessed by measuring changes in lipid
peroxidation and glutathione contents in the host cell line or
tissues. In addition, the activity of the antioxidant systems can
be evaluated by measuring activities of superoxide dismutase,
catalase, and glutathione peroxidase. (Microbial Pathogenesis,
Volume 32, Issue 1, January 2002, pages 27-34).
[0200] If conditions leading to activation of virulence are known,
or for example once they are identified using methods described in
this application, a screen may be conducted for agents that reduce
virulence. These agents may be small molecules, proteins,
antibodies, etc. There are numerous applications to humans,
livestock, plants, and the like. The method is especially useful
for infections which remain dormant for long periods of time and
then cause problems periodically when virulence is induced such as
TB, malaria, and herpes. Some P. vivax and P. ovale sporozoites do
not immediately develop into exoerythrocytic-phase merozoites, but
instead produce hypnozoites that remain dormant for periods ranging
from several months (6-12 months is typical) to as long as three
years. After a period of dormancy, they became reactivate and
produce merozoites. Hypnozoites are responsible for long incubation
and late relapses in these two species of malaria.
[0201] Drugs may be compounds or entities that alter, inhibit,
activate, or otherwise affect biological or chemical events. For
example, drugs may include, but are not limited to, anti-AIDS
substances, anti-cancer substances, antibiotics,
immunosuppressants, anti-viral substances, enzyme inhibitors,
including but not limited to protease and reverse transcriptase
inhibitors, fusion inhibitors, neurotoxins, opioids, hypnotics,
anti-histamines, lubricants, tranquilizers, anti-convulsants,
muscle relaxants and anti-Parkinson substances, anti-spasmodics and
muscle contractants including channel blockers, miotics and
anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or
anti-protozoal compounds, modulators of cell-extracellular matrix
interactions including cell growth inhibitors and anti-adhesion
molecules, vasodilating agents, inhibitors of DNA, RNA or protein
synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal
and non-steroidal anti-inflammatory agents, anti-angiogenic
factors, anti-secretory factors, anticoagulants and/or
antithrombotic agents, local anesthetics, ophthalmics,
prostaglandins, anti-depressants, anti-psychotic substances,
anti-emetics, and imaging agents.
[0202] Possible applications include screening for drugs that
reduce virulence/infectivity of a strain of bacteria and testing
bacterial isolates from patients for virulence. For example, a
hybrid screen and/or cartridge system pre-loaded with several drugs
may be injected into plugs at the same time as combining infectable
host cells with bacteria. Preventing organisms from virulence is
less likely to cause evolution of resistance than simply killing
them. Importantly, confinement may change virulence. Confinement
may induce virulence not observed in well-plates, so the method may
have advantages over traditional well-plate methods.
[0203] The sensitivity of a bacterial strain to many antibiotics
can be screened in a single experiment by using plug-based
microfluidics. A single bacterial sample can be combined with many
antibiotics to generate an antibiogram, or chart of drug
susceptibilty. A pre-formed array of plugs of six antibiotics--two
beta-lactams (ampicillin, AMP, and oxicillin, OXA); a cephalosporin
(cefoxitin, CFX); a fluoroquinolone (levofloxicin, LVF);
vancomycin, VCM; and a macrolide (erythromycin, ERT)--was generated
by aspiration. Antibiotics were tested at the breakpoint
concentration, (British Society for Antimicrobial Chemotherapy,
BSAC Methods for Antimicrobial Susceptiblity Testing, 2007) the
accepted concentration of antibiotic at which bacterial
susceptibility is determined (FIG. 2d). Since stochastic
confinement of the bacterium into nanoliter-sized plugs generates
many empty plugs, 50 plugs of each antibiotic were generated such
that it was statistically likely that each condition would contain
several plugs each occupied by a single bacterium. In total,
400-500 plugs were formed for each screen, which consisted of 6
drug conditions and 2 blank conditions. All 400-500 plugs were
collected in the same coil of tubing. A blank condition was located
at the beginning and end of the array to ensure that the position
in the array did not affect assay results. The plugs in this
antibiotic array were merged with Methicillin Resistant S. aureus
(MRSA, ATCC#43300) at an initial cell density of 4.times.10.sup.5
CFU/mL and the viability indicator on-chip to form plugs
approximately 4 mL in volume, as illustrated in FIG. 2a. The merged
plugs were collected and incubated for 7 h at 37.degree. C. After
incubation, the fluorescence intensity of the plugs was
measured.
[0204] Occupied plugs containing an antibiotic to which the
bacterial strain was resistant showed increased fluorescence
intensity, whereas plugs containing an antibiotic to which the
bacterial strain was sensitive showed no significant increase in
fluorescence intensity (FIG. 2c). Plugs containing VCM were used as
a negative control, because VCM inhibited this S. aureus strain in
macro-scale experiments, in agreement with expectations (B. T.
Tsuji, M. J. Rybak, C. M. Cheung, M. Amjad and G. W. Kaatz, Diagn.
Microbiol. Infect. Dis., 2007, 58, 41-47). The average increase in
fluorescence from all plugs containing VCM was used a baseline to
which the increase in fluorescence intensity of all other plugs was
compared (FIG. 2b, .DELTA. VCM). Four out of 49 (12%) control plugs
with no antibiotic (FIG. 2b, +blank 1) showed an increase in
fluorescence intensity more than three times greater than the VCM
baseline, indicating that they were occupied by bacteria. However,
the other plugs with no antibiotic showed an increase in
fluorescence intensity similar to the baseline, indicating that
they were unoccupied (FIG. 2b, +blank 1).
[0205] By comparing the fluorescence increase in each plug to the
VCM baseline, it can determine which antibiotics were toxic to the
bacteria. Plugs occupied with a viable bacterium showed an increase
in fluorescence intensity greater than three times the VCM
baseline. FIG. 2c shows the average intensities of plugs that
showed an increase in fluorescence intensity greater than 3 times
the baseline (black bars) and plugs that showed an increase in
fluorescence intensity less than 3 times the baseline (hatched
bars). No plugs containing VCM or LVF had a fluorescence increase
greater than 3 times the baseline, indicating that MRSA was
sensitive to these antibiotics. Poisson statistics (Eq. 2) can be
used to predict the probability of not loading a bacterium into any
of plugs in the conditions LVF or VCM. In other words, Eq. 2
predicts the possibility of the LVF or VCM results being
false-negative.
f ( k , .lamda. ) = .lamda. k - .lamda. k ! ( 2 ) ##EQU00001##
[0206] In Eq. 2, f is the probability of having k bacteria in a
plug given an average bacterial loading of .lamda. bacteria per
plugs. The experimentally determined .lamda. was 0.12, as 12% of
control plugs with no antibiotics received bacteria (FIG. 2b,
+blank plugs). For k=0 and .lamda.=0.12, we calculated the
probability of having an unoccupied plug to be 0.887. The
probability of having 49 unoccupied plugs is 0.887.sup.49, or
0.0028. Given that LVF and VCM had at least 49 plugs, the
probability of a false-negative due to loading is less than
0.3%.
[0207] The results from the MRSA antibiotic screen were used to
make the antibiogram in FIG. 2d. The antibiotics were tested at the
breakpoint concentration, and the fluorescence data was used to
determine if the bacterial strain was sensitive (S) or resistant
(R) to the antibiotic. Sensitive means that no plugs containing a
specific antibiotic showed an increase in fluorescence intensity
greater than 3 times the VCM baseline. Resistant means that at
least one plug containing a specific antibiotic showed increased
fluorescence intensity greater than 3 times the VCM baseline. The
susceptibility profile generated for MRSA by using the microfluidic
screen was identical to the profile generated by using
Mueller-Hinton agar plate tests and similar to previous reports in
the literature for MRSA. (B. T. Tsuji, M. J. Rybak, C. M. Cheung,
M. Amjad and G. W. Kaatz, Diagn. Microbiol. Infect. Dis., 2007, 58,
41-47) However, antibiotic sensitivity testing is influenced by
many factors, including bacterial load, culturing conditions,
temperature, bacterial strain, and type of assay used to detect
sensitivity. In addition, a cell population may contain
sub-populations of cells with variable sensitivity to a given
antibiotic. All of these factors should be considered and further
characterized before formulating guidelines for implementing
plug-based antibiotic sensitivity assays.
[0208] Plug-based methods can also be used to determine the minimal
inhibitory concentration (MIC) of an antibiotic against a bacterial
sample.
[0209] Next, this microfluidic approach was used to determine the
MIC of the antibiotic cefoxitin (CFX) for MRSA and MSSA (FIG. 6).
This assay was similar to the antibiotic screening assay described
above, except that the pre-formed array of antibiotic plugs all
contained the same antibiotic and the concentration of that
antibiotic in each plug of the pre-formed array was different.
Again, plugs containing saline solution were included at the
beginning and end of the array to serve as negative controls and to
ensure that the first and last plugs of the array gave similar
assay results. The positive control plugs consisted of CFX at a
concentration of 24 mg/L, as both strains are known to be inhibited
by CFX at this concentration. Plugs of the antibiotic array were
merged with bacteria and the fluorescent viability indicator as
illustrated in FIG. 6a and incubated at 32.degree. C. Plugs with
MRSA were incubated for 6.75 h and plugs with MSSA were incubated
for 6.5 h. It should be noted that temperature can affect the
results of antibiotic sensitivity assays. Here, the difference in
MIC of MRSA and MSSA was discerned by assays conducted at
32.degree. C.
[0210] After incubation, the fluorescence intensity of the plugs
was measured. Here, the average increase in fluorescence intensity
of plugs containing 24 mg/L CFX was used as the baseline to which
the increase in fluorescence intensity of other plugs was compared.
Because MRSA is resistant to many beta-lactam antibiotics, CFX
should be less effective against the strain MRSA. As expected, the
MIC of CFX was higher for MRSA (<8 mg/L) than the MIC of CFX for
MSSA (<4.0 mg/L) (FIG. 6b). These results validate the use of
this plug-based technology for screening both the susceptibility
and the minimal inhibitory concentration of many antibiotics
against a single bacterial sample.
[0211] The issue of screening media conditions along with drugs is
of interest. The outcome of a drug screen can be heavily influenced
by the media and culturing conditions used for the screen. To
eliminate false negatives in a drug screen, it would be useful to
screen the same drug condition in a variety of media and culturing
conditions. For instance, an unknown sample of bacteria may be
screened against the drug oxicillin at 30 and 37.degree. C., 100
and 150 mM NaCl, and in Luria Bertani media and soy trypticase
media. The media may: 1) influence the growth rate of bacteria
regardless of drug condition (if the media condition is such that
bacteria grow very slowly, the assay would falsely determine that
the drug is killing the bacteria); or 2) influence the interaction
between the bacteria and the drug.
[0212] Stochastic confinement combined with plug-based microfluidic
handling methods accelerates bacterial detection and enables rapid
functional antibiotic screening. By using this method, assays may
be performed on a single bacterium, potentially eliminating the
need for pre-incubation. By confining and analyzing single
bacterium in plugs, detection time is now determined by plug
volume. We were able to achieve detailed functional
characterization of a bacterial sample in less than 7 hours. We
also demonstrated that a bacterium in a 1 mL plug may be detected
in as little as 2 hours. The detection time is limited by the
formation and measurement of plugs of small volume and is less
dependent on the initial concentration and growth rate of bacteria
in the sample. This feature may be potentially important for
accelerated detection of slowly-growing species such as M.
tuberculosis, a pathogen of significant importance world-wide. (E.
Keeler, M. D. Perkins, P. Small, C. Hanson, S. Reed, J. Cunningham,
J. E. Aledort, L. Hillborne, M. E. Rafael, F. Girosi and C. Dye,
Nature, 2006, 444 Suppl 1, 49-57) Here, we have demonstrated a
screen with 400-500 mL plugs. High-throughput screens with more
conditions and increased concentration of the sample would require
methods that can handle larger numbers of smaller plugs, including
methods for automated sorting and analysis. (Y. C. Tan, Y. L. Ho
and A. P. Lee, Microfluid. Nanofluid., 2008, 4, 343-348; D. Huh, J.
H. Bahng, Y. B. Ling, H. H. Wei, O. D. Kripfgans, J. B. Fowlkes, J.
B. Grotberg and S. Takayama, Anal. Chem., 2007, 79, 1369-1376; K.
Ahn, C. Kerbage, T. P. Hunt, R. M. Westervelt, D. R. Link and D. A.
Weitz, Appl. Phys. Lett., 2006, 88, 024104; M. Chabert and J.-L.
Viovy, Proceedings of the National Academy of Sciences, 2008, 105,
3191-3196) Upon incorporating such methods for handling and sorting
large numbers of plugs of small volume, this technique may be used
for the detection of bacteria in a sample at a cell density much
lower than 10.sup.5 CFU/mL. Since the activity of single cells is
being measured, it is conceivable that detecting the presence of
even a single bacterium in a sample may be feasible.
[0213] Given that a typical 5 mL blood sample from a patient with
bacteremia contains a cell density of 100 CFU/mL, (L. G. Reimer, M.
L. Wilson and M. P. Weinstein, Clin. Microbiol. Rev., 1997, 10,
444-7) this method is capable of performing dozens of functional
tests on such a sample. Patient-specific characterization of
bacterial species would not only lead to more rapid and effective
treatment, but such an advance would also enable in-depth
characterization of bacterial infections at the population level.
Such detailed characterization may aid in tracking and identifying
new resistance patterns in bacterial pathogens. (S. K. Fridkin, J.
R. Edwards, F. C. Tenover, R. P. Gaynes and J. E. McGowan, Clin.
Infect. Dis., 2001, 33, 324-329; R. T. Horvat, N. E. Klutman, M. K.
Lacy, D. Grauer and M. Wilson, J. Clin. Microbiol., 2003, 41,
4611-4616) The principles of these methods, stochastic single-cell
confinement and multiple functional assays without sample
pre-incubation, may also be applied to other areas, including
performing functional tests on field samples, detecting
contamination of food or water, separating and testing samples with
mixtures of species, measuring functional heterogeneity in
bacterial populations, and monitoring industrial bioprocesses.
[0214] Other Applications
[0215] Confinement affects may be of particular use in the
detection of slow growing bacterial strains such as Mycobacterium
tuberculosis (the strain which causes tuberculosis, TB). To grow a
culture of M. tuberculosis using traditional methods takes several
weeks. Current tests for TB are based on an immune response, but
cannot distinguish between someone with an active infection and
someone who has previous immunization. Other current tests involve
PCR based methods. Stochastic confinement should increase the
sensitivity of detection based assays and also remove the need for
a lengthy preincubation step before running the detection assay
since detection may be done with a single confined cell.
[0216] Confinement effects may be used to screen natural sources
for candidate organisms or their genes that perform functions of
interest or generate molecules of interest. Functions of interest
include nitrogen fixation, carbon recycling, hydrocarbon
production, pollutant degradation, solar energy conversion, forming
a symbiotic relationship with other microbes, producing a toxin
that kills other organisms, produces light, produces an odor,
generates electricity. Molecules of interest include drug
candidates, small molecule inhibitors, enzymes which degrade
cellulose, enzymes which degrade pollutants, adhesives, electron
transport molecules, metal chelators, selective inhibitors of small
molecules, catalysts, plasticizing agents, and proteases.
[0217] Some of these functions may not occur in large volume (low
density of cell type which performs function) samples or samples
with mixtures of microbes. One advantage of confinement is that
individual microbes will be able to function under high density
conditions. This would be useful for rare cell types in a sample,
as high density functions would not be occurring in the original
sample due to the rare cells being at a low cell density. Such
functions would not be occurring in a macroscale sample.
Confinement may also enable rare cell types of initiate growth or
increase the growth rate due to the high concentration of rare
cells in a small volume plug after confinement. This type of
approach can be extended to rare cell types from mixed species
samples by confining rare cell at high densities to enable more
rapid growth of the rare cells due to both increased inoculation
density and eliminating competition with other strains in the
original mixed sample. The effect of confinement in small volumes
is increased if the molecules accumulating in the plug are
immiscible in the carrier fluid (the fluid surrounding the plug).
Using immiscible fluids around the plug will prevent released
microbial products from diffusing out of the plug and hence the
released products will more quickly accumulate and achieve a higher
concentration in the plug.
[0218] Other applications include detecting bacteria for
applications in homeland security and safety of the food chain and
water. It is also possible to apply these methods of detection to
the areas of sepsis, bioenergy, proteins, enzyme engineering, blood
clotting, biodefense, food safety, safety of water supply, and
environmental remediation.
[0219] The following patents and patent applications are hereby
entirely incorporated by reference in their entirety: WO
05-010169A2, U.S. Pat. No. 6,500,617, WO 2007-009082 A1.
[0220] A process of collecting a useful product from stochastically
confined cells may comprise: confining organisms, cells or
particles (by themselves, or with their enemies such as other
bacterial or other cells), or with addition of stimulating
chemicals; accumulating their products (antibiotics or other
potentially valuable enzymes); using these products for detection;
using these products for further screening (for example, dilute and
different concentrations and merging with suspensions of other
bacteria to see if those bacteria get killed off, or drip those
dilutions into standard growth assay plates); using the accumulated
enzyme and assaying for function (as described in this application,
including but not limited to cellulose degradation, catalysts for
synthesis, disruption of biofilms); and adding other assays. When
plugs are used for stochastic confinement, allowing an organism to
multiply inside a plug and then splitting the plug into daughter
plugs (where at least two daughter plugs contain daughter
organisms) provides an opportunity to perform multiple assays on
clones of the organism (including assays that cannot be performed
on a single organism or in a single volume).
[0221] All of the methods and applications described herein may be
done under controlled atmosphere using plugs/droplets, where
fluorocarbon can enable transport of gases to the plug, and
massively parallel small-scale incubations under controlled
atmosphere can be performed. This may be useful for control of
virulence, for hydrogen generation and for using organisms that
require controlled atmosphere (anaerobes, organisms that consume or
produce methane or other hydrocarbons or H.sub.2S, etc).
[0222] The methods described herein may also be used to detect
fungi, archaea and other organisms in a sample.
[0223] Test Strips
[0224] General Components of Test-Strips
[0225] A test strip comprises an amplification layer and may
comprise one or more of the following layers: a filtration wetting
layer with selectivity, a detection layer with threshold and a
layer of substrate for signal output. A signal produced by target
bacteria, for example an enzyme, will turn on the amplification
reactions in the amplification layer. Multiple amplification layers
may be applied to achieve a high magnitude of amplification. A
substrate is used to detect the generation of control molecule or
the output of the amplification in the system.
[0226] The amplification region may be regulated by a threshold
mechanism. The methods for amplification may be chosen from those
described in the section entitled "Amplification." A detailed
definition of threshold may be found below.
[0227] The techniques described in this section aim to detect a
small number of molecules or particles in a short time with a high
resistance to noise and background signals. To achieve such goals,
each of these techniques consists of multiple modules. The two most
important modules are the amplification process with positive
feedback that produces a large amount of substances in short time
and the inhibitory mechanism. The interplay between these two
processes sets up a threshold or a threshold-like behavior. In an
ideal system, a threshold is the concentration below which an input
gives a background output and above which an input gives a the
signal output, where the two outputs are easily distinguishable. To
achieve most useful amplification, the signal output must be
significantly (often by two orders of magnitude or more) different
than the background output. The transfer function, the function of
output versus input, may be a shifted ideal step function. However,
in many cases, it is impossible to achieve an ideal threshold, but
possible to achieve a threshold-like behavior, with which the
transfer function is similar to a step function but has a finite
slope at the transition region. A sigmoidal function or a similar
function may be used to describe such threshold-like behaviors.
Another way to look at threshold is the time to reach maximum
possible output as a function of input. With an ideal threshold,
this function is infinite when the input is below the threshold and
reaches a constant small positive value when the input is above the
threshold. With a threshold-like behavior, this function is very
large when the input is below the threshold. As the amount of input
increases from the threshold, this function decreases rapidly and
reaches a very small value (such as 10% or less of that at the
threshold). In the most ideal case, at threshold, a change in the
number of input molecules of 1 unit leads to a drastic change in
the output.
[0228] As long as the threshold is tuned properly, the
amplification process may selectively respond to only the active,
target particles (molecules) even in the presence of a large excess
of interfering particles (molecules). One or multiple amplification
layers with threshold may be added to increase the degree of
amplification. Threshold response may be incorporated in the
detection layer to limit false positives and false negatives.
Diffusion of signal molecules and control molecules may be
restricted on each layer by choosing the appropriate material.
Thus, stochastic confinement may be applicable to test strips. For
example, a bacterium on the strip is confined by limited diffusion
(the reagents and products are not mixed on purpose), or the test
strip may be structured to restrict diffusion, for example when
based on alumina or track-etched membranes.
[0229] FIG. 8 is a schematic description of a test strip with an
amplification system. Bacteria are brought into contact with a
filtration wetting layer. Enzymes (E) produced by target bacteria,
for example in the scale of pM or even lower, enter a detection
layer with threshold and turn on the reactions to generate control
molecules (C). After proceeding to amplification layer, the signal
will be amplified and the concentration of control molecule
increased to .mu.M or mM. The control molecules will react with
chromogenic, fluorogenic or other substrate in the substrate layer
to give a strong output signal. Output signal can also be generated
in any other ways.
[0230] A timer region may be added to the test strip as shown in
FIG. 9. This timer region is an analogous reaction which is not
detecting the analyte, but instead demonstrating the reaction is
running correctly under the current conditions (age of strip,
temperature, pressure, humidity, presence of certain impurities,
etc.). It also gives the user the assurance that the strip is
working and that they have waited long enough for the results.
There may be one or multiple timer regions to indicate different
parameters (age of strip, temperature, pressure, humidity, presence
of certain impurities, etc.)
[0231] The timer region may have the same amplification system with
reactions of the same sensitivity as the detection region. It may
be used to test for false positive. However, the timer regions may
utilize other techniques for specific purposes. Some amplification
systems may be previously loaded with a known amount of analyte
which may be activated upon the beginning of the test (e.g. by
wetting).
[0232] Amplification
[0233] Amplification schemes using enzyme cascades are known. In
particular, enzyme cascades may be modified to detect the type of
molecule not naturally associated with the enzymes. For example,
the crab blood cascade may be used to detect air pollutants by
modifying enzymes at the beginning of the cascade to detect a new
type of input.
[0234] Cascade assay formats may be used. These detect an analyte
through a process wherein, a first signal generating compound
(i.e., SGC #1) produces a product that may be utilized by a second
SGC #2 to produce a product which, e.g., may be utilized by a third
SGC #3. The subject cascade of products from SGC #1-3 results in
amplification which results in a greater overall signal than may be
achieved by any single SGC.
[0235] Many substances could be detected by a scheme which uses the
same (generic) amplification mechanism. For different analyte, a
different starter reaction is designed, but all starter reactions
produce the same output. For example, a detection scheme with the
first step which is designed to detect a specific activity and the
second steps which is able to amplify the product of the first
step. An example is using the coagulation cascade as the generic
amplification mechanism, and a set of started reactions that all
produce an activator of thrombin. One would only have to redesign
the starter reaction to detect a new analyte
[0236] Alternatively, selection may be performed indirectly by
coupling a first reaction to subsequent reactions that take place
in the same plug. There are two general ways in which this may be
performed. In the first method, the product of the first reaction
is reacted with, or bound by, a molecule which does not react with
the substrate of the first reaction. In a second, the coupled
reaction will only proceed in the presence of the product of the
first reaction. For example, a genetic element encoding a gene
product with a desired activity may then be purified by using the
properties of the product of the second reaction to induce a change
in the detectable properties of the genetic element.
[0237] Alternatively, the product of the reaction being selected
may be the substrate or cofactor for a second enzyme-catalyzed
reaction. The enzyme to catalyze the second reaction may either be
translated in situ in the plug or incorporated in the reaction
mixture prior to incorporation into a plug. Only when the first
reaction proceeds will the coupled enzyme generate a product which
may be used to induce a change in the detectable properties of the
genetic element. Stochastic confinement could be used to increase
local concentration and signal to noise ratio and to give
advantages described elsewhere herein. The product of the first
reaction could be a substrate, enzyme, or cofactor of the second
reaction, or promote the release of inhibition of the second
reaction. More reaction steps could be used.
[0238] The concept of coupling may be elaborated to incorporate
multiple enzymes, each using as a substrate which is the product of
the previous reaction. This allows for selection of enzymes that
will not react with an immobilized substrate. It may also be
designed to give increased sensitivity by signal amplification if a
product of one reaction is a catalyst or a cofactor for a second
reaction or series of reactions leading to a selectable product.
Furthermore an enzyme cascade system may be based for the
production of an activator for an enzyme or the destruction of an
enzyme inhibitor. Coupling also has the advantage that a common
selection system may be used for a whole group of enzymes which
generate the same product and allows for the selection of
complicated chemical transformations that cannot be performed in a
single step.
[0239] Previously developed methods of detection include those
using nucleotide-based amplification (such as immuno-polymerase
chain reaction (iPCR), (Adler, M.; Wacker, R.; Niemeyer, C. M.,
Sensitivity by combination: immuno-PCR and related technologies.
Analyst 2008, 133, (6), 702-718.) amplification based on allosteric
catalysis, (Zhu, L.; Anslyn, E. V., Signal amplification by
allosteric catalysis. Angewandte Chemie-International Edition 2006,
45, (8), 1190-1196.) biobarcode, (Nam, J. M.; Stoeva, S. I.;
Mirkin, C. A., Bio-bar-code-based DNA detection with PCR-like
sensitivity. Journal of the American Chemical Society 2004, 126,
(19), 5932-5933; Nam, J. M.; Thaxton, C. S.; Mirkin, C. A.,
Nanoparticle-based bio-bar codes for the ultrasensitive detection
of proteins. Science 2003, 301, (5641), 1884-1886.) molecular
beacon, (Li, J. W. J.; Chu, Y. Z.; Lee, B. Y. H.; Xie, X. L. S.,
Enzymatic signal amplification of molecular beacons for sensitive
DNA detection. Nucleic Acids Research 2008, 36, (6).)
liposome-based amplification, (Edwards, K. A.; Baeumner, A. J.,
Liposomes in analyses. Talanta 2006, 68, (5), 1421-1431.) and
nanowire sensor. (Zheng, G. F.; Patolsky, F.; Cui, Y.; Wang, W. U.;
Lieber, C. M., Multiplexed electrical detection of cancer markers
with nanowire sensor arrays. Nature Biotechnology 2005, 23, (10),
1294-1301; Cui, Y.; Wei, Q. Q.; Park, H. K.; Lieber, C. M.,
Nanowire nanosensors for highly sensitive and selective detection
of biological and chemical species. Science 2001, 293, (5533),
1289-1292.) Each of these techniques has one or multiple
disadvantages: slow response, vulnerability to noise and
environmental degradation, low detection limit, and requirement for
highly sophisticated and expensive fabrication.
[0240] The techniques described in this section aim to detect a
small number of molecules or particles in a short time with a high
resistance to noise and background signals. To achieve such goals,
each of these techniques consists of multiple modules. The two most
important modules are the amplification process with positive
feedback that produces a large amount of substances in short time
and the inhibitory mechanism. The interplay between these two
processes sets up a threshold or a threshold-like behavior. In an
ideal system, a threshold is the concentration below which an input
gives a background output and above which an input gives a the
signal output, where the two outputs are easily distinguishable. To
achieve most useful amplification, the signal output must be
significantly (often by two orders of magnitude or more) different
than the background output. The transfer function, the function of
output versus input, may be a shifted ideal step function. However,
in many cases, it is impossible to achieve an ideal threshold, but
possible to achieve a threshold-like behavior, with which the
transfer function is similar to a step function but has a finite
slope at the transition region. A sigmoidal function or a similar
function may be used to describe such threshold-like behaviors.
Another way to look at threshold is the time to reach maximum
possible output as a function of input. With an ideal threshold,
this function is infinite when the input is below the threshold and
reaches a constant small positive value when the input is above the
threshold. With a threshold-like behavior, this function is very
large when the input is below the threshold. As the amount of input
increases from the threshold, this function decreases rapidly and
reaches a very small value (such as 10% or less of that at the
threshold). In the most ideal case, at threshold, a change in the
number of input molecules of 1 unit leads to a drastic change in
the output.
[0241] An elementary amplification method for detecting molecules
includes: a) a set of molecules or materials that constitute an
amplification process that is capable of amplifying one or multiple
components (termed output); b) a set of molecules or materials that
provides an inhibitory mechanism to stop or slow down amplification
by the amplification process described in a) when the amount of the
analyte is insufficient; c) an activating mechanism which, once
sufficient analyte (termed input) is present, triggers the
amplification process described in a); d) a readout process to
provide an apparent signal (with visual signal as one example).
[0242] Inhibition in part (b) and the amplification process in part
(a) set up a threshold or threshold-like behavior which gives very
little or no output if the input is below the threshold and gives
fast and abundant output if the input is above the threshold. This
case is termed positive contrast.
[0243] Alternatively, the system may be set up with the input
providing a mechanism to inhibit the amplification process. In such
cases, with a below-threshold amount of input, a lot of the output
is produced, while an above-threshold amount of input would give
little or no output. This phenomenon is called negative contrast.
The roles of part (b) and part (c) are swapped. The general feature
is a big contrast (be it positive or negative) between the output
of a below- or above-threshold input.
[0244] Elementary amplification methods may be coupled in such a
way that outputs of one may be used as inputs for another. This is
also called a cascade. The number of elementary amplification
methods in a cascade may be varied depending on how much more
amplification is needed in comparison to what is provided by each
elementary amplification method.
[0245] The analyte may be any molecules such as enzymes, DNA, RNA,
small molecules, or any other molecules. It may come from any
sources including bacterial components, human fluids, water
samples, etc.
[0246] The amplification processes may be any reaction that is
autocatalytic or a reaction network with one or more positive
feedback loops. The processes may involve enzymes such as those in
the blood clotting cascade, apoptosis, and Limulus amebocyte
(horseshoe crab) lysate (LAL), or any other enzymes. The processes
may involve inorganic chemicals such as the Co(III)-Co(II)-oxone
system, (Endo, M.; Abe, S.; Deguchi, Y.; Yotsuyanagi, T., Kinetic
determination of trace cobalt(II) by visual autocatalytic
indication. Talanta 1998, 47, (2), 349-353; Endo, M.; Ishihara, M.;
Yotsuyanagi, T., Autocatalytic decomposition of cobalt complexes as
an indicator system for the determination of trace amounts of
cobalt and effectors. Analyst 1996, 121, (4), 391-394; Tsukada, S.;
Miki, H.; Lin, J. M.; Suzuki, T.; Yamada, M., Chemiluminescence
from fluorescent organic compounds induced by cobalt(II) catalyzed
decomposition of peroxomonosulfate. Analytica Chimica Acta 1998,
371, (2-3), 163-170.) Ag.sup.+-Ag(0) system, systems in which a
metal surface catalyzes reduction of Ag+ or other ions, producing
more metal surface available for catalysis the chlorite-iodide
system, (Dateo, C. E.; Orban, M.; Dekepper, P.; Epstein, I. R., J.
Am. Chem. Soc. 1982, 104, 2, 504-509) or
thiosulfate-chlorite-hydronium system. (Runyon, M. K.;
Johnson-Kerner, B. L.; Ismagilov, R. F., Minimal functional model
of hemostasis in a biomimetic microfluidic system. Angewandte
Chemie-International Edition 2004, 43, (12), 1531-1536; Horvath, A.
K.; Nagypal, I.; Epstein, I. R., Kinetics and mechanism of the
chlorine dioxide-tetrathionate reaction. Journal of Physical
Chemistry A 2003, 107, (47), 10063-10068; Horvath, A. K.; Nagypal,
I.; Epstein, I. R., Oscillatory photochemical decomposition of
tetrathionate ion. Journal of the American Chemical Society 2002,
124, (37), 10956-10957; Nagypal, I.; Epstein, I. R., Systematic
Design of Chemical Oscillators 0.37. Fluctuations and Stirring Rate
Effects in the Chlorite Thiosulfate Reaction. Journal of Physical
Chemistry 1986, 90, (23), 6285-6292.). They may also involve
organic reactions, such as those with acid as autocatalysts.
(Ichimura, K., Nonlinear organic reactions to proliferate acidic
and basic molecules and their applications. Chemical Record 2002,
2, (1), 46-55.) They may also be combinations of different types of
chemicals.
[0247] Phenomena in which nucleation is involved may also be used
as an amplification process, in which a crystal of aggregate
produced by nucleation may serve as a nucleus to promote more
production of such crystal or aggregate.
[0248] An amplification process may also be achieved by using
materials that release substances that catalyze the release of more
of such substances.
[0249] There are many inhibitory mechanisms. Each mechanism may
occur directly or indirectly through more than one reaction or
processes.
[0250] The first mechanism may use chemical inhibitors. These
chemical inhibitors may be stoichiometric, such as those that bind
to enzymes and block the active sites or ligands that bind to and
sequester metal cations. The chemical inhibitors may also be
catalytic, such an enzyme that cleaves an active enzyme that is
important in positive feedback loops.
[0251] The second mechanism may use materials to mechanically
separate important components of the amplification process. These
materials include, but are not limited to, vesicles containing
liquid, particles of solid or gel, and any combination of
single-layer or multi-layer particles or vesicles of one type or
multiple types.
[0252] There are many activating mechanisms. For example, the input
may be one or multiple types of the substances that are inhibited
chemically or mechanically by materials. The input may directly or
indirectly produce more of the substances that are inhibited
chemically or mechanically in the inhibitory mechanism. The input
may directly or indirectly interfere with the chemical or
mechanical inhibition by competition with the substances being
inhibited. The input may directly or indirectly interfere with the
chemical or mechanical inhibition by chemically modifying the
chemical inhibitors or the materials used to mechanically separate
the components of the amplification process.
[0253] The input generates a high local concentration of substances
that may have functions described above. Stochastic confinement is
an example of this category.
[0254] With such flexibility of activating mechanism, the methods
may be designed to be applicable to many kinds of analytes with
different desired degrees of specificity. For example, if an
existing method needs modifying to be applicable for detection of a
different analyte, the activating mechanism may be changed
completely or may be adapted with single of multiple steps to use
the analyte of interest to promote the production of the analyte of
the existing method.
Readout Processes
[0255] One or more substances that are amplified or activated by
the amplification process may promote the production of some form
of easily detectable readout. This readout may be a visual signal
based on color. The color may come from any reaction that can
generate color when the input (over the threshold) is present. For
example, an enzyme amplified in the amplification process cleaves a
fluorogenic substrate or a chromogenic substrate to give a
fluorescent signal or color. Some other examples are pH indicators,
reduction-oxidation potential indicators, and indicators for
specific cations.
[0256] Alternatively, the readout may be a visual signal based on
production of aggregates (precipitates) or crystals from any method
when the input (over the threshold) is present. These aggregates
(precipitates) or crystals may be the result of any process such as
chemical reaction or production of paramagnetic substances that
come together as solid in a magnetic field.
[0257] Examples of reactions that produce paramagnetic solids that
may be used in an amplification process include: 1)
Ag.sup.+->Ag:Ag+ has electron configuration of d.sup.10, so it
is diamagnetic. Ag has electron configuration of s.sup.1d.sup.10,
so it is paramagnetic. The autocatalyst is Ag (or its surface to be
more detailed);
2) Guyard reaction: Mn(VII) (such as, KMnO.sub.4) reacts with
Mn(II) (some soluble salt such as nitrate or chloride) and makes
MnO.sub.2 which is a black paramagnetic powder. The autocatalyst is
MnO.sub.2 solid (its surface). (Polissar, M. J. Journal of Physical
Chemistry 1935, 39, 1057.)
[0258] Further still, the readout may be any other detectable
signal, such as an electrical signal that gives analog or digital
readout or any other kind.
[0259] Amplification Process Involving Enzymes with Chemical
Amplification Processes
[0260] The amplification process may be an autocatalytic reaction
or reaction network that has a positive feedback. This positive
feedback may be achieved through one step (FIG. 10). In such case,
an autocatalytic enzyme may catalyze the cleavage of its precursor.
(A chemical inhibitor may also used to inhibit this autocatalytic
enzyme, and a fluorogenic substrate may be used to detect this
autocatalytic enzyme). Positive feedback may also be achieved
through two steps (FIG. 11). In such case, enzyme 1 may catalyze
the cleavage of enzyme precursor 2 to produce enzyme 2, which may
catalyze the cleavage of enzyme precursor 1 to produce enzyme 1.
(Common or specific inhibitors and fluorogenic substrates for
enzyme 1 and enzyme 2 may be included). Generally, positive
feedback may be achieved through any number of steps, in which one
substance, called substance 1, may catalyze directly the production
of itself or indirectly by catalyzing the production of another
substance that may directly or indirectly catalyze the production
of the substance 1. Many positive feedback loops may also be
coupled with each other into a cascade, in which the output of one
positive feedback loop is used as the input of another (FIG. 12).
In such case, the number of positive feedback loops with threshold
must be at least 1 and may vary from 1 to all.
[0261] In certain embodiments at least one enzyme in the positive
feedback loop is inhibited by an inhibitor. Not every enzyme is
necessarily inhibited, but many may be. The inhibitors may be those
found naturally or synthesized. Antibodies may also be used as
inhibitors.
[0262] If there is only one enzyme involved in the positive
feedback loops (FIG. 10), the input may be that enzyme. If there
are multiple enzymes involved in the positive feedback loops (FIG.
11), the input may be single ones or combinations of the enzymes.
The input may produce single or combinations of enzymes from the
same or different precursors as in the amplification process,
through one or many steps. The input may also be much more strongly
binding substrate for the inhibitor, or promotes the production of
much more strongly binding substrate for the inhibitor. The input
may chemically alter the inhibitors and disable the inhibitory
effect, or promote the production of substances with such
function.
[0263] If fluorogenic substrates are used, one or multiple
fluorogenic substrates may be cleaved by one or multiple enzymes in
the amplification process, through one or multiple steps. In
certain embodiments, there has to be at least one cleavage reaction
but one cleavage reaction may or may not be sufficient. For methods
with plasma or whole blood, blood clotting may also be used as a
visual readout.
[0264] Amplification Using Materials
[0265] Amplification process: The unit of the material contains
molecules that once released can promote the release of more
molecules from other units of the material. The units may be
vesicles containing liquid (such as liposomes) or particles (of
solid or gel).
[0266] Amplification using materials may be coupled with chemical
amplification. In other words, besides effects on the material, the
released molecules may also undergo chemical amplification.
[0267] The molecules enclosed in the materials may be inactive and
get activated when released by chemical reactions with substances
in the bulk or by any other ways such as change in conformation,
release of self-inhibition by an attached inhibitor, etc. They may
also be in the same form after release, but the breakage of the
material is very slow when they are enclosed, such as when the
material is designed to have different reactivity with the
molecules in the enclosed environment or in the bulk.
[0268] Inhibitory mechanism: These molecules may also be inhibited
by some substances in the bulk when they are released.
[0269] Activating mechanism: The input may also be or produce
(through one or multiple steps) a substance that releases the
molecules from the materials (this substance may be similar to or
different from the enclosed) or releases the molecules from the
inhibitor (by competing with the inhibitor or by inactivating the
inhibitor).
[0270] Readout process: readout processes described elsewhere
herein may be used, depending on specific situations.
[0271] Activating mechanism via generating a high concentration of
substances.
[0272] In certain embodiments, the input generates a high local
concentration of substances.
[0273] This technique can be used in combination with amplification
processes using chemical reactions or materials.
[0274] Stochastic Confinement
[0275] Stochastic confinement as described above is a powerful tool
to generate high local concentrations from a solution of low bulk
concentration. This technique may also be used to distinguish
interfering particles (molecules) with much weaker activity but
much larger number than those of interest. When interfering
particles (molecules, etc.) are in a large excess, the total
activity of interfering particles may be higher than the total
activity of active particles (molecules) of interest. Even with
kinetic amplification via a threshold response, it is impossible to
detect the signal from the particles of interest in the presence of
overwhelming background signal from the interfering particles
unless separation is used to isolate the particles of interest.
This situation may be commonly encountered in the microbiological
analysis of environmental samples. For example, human samples,
including skin, saliva, and stool samples, being analyzed for
microorganisms are routinely contaminated with bacteria or other
cells (blood). Stochastic confinement is a powerful method to
compartmentalize the active particles separately from the
interfering particles. Therefore, as long as the threshold is tuned
properly, the amplification process can selectively respond to only
the active, target particles (molecules) even in the presence of a
large excess of interfering particles (molecules).
[0276] Immobilizing Molecules onto a Limited Surface
[0277] Surfaces may be the interfaces of a plug, surface of a
particle, surface of a microbe, and patterns on a surface which
allows it to adsorb specific molecules. Many techniques for
immobilizing molecules onto a surface may be used. These techniques
include but are not limited to using antibodies, biotin/avidin
interaction, and His-tag/Ni.sup.2+/nitrolotriacetic acid
(His-tag/Ni/NTA) interaction.
[0278] Using Particles Containing Molecules
[0279] In some embodiments, the molecules are enclosed in the
particles. When needed the particles release the molecules
providing a burst of high concentration of these molecules in
solution. This process may or may not be autocatalytic. In other
words, the released molecules do not have to promote the release of
more molecules, although they may.
[0280] Stochastic confinement, immobilizing molecules onto a
limited surface and using particles containing molecules, may be
used in different combinations to generate even higher local
concentrations. For example, if the analytes are bacteria (with or
without background interfering bacteria), the specific antibodies
for the bacteria of interest can be used to concentrate a reporter
enzyme on the surface of the bacteria. The bacteria tagged with
reporter enzymes are then stochastically confined into plugs. These
plugs are then merged with plugs containing vesicles containing
secondary reporter enzymes. The reporter enzymes tagged on the
bacteria promotes the breakage of the vesicles, releasing an
amplified number of secondary reporter enzymes. The reporter
enzymes then act as input for one of the amplification
processes.
[0281] Using Membranes
[0282] To generate a high local concentration, a membrane can be
used to keep one particular component on one side. For example, a
membrane that is impermeable to H.sup.+ but permeable to
R.sup.---H.sup.+ can be used. On one side of the membrane, an
amplification process uses R.sup.---H.sup.+ (which diffuses in from
the other side of the membrane) as input to initiate the production
of H.sup.+ and amplifies the amount of H. Other substances and
amplification processes may be used with appropriate membranes as
well.
[0283] Using Phase for Separation
[0284] Two sets of substances are separated into insoluble or
immiscible phases (solid/solid, solid/liquid, or liquid/liquid). A
shuttle substance may transfer between two phases and speeds up the
reaction in one or both phases by fast reactions with positive
feedback. For example, the system contains solid oxidant and iron
particles in water. If the analyte is Fe.sup.2+ (or Fe.sup.3+) or
produces Fe.sup.2+ (or Fe.sup.3+), Fe.sup.2+ is oxidized by the
solid oxidant to Fe.sup.3+, which oxidizes Fe(0) (from the iron
particles) to Fe.sup.2+. Eventually, the solution contains a lot of
Fe.sup.2+ (if the metal particles are in excess) or Fe.sup.3+ (if
the solid oxidant is in excess) which may be detected by strongly
colored indicators for Fe.sup.2+ or Fe.sup.3+.
EXPERIMENTAL
Microfluidic Device Design and Fabrication
[0285] Microfluidic devices were fabricated by using soft
lithography (Y. N. Xia and G. M. Whitesides, Annu. Rev. Mater.
Sci., 1998, 28, 153-184.) as described previously. (L. S. Roach, H.
Song and R. F. Ismagilov, Anal. Chem., 2005, 77, 785-796; H. Song
and R. F. Ismagilov, J. Am. Chem. Soc., 2003, 125, 14613-14619; L.
Li, D. Mustafi, Q. Fu, V. Tereshko, D. L. L. Chen, J. D. Tice and
R. F. Ismagilov, Proc. Natl. Acad. Sci. U.S.A., 2006, 103,
19243-19248.) Except where noted below, plugs were collected in PFA
or PTFE Teflon tubing (Zeus, Orangeburg, S.C.) with 150 .mu.m or
200 .mu.m inner diameter (I.D.). The tubing was cut at a 45 degree
angle, inserted into the outlet of the microfluidic device up to
the inlet junction, and sealed into the device by using PDMS
prepolymer (10:1 elastomer to curing agent). To aid in imaging of
the plugs, the Teflon tubing was wound in a spiral on a glass
slide, and PDMS prepolymer was poured over the tubing to fix it in
place. The device with attached tubing was then autoclaved at
135.degree. C. for 10 min to sterilize. Once sterilized, the glass
slide containing the tubing was transferred to a sterile Petri
dish.
[0286] Flowing Solutions into the Microfluidic Devices
[0287] All solutions were loaded into 1700 series Gastight syringes
(Hamilton, Reno, Nev.) with removable 27 gauge needles and 30 gauge
Teflon tubing (Weico, Wire & Cable, Edgewood, N.Y.). To
maintain sterility, the syringes were filled and attached to the
device within a biosafety cabinet. Syringes were connected to the
microfluidic devices by using 30 gauge Teflon tubing. Solutions
where flowed into the microfluidic devices by using previously
described methods. (L. Li, D. Mustafi, Q. Fu, V. Tereshko, D. L. L.
Chen, J. D. Tice and R. F. Ismagilov, Proc. Natl. Acad. Sci.
U.S.A., 2006, 103, 19243-19248) Flow rates were controlled by using
PHD 2000 infusion syringe pumps (Harvard Apparatus, Holliston,
Mass.).
[0288] Bacterial Cell Culture
[0289] Cells were obtained from ATCC (Staphylococcus aureus
ATCC#25923 (MSSA) and Staphylococcus aureus ATCC#43300 (MRSA)).
Stock solutions of the cells were made by using Luria-Bertani media
Miller formulation (LB) (BD, Sparks, Md.) containing 30% (v/v)
glycerol and stored at -80.degree. C. For each experiment, a vial
of frozen stock was brought to room temperature and streaked onto a
Modified Trypticase Soy Agar (TSA II, BD, Sparks, Md.) plate and
incubated overnight at 30.degree. C. Colonies from the plates were
transferred to LB and cultured at 37.degree. C., 140 rpm for 3 h at
which point OD.sub.600 was 1.5-2.0. Cell densities were then
adjusted by diluting in LB. To maintain sterility, all procedures
were performed in a biosafety cabinet and all tubing, devices,
syringes, and solutions used were either autoclaved, sterilized by
EtOH, packaged sterile, or filtered through a 0.45 .mu.m PES or
PTFE filter.
[0290] Antibiotic Preparation
[0291] Antibiotic stock solutions of ampicillin (AMP), oxacillin
(OXA), cefoxitin (CFX), levofloxacin (LVF), vancomycin (VCM),
erythromycin (ERT) were made by using 150 mM NaCl.sub.aq at a
concentration of 4000 times greater than the final concentration in
the plugs, filter sterilized, and then frozen at -80.degree. C.
(AMP, Fisher Bioreagents, Fair Lawn. NJ; OXA, LVF, Fluka, Buchs,
Switzerland; CFX, VCM, ERT, Sigma, St. Louis, Mo.). For example,
AMP was tested at the breakpoint concentration of 0.25 mg/L,
meaning that the stock solution was prepared at a concentration of
1000 mg/L. In the case of erythromycin (ERT), a stock solution was
prepared at 1000 times the final concentration in plugs. Before
each experiment, vials of the antibiotics were thawed and diluted
1000.times.(250.times. for ERT) with saline containing 80 .mu.M
fluorescein carboxylate. Fluorescein carboxylate was used to aid in
indexing the resultant array of plugs. The plugs in FIG. 7 contain
no fluorescein carboxylate, since indexing was not required. The
blank conditions consisted of 150 mM NaCl. Antibiotic solutions
were further diluted on chip 1:3 (v/v) during plug formation. 20
.mu.M fluorescein carboxylate did not interfere with the viability
assay, the activity of the cells, or effectiveness of antibiotic in
tests performed on 96 well plates.
[0292] Antibiotic Testing on Plates
[0293] Plates were made from Mueller Hinton Agar (Fluka,
Switzerland). After autoclaving, the agar was cooled and
antibiotics were added and 20 mL plates were poured. For CFX and
OXA testing, 50 .mu.L of MRSA and MSSA bacterial culture at
4.times.10.sup.3 CFU/mL was spread onto separate TSA plates. The
plates were incubated at 30.degree. C. After 16.5 h and 40 h the
plates were examined for colonies. MRSA colonies appeared on CFX
after 16.5 h and on the OXA plates after 40 h. Even after 40 h,
MSSA colonies did not appear on the CFX or OXA plates. For AMP,
ERT, LVF, and VCM, 5 .mu.L of culture at 2.times.10.sup.4 CFU/mL
were spread onto plates, and the plates were incubated at
37.degree. C. for 12 h. After 12 h, growth of colonies on the
plates was considered resistance to the antibiotic and no colonies
on the plates were considered sensitivity to the antibiotic. For
all tests, control plates with no antibiotic were inoculated to
ensure that each plate tested received many CFU during
inoculation.
[0294] Comparing Detection Times of Bacteria in Nanoliter Plugs and
Milliliter-Scale Culture
[0295] Plugs were formed by using the general methods described
previously. (L. Li, D. Mustafi, Q. Fu, V. Tereshko, D. L. L. Chen,
J. D. Tice and R. F. Ismagilov, Proc. Natl. Acad. Sci. U.S.A.,
2006, 103, 19243-19248; D. N. Adamson, D. Mustafi, J. X. J. Zhang,
B. Zheng and R. F. Ismagilov, Lab Chip, 2006, 6, 1178-1186) Plugs
were formed in a 3 inlet PDMS device with 100 .mu.m wide channels
by flowing S. aureus culture in LB at 2.times.10.sup.5 CFU/mL at 1
.mu.L/min, a 20% alamarBlue solution in saline at 1 .mu.L/min, and
fluorinated carrier fluid at 5 .mu.L/min. 25 plugs were collected
in the channel. Inlets and the outlet were sealed with silicon
grease and the device was placed in a Petri dish containing LB for
incubation. The same aqueous solutions were mixed 1:1 (total volume
0.6 mL) in a 14 mL polypropylene round-bottom tube (BD Falcon,
Franklin Lakes, N.J.). After 2.8 h, plugs were made from the
milliliter-scale culture by using the same method, with the cell
culture containing alamarBlue for both aqueous inlets. Both sets of
plugs were immediately imaged by using a epi-fluorescence
microscope (IRE2, Leica) with a Cy3 (Chroma 41007, Cy3) filter and
a 10.times.0.3 NA objective for a 5 ms exposure time with binning
set to 4 and gain set to 200. Fluorescent images of plugs were
processed by subtracting the average background intensity from all
images. Line scans (FIG. 1b) with a width of 25 pixels were taken
along the long axis of each plug.
[0296] Experiment to Compare Plug Size to Detection Time
[0297] PDMS devices with channel widths ranging from 200 to 800
.mu.m were prepared. Teflon tubing with diameter similar to that of
the channel was cut at a 45.degree. angle, inserted into the device
up to the inlet junction, and sealed in place using PDMS. For FIGS.
1c and d, plugs were formed as above, with the exception that the
.about.1500 mL plugs were made via aspiration by using a manual
aspirator. In addition, 1 mL plugs were formed in PTFE tubing with
an outer diameter (OD) of 200 .mu.m and an inner diameter (ID) of
90 .mu.m, 690 mL plugs were formed in PTFE tubing with an OD of 700
.mu.m and an ID of 600 .mu.m, 100 and 120 mL plugs were formed in
PTFE tubing with an OD of 800 .mu.m and an ID of 400 .mu.m, and
1500 mL plugs were formed in PTFE tubing with an OD of 1100 .mu.m
and an ID of 1000 .mu.m. Plugs were collected in the Teflon tubing,
the tubing was sealed with wax, and the tubing was placed in a
Petri dish containing LB for incubation and imaging. Incubation and
imaging was performed in a microscope incubator (Pecon GmbH,
Erbach, Germany). Time zero is defined as the time at which the
sample entered the incubator, which was less than 20 min after
sample preparation. Fluorescence measurements were taken with 5 ms
exposure times with a 5.times.0.15 NA objective using a 1.times.
camera coupler for plug sizes 1, 12.6, 100, and 690 mL plugs and a
0.63.times. camera coupler for 1500 mL plugs. Plugs 125 mL in
volume were imaged with 10 ms exposure times with a 5.times.0.15 NA
and a 0.63.times. camera coupler.
[0298] Plugs were analyzed by first separating them from the
background by thresholding to exclude intensity below 250. The
average intensity of the thresholded plugs was measured. Over time,
the intensity of the plugs diverged into 2 groups, occupied plugs
and unoccupied plugs. All occupied plugs had a change in intensity
more than 2 fold unoccupied plugs. Detection time is defined as the
time at which the fold change of the occupied plugs compared to the
intensity of unoccupied plugs reaches a maximum. Fold change in
intensity is defined as the change in intensity of an occupied
plugs divided by the average change in intensity of unoccupied
plugs (Eq. 1).
Fold change.sub.(t=ti)=Occupied
plug(I.sub.t=ti-I.sub.t=1)/Unoccupied plugs(I.sub.t=1-I.sub.t=1)
(1)
[0299] In Eq. 1, I.sub.t=ti is intensity at time point i. The
intensity of the empty plugs is the average of all empty plugs in
each experiment.
[0300] Comparing Detection Times of Bacteria in Nanoliter Plugs and
96 Well Plates
[0301] For FIG. 1d, 96 well plates results for FIG. 1d were
acquired in a Tecan Safire II plate reader (MTX Lab Systems,
Vienna, Va.) with Ex/Em 560/630 nm, gain 25, and 40 .mu.s
integration time. 200 .mu.L of cell culture suspended in LB with
10% alamarBlue was added to wells of a Costar 96 well assay plate
with black sides and a clear, flat bottom (Corning, Corning, N.Y.).
Each data point represents triplicate measurements taken at
37.degree. C. Fold change in intensity from 96 well plate results
were calculated by using Eq. 1 where the well with LB and
alamarBlue only was the unoccupied plug condition.
[0302] Screening Susceptibility of Bacteria to Many Antibiotics
[0303] For antibiotic screening experiments (FIG. 2), an array of
.about.50 nL antibiotic plugs was aspirated into Teflon tubing (200
.mu.m ID) using a manual aspirator. Air spacers were included
between each antibiotic plug to prevent merging of adjacent
antibiotic plugs and to enable indexing of plugs in the output
array. Plugs of saline solution were included as the first and last
plugs in the preformed array to serve as positive controls. The
Teflon tubing containing the array of antibiotic plugs was sealed
into a device inlet by using wax (Hampton Research, Aliso Viejo,
Calif.). To screen the susceptibility of MRSA and MSSA to each
antibiotic, bacterial samples and indicator were merged with the
preformed array of antibiotic. Bacterial samples were at a density
of 4.times.10.sup.5 CFU/mL in LB, and viability indicator solution
was made by mixing 4 parts alamarBlue solution (AbD Serotec,
Oxford, UK) with 6 parts 150 mM NaCl., The flow rate of the
antibiotic array was 0.25 .mu.L/min; the flow rate of the bacterial
solution was 0.5 .mu.L/min, and the flow rate of the viability
indicator was 0.25 .mu.L/min. The carrier fluid was FC40 (Acros
Organics, Morris Plains, N.J.) with a flow rate of 1.6 .mu.L/min.
For each antibiotic plug in the preformed array, approximately 50
smaller plugs (4 mL in volume) were formed, each potentially
containing a single bacterium. The resulting plugs were collected
in the coil PTFE Teflon tubing (I.D.=150 .mu.m).
[0304] After plug formation, the tubing was disconnected from the
PDMS device, and the ends were sealed with wax. The Petri dish
containing the tubing was filled with 20 mL of LB solution to
prevent evaporation of the plugs during incubation. The plugs were
immediately transferred to a microscope incubator (Pecon GmbH,
Erbach, Germany). Time zero is defined as the time which the plugs
entered the incubator, which was about 20 minutes after plugs were
formed. Fluorescence measurements for plugs were recorded by using
an inverted epi-fluorescence microscope (DMI6000, Leica,
Bannockburn, Ill.) with a 10.times.0.3 NA objective (HCX PL
Fluotar) coupled to a CCD camera ORCA ERG 1394 (12-bit,
1344.times.1024 resolution) (Hamamatsu Photonics) by using a
0.63.times. camera coupler. Images were taken of each plug using
Metamorph Imaging Software (Molecular Devices, Sunnyvale, Calif.)
every 30 min with exposure times of 5 ms. Plugs were analyzed by
first separating them from the background by thresholding to
exclude intensity below 250. The average intensity of the
thresholded plugs was measured. The change in intensity at time
point ti is I.sub.t=ti-I.sub.t=1. In the experiment described in
FIG. 2c, fluorescence intensity of plugs was normalized by setting
the intensity of the brightest plug to 100.
[0305] Determining the Minimal Inhibitory Concentration of a Drug
Against a Bacterial Sample
[0306] For MIC determination in plugs (FIG. 3), a procedure similar
to screening susceptibility of many antibiotics was used. The input
array of antibiotics consisted of plugs of CFX at a range of
concentrations. Bacterial samples were MRSA or MSSA in LB at cell
densities near 10.sup.6 CFU/mL. In FIGS. 3b and c, fluorescence
intensity of plugs was normalized as described for FIG. 2c.
[0307] Statistical Analysis of Antibiotic Screening Results
[0308] Unpaired t-tests were performed to compare antibiotic
screening results to positive and negative controls. For FIG. 2c:
VCM and LVF are statistically different than positive controls and
AMP, CFX, OXA, ERT, and blank conditions were all statistically
different than the negative control. For FIG. 3b: 8 and 24 mg/L CFX
were statistically different than positive controls and 0, 0.2, 1,
2, and 4 mg/L were statistically different than the negative
control. For FIG. 3c: 4, 8, and 24 mg/L CFX were statistically
different than positive controls and 0, 0.2, 1, and 2 mg/L were
statistically different than the negative control. P values are
two-tailed.
[0309] Detection and Drug Screening of MRSA and MSSA in Human Blood
Plasma
[0310] For FIG. 4, cells were suspended in a 1:1 mixture of human
blood plasma (Pooled normal plasma George King Bio-Medical,
Overland Park, Kans.) and LB containing 40% alamarBlue. Plugs were
formed and collected in Teflon tubing (200 .mu.m ID). Images were
taken with a 5.times.0.15 NA objective with a 0.63.times. camera
coupler. Texas red pictures were taking every 10 minutes with
exposure times of 25 ms. A bright-field image was taken at
beginning and end of experiment. Linescans of original plug images
were taken at time 0 and time 7.5 h. Adobe Photoshop was used to
enhance contrast of plugs shown in FIG. 4.
[0311] Microfluidic bacterial detection and drug screening are
applicable to complex, natural matrices, including human blood
plasma.
[0312] To validate the applicability of this method to detecting
bacteria in natural matrices, this method was used to detect
bacteria in a sample of human blood plasma. Bacterial strains MSSA
or MRSA were inoculated into pooled human blood plasma at a
concentration of 3.times.10.sup.5 CFU/mL. To test the sensitivity
of the bacteria to beta-lactams, the antibiotic ampicillin (AMP)
was added to the culture at the breakpoint concentration. The
inoculated plasma was then combined on-chip with viability
indicator as illustrated FIG. 4a. After 7.5 h of incubation at
37.degree. C., plasma samples infected with MRSA were
distinguishable from samples infected by MSSA by screening the
samples against AMP at the breakpoint concentration. While plugs
containing MRSA and AMP showed a similar increase in fluorescence
intensity to plugs containing MRSA and no AMP (FIGS. 4b and c),
plugs containing MSSA and AMP showed no increase in fluorescence
intensity (FIGS. 7d and e).
[0313] Amplification examples 1-6 involve blood coagulation
proteins. Amplification example 7 involves proteins in apoptosis.
Amplification example 8 involves Limulus amebocyte lysate from
Horseshoe crabs.
Amplification Example 1
[0314] Amplification process: The enzyme precursor is engineered
prothrombin that may be cleaved by thrombin to produce more
thrombin. The potential of this engineering approach is supported
by a method to make an engineered factor X that may be cleaved by
thrombin. (Louvain-Quintard, V. B.; Bianchini, E. P.;
Calmel-Tareau, C.; Tagzirt, M.; Le Bonniec, B. F.,
Thrombin-activable factor X re-establishes an intrinsic
amplification in tenase-deficient plasmas. Journal of Biological
Chemistry 2005, 280, (50), 41352-41359.)
[0315] Inhibitory mechanism: The inhibitor is hirudin (or other
inhibitors or antibodies of thrombin such as antithrombin III,
heparin, or anophelin), which binds to thrombin and prevent the
cleavage of prothrombin by thrombin.
[0316] Activating mechanism: The analyte, bacterial phosphatase,
cleaves a tag attached to an inhibitor of hirudin (anti-hirudin),
allowing this activated molecule to inhibit hirudin and release
thrombin from hirudin. Alternatively, the bacterial phosphatase
cleaves a tag attached to thrombin. In both cases, thrombin then
cleaves prothrombin and produces more thrombin. The specificity of
this method may be designed to match expectation by changing the
specificity of the cleavage of the tag by bacterial phosphatase. If
detection of an enzyme different from phosphatase is needed, a
different tag is used.
[0317] Readout process: Thrombin enzymatically cleaves a
fluorogenic substrate (such as Boc-Asp(OBzl)-Pro-Arg-MCA) to give
fluorescent signal.
[0318] This method is predicted to be feasible with concentration
of prothrombin from 1 nM to 1 .mu.M, be activated with
concentration of thrombin input after concentrating techniques of
as low as 10 .mu.M, give response after 1-10 minutes, and give
amplification gain of 3 to 9 orders of magnitude.
Amplification Example 2
[0319] Amplification process: The enzyme precursor is factor XII
that may be cleaved by factor XIIa to produce more factor XIIa in
the presence of dextran sulfate or negatively charge surface in
general. (Tankersley, D. L.; Finlayson, J. S., Kinetics of
Activation and Autoactivation of Human Factor-Xii. Biochemistry
1984, 23, (2), 273-279.)
[0320] Inhibitory mechanism: The inhibitor is ecotin (or other
inhibitors or antibodies of factor XIIa).
[0321] Activating mechanism: the analyte, bacterial phosphatase,
cleaves a tag attached to an inhibitor of ecotin, allowing this
activated molecule to inhibit ecotin and release factor XIIa from
ecotin. The system may also be designed so that the analyte cleaves
a tag attached to factor XIIa or kallikrein. Factor XIIa or
kallikrein then cleaves factor XII and produces more factor XIIa.
The specificity of this method may be designed to match expectation
by changing the specificity of the cleavage of the tag by bacterial
phosphatase. If detection of an enzyme different from phosphatase
is needed, a different tag is used.
[0322] Readout process: Factor XIIa enzymatically cleaves a
fluorogenic substrate (such as Boc-Gln-Gly-Arg-MCA) to give
fluorescent signal.
[0323] This method is predicted to be feasible with concentration
of factor XII from 0.1 .mu.M to 10 .mu.M, be activated with
concentration of kallikrein input after concentrating techniques of
as low as 1 nM, give response after 1-10 minutes, and give
amplification gain of 3 to 5 orders of magnitude.
Amplification Example 3
[0324] Amplification process: The enzyme precursor is factor XI
that may be cleaved by factor XIa to produce more factor XIa in the
presence of dextran sulfate or negatively charge surface in
general. (Gailani, D.; Broze, G. J., Factor-Xi Activation in a
Revised Model of Blood-Coagulation. Science 1991, 253, (5022),
909-912; Naito, K.; Fujikawa, K., Activation of Human
Blood-Coagulation Factor-Xi Independent of Factor-Xii-Factor-Xi Is
Activated by Thrombin and Factor-Xia in the Presence of Negatively
Charged Surfaces. Journal of Biological Chemistry 1991, 266, (12),
7353-7358.)
[0325] Inhibitory mechanism: The inhibitor is aprotinin (or other
inhibitors or antibodies of factor XIa).
[0326] Activating mechanism: the analyte, bacterial phosphatase,
cleaves a tag attached to an inhibitor of aprotinin, allowing this
activated molecule to inhibit aprotinin and release factor XIa from
aprotinin. The system may also be designed so that the analyte
cleaves a tag attached to factor XIa. Factor XIa then cleaves
factor XI and produces more factor XIa. The specificity of this
method may be designed to match expectation by changing the
specificity of the cleavage of the tag by bacterial phosphatase. If
detection of an enzyme different from phosphatase is needed, a
different tag is used.
[0327] Readout process: Factor XIa enzymatically cleaves a
fluorogenic substrate (such as Boc-Glu(OBzl)-Ala-Arg-MCA) to give
fluorescent signal.
[0328] This method is predicted to be feasible with concentration
of factor XI from 0.1 .mu.M to 10 .mu.M, be activated with
concentration of factor XIa input after concentrating techniques of
as low as 1 nM, give response after 1-10 minutes, and give
amplification gain of 3 to 5 orders of magnitude.
Amplification Example 4
[0329] Amplification process: Factor XII and prekallikrein are
precursors of factor XIIa and kallikrein, respectively. In the
presence of dextran sulfate or a negatively charged surface in
general, factor XIIa cleaves both factor XII and prekallikrein to
produce factor XIIa and kallikrein, respectively, while kallikrein
cleaves factor XII to produce factor XIIa. (Tankersley, D. L.;
Finlayson, J. S., Kinetics of Activation and Autoactivation of
Human Factor-Xii. Biochemistry 1984, 23, (2), 273-279.)
[0330] Inhibitory mechanism: Inhibitors or antibodies that are
common to both factor XIIa and kallikrein (such as ecotin) or
different inhibitors specific to each may be used.
[0331] Activating mechanism: the analyte, bacterial phosphatase,
cleaves a tag attached to an inhibitor of ecotin, allowing this
activated molecule to inhibit ecotin and release factor XIIa and
kallikrein from ecotin. The system may also be designed so that the
analyte cleaves a tag attached to factor XIIa and/or kallikrein,
which then activate the amplification process. The specificity of
this method may be designed to match expectation by changing the
specificity of the cleavage of the tag by bacterial phosphatase. If
detection of an enzyme different from phosphatase is needed, a
different tag is used.
[0332] Readout process: Factor XIIa or kallikrein or both
enzymatically cleave fluorogenic substrates (such as
Boc-Gln-Gly-Arg-MCA for factor XIIa and Pro-Phe-Arg-MCA for
kallikrein) to give fluorescent signal.
[0333] This method is predicted to be feasible with concentration
of factor XII from 0.1 .mu.M to 10 .mu.M and concentration of
kallikrein from 10 .mu.M to 10 nM, be activated with concentration
of kallikrein input after concentrating techniques of as low as 1
nM, give response after 1-10 minutes, and give amplification gain
of 3 to 5 orders of magnitude.
Amplification Example 5
[0334] Amplification process: The reaction network with positive
feedback is shown in FIG. 13. This is an example of cases in which
positive feedback loops may be achieved through multiple steps and
many positive feedback loops may be coupled with each other to form
a cascade. In this network, there are two positive feedback loops.
The first one includes the production of factor Xa from factor X
catalyzed by the input (InhA1), the production of factor VIIIa from
factor VIII, the binding of factor VIIIa to factor IXa to form a
complex, and the production of Xa from factor X catalyzed by the
VIIIa:IXa complex. The output of this first loop is factor Xa. The
second positive feedback loop includes the production of factor Va
from factor V catalyzed by the input (factor Xa), the binding of
factor Va to the input (factor Xa), the production of thrombin from
prothrombin catalyzed by the Xa:Va complex or the input of the
first loop (InhA1), and the production of factor Va from factor V
catalyzed by thrombin. The output of this loop is thrombin.
Thrombin is detected by the cleavage of a fluorogenic substrate to
release fluorescent molecules catalyzed by thrombin. ATIII/heparin
is used to inhibit factor Xa, the VIIIa:IXa complex, and
thrombin.
[0335] Inhibitory mechanism: The chemical inhibitors are
ATIII/heparin that may inhibit factor Xa, the VIIIa:IXa complex,
and thrombin. Other common or specific inhibitors for these
enzymes, of for factor VIIIa, Va, and IXa may be used as well.
[0336] Activation mechanism: Similar to examples 1-4, the analyte,
bacterial phosphatase, may cleave tagged molecules and release
them. These molecules may be the activated factors shown in FIG. 13
or inhibitors of the inhibitors of those activated factors used in
the inhibitory mechanism. The specificity of this method may be
designed to match expectation by changing the specificity of the
cleavage of the tag by bacterial phosphatase. If detection of an
enzyme different from phosphatase is needed, a different tag is
used.
[0337] Readout process: Thrombin enzymatically cleaves fluorogenic
substrates (such as Boc-Asp(OBzl)-Pro-Arg-MCA) to give fluorescent
signal as shown in FIG. 13. Other single of combinations of
activated factors may be used to cleave fluorogenic substrates as
well.
[0338] Using the techniques described in this application, this
method is predicted to be feasible with concentration of enzyme
precursors from 0.1 .mu.M to 1 .mu.M, be activated with
concentration of InhA1 input after concentrating techniques of as
low as 1 nM, give response after 1-10 minutes, and give
amplification gain of 3 to 6 orders of magnitude.
Amplification Example 6
[0339] This example is similar to amplification example 5, but is
more complicated. Here the amplification process contains most of
the components in the natural blood clotting network, as shown in a
review. (Kastrup, C. J.; Runyon, M. K.; Lucchetta, E. M.; Price, J.
M.; Ismagilov, R. F., Using chemistry and microfluidics to
understand the spatial dynamics of complex biological networks.
Accounts of Chemical Research 2008, 41, (4), 549-558.) Positive
feedback loops are achieved through multiple steps. Inhibitory
mechanism, activating mechanism, and readout process are also
similar to amplification example 5, and can be done with single of
combinations of enzymes. Additionally, blood clotting may also be
visualized by eyes. (Song, H.; Li, H. W.; Munson, M. S.; Van Ha, T.
G.; Ismagilov, R. F., On-chip titration of an anticoagulant
argatroban and determination of the clotting time within whole
blood or plasma using a plug-based microfluidic system. Analytical
Chemistry 2006, 78, (14), 4839-4849.)
[0340] Using the techniques described in this application and the
reaction conditions previously described, (Kastrup, C. J.; Runyon,
M. K.; Shen, F.; Ismagilov, R. F., Modular chemical mechanism
predicts spatiotemporal dynamics of initiation in the complex
network of hemostasis. Proceedings of the National Academy of
Sciences of the United States of America 2006, 103, (43),
15747-15752.) the predicted response is in 1-5 minutes.
[0341] Results from simulations and experiments (Kastrup, C. J.;
Runyon, M. K.; Shen, F.; Ismagilov, R. F., Modular chemical
mechanism predicts spatiotemporal dynamics of initiation in the
complex network of hemostasis. Proceedings of the National Academy
of Sciences of the United States of America 2006, 103, (43),
15747-15752.) shown in FIGS. 14 and 15 support the ideas in
amplification examples 1-6 discussed above. In general, they show a
threshold-like behavior in which the time of response (time for
amount of some certain substance to reach a detectable value)
drastically reduces as the amount of input increases over a certain
value. Although only amplification examples 1, 2, and 4 were
considered in FIG. 14, and amplification example 6 in FIG. 15, the
reaction network of amplification example 3 is similar to that of
amplification example 1 and the reaction network of amplification
example 5 has complexity between those of amplification examples 4
and 6. Therefore, amplification examples 3 and 5 is expected to
work as well.
[0342] FIG. 14 is the time to get response versus amount of input
obtained by simulation using previously found rate constants
(Tankersley, D. L.; Finlayson, J. S., Kinetics of Activation and
Autoactivation of Human Factor-Xii. Biochemistry 1984, 23, (2),
273-279; Kuharsky, A. L.; Fogelson, A. L., Surface-mediated control
of blood coagulation: The role of binding site densities and
platelet deposition. Biophysical Journal 2001, 80, (3), 1050-1074;
Ulmer, J. S.; Lindquist, R. N.; Dennis, M. S.; Lazarus, R. A.,
Ecotin Is a Potent Inhibitor of the Contact System Proteases Factor
Xiia and Plasma Kallikrein. Febs Letters 1995, 365, (2-3), 159-163.
Kawabata, S. I.; Miura, T.; Morita, T.; Kato, H.; Fujikawa, K.;
Iwanaga, S.; Takada, K.; Kimura, T.; Sakakibara, S., Highly
Sensitive Peptide-4-Methylcoumaryl-7-Amide Substrates for
Blood-Clotting Proteases and Trypsin. European Journal of
Biochemistry 1988, 172, (1), 17-25; Stone, S. R.; Hofsteenge, J.,
Kinetics of the Inhibition of Thrombin by Hirudin. Biochemistry
1986, 25, (16), 4622-4628.)
[0343] FIG. 1(a) is the simulation of the amplification process
used in amplification example 1, with set initial concentration of
the engineered prothrombin (1.4*10.sup.-6 M), thrombin
(1.4*10.sup.-10 M), and hirudin (1*10.sup.-8 M), and varied
concentration of input which is thrombin. Time of response for each
concentration of input was defined as the time when concentration
of thrombin reaches 80% of the initial concentration of prothrombin
(if this time is larger than 10000 seconds, it was set to 10000
seconds). Rate of cleavage of engineered prothrombin by thrombin
was taken to be the same as the rate of cleavage of factor V by
thrombin.
[0344] FIG. 1(b) is the simulation of the amplification process
used in example 2, with set initial concentration of the factor XII
(1.times.10.sup.-6 M), factor XIIa (1.times.10.sup.-10 M), ecotin
(1.times.10.sup.-7 M), and Boc-Gln-Gly-Arg-MCA (fluorogenic
substrate for factor XIIa), and varied concentration of input which
is kallikrein. Time of response for each particular concentration
of input was defined as the time when concentration of the
fluorescent molecules reaches 80% of the initial concentration of
the fluorogenic substrate. If this time is larger than 10000
seconds, it was set to 10000 seconds.
[0345] FIG. 1(c) Simulation of the amplification process used in
example 4, with set initial concentration of the factor XII
(1.times.10.sup.-6 M), factor XIIa (1.times.10.sup.-10 M),
prekallikrein (1.times.10.sup.-10 M), kallikrein
(1.times.10.sup.-14 M), ecotin (1.times.10.sup.-7 M), and
Boc-Gln-Gly-Arg-MCA (fluorogenic substrate for factor XIIa), and
varied concentration of input which is kallikrein. Time of response
for each particular concentration of input was defined as the time
when concentration of the fluorescent molecules reaches 80% of the
initial concentration of the fluorogenic substrate. If this time is
larger than 10000 seconds, it was set to 10000 seconds.
[0346] FIG. 15 is the experimental results showing how blood
clotting time varied with size of patch of tissue factor, an input
for the blood clotting network discussed in example 6. (Kastrup, C.
J.; Runyon, M. K.; Shen, F.; Ismagilov, R. F., Modular chemical
mechanism predicts spatiotemporal dynamics of initiation in the
complex network of hemostasis. Proceedings of the National Academy
of Sciences of the United States of America 2006, 103, (43),
15747-15752.) The patch size in these experiments correlated with
local concentration of tissue factor.
Amplification Example 7
[0347] Amplification process: This reaction network with positive
feedback involves proteins in apoptosis. This network includes the
production of caspase-9 from procaspase-9 catalyzed by cytochrome C
and Apaf1, the dimerization of caspase-9, the production of
caspase-3 from procaspase-3 catalyzed by caspase-9-dimer, the
production of caspase-9 from procaspase-9 by catalyzed by either
caspase-9 dimer or caspase-3.
[0348] Inhibitory mechanism: Inhibitors or antibodies for caspase-3
and/or caspase-9 are used.
[0349] Activation mechanism: Similar to examples 1-5, the analyte,
bacterial phosphatase, may cleave tagged molecules and release
them. These molecules may be caspase-3 and/or caspase-9, or
inhibitors of the inhibitors used in the inhibitory mechanism. The
specificity of this method may be designed to match expectation by
changing the specificity of the cleavage of the tag by bacterial
phosphatase. If detection of an enzyme different from phosphatase
is needed, a different tag is used.
[0350] Readout process: caspase-3 and/or caspase-9 enzymatically
cleave fluorogenic substrates to give fluorescent signal.
[0351] Using the techniques described in this patent, this method
is predicted to be feasible with concentrations of enzyme
precursors from 0.1 .mu.M to 10 .mu.M, be activated with
concentration of input after concentrating techniques of as low as
1 nM, give response after 1-10 minutes, and give amplification gain
of 3 to 5 orders of magnitude.
Amplification Example 8
[0352] Limulus amebocyte lysate (LAL) is known to coagulate when
bacterial lipopolysaccharide (LPS) is present. One mechanism is the
binding of LPS to an 82-kDa protein (termed LPS-binding protein
(LBP)), which normally negatively regulates coagulation. (Roth, R.
I.; Tobias, P. S., Lipopolysaccharide-Binding Proteins of Limulus
Amebocyte Lysate. Infection and Immunity 1993, 61, (3),
1033-1039.)
[0353] Amplification process: The clotting network of LAL.
[0354] Inhibitory mechanism: A small excess of LBP.
[0355] Activating mechanism: Generally, the input may be or promote
the production of some substance that binds to LBP. The input may
be bacterial LPS or bacterial phosphatase that may cleave tagged
(and inactive) LPS.
[0356] Readout process: Clotting or absorbance at 405 nm may be
used.
[0357] The variations below can be applied individually or in
combinations with other variations to all of amplification examples
1-8 shown above.
[0358] Variation 1:
[0359] Negative contrast is used instead of positive contrast.
[0360] Amplification processes: The processes used in examples 1-8
are used here.
[0361] Inhibitory mechanism: There is no inhibitory mechanism.
[0362] Activating mechanism: Inhibitory mechanism used in examples
1-8 are used as input to see the contrast.
[0363] Readout process: The processes used in examples 1-8 are used
here.
[0364] Variation 2:
[0365] An enzyme precursor (inactive form) and an enzyme (active
form) do not have to be two totally different molecules. They only
need to have different reactivity.
[0366] The inactive form may have an inactive conformation while
the active form has an active conformation. A conformation change
may be facilitated by binding of a small molecule to an enzyme, an
enzyme to and enzyme, a small molecule to a DNA or RNA molecule, an
enzyme to a DNA or RNA molecule, or binding of more than two
substances.
[0367] The inactive form may be tagged with an inhibitor, thus
being self-inhibitory. When the linker to the inhibitor is cleaved,
the molecule is now active. Positive feedback can be incorporated
in these variations because the active enzyme can catalyze the
change of conformation or the cleavage of the self-inhibiting tag
of another enzyme of the same kind or of different kind.
[0368] Variation 3:
[0369] Detection of molecules other than enzymes may be achieved as
well. To the systems in examples 1-8 without or with any single or
combination of variations, one or multiple steps is added. The
analyte, which may not be an enzyme, promotes the activation of an
enzyme that may cleave the tag from the substrate used in
activating mechanisms in examples 1-8, through one or multiple
reactions. For example, activation of enzymes may be done through
change of conformation (a result of binding of some molecule to an
enzyme), linking two inactive components to make an active
substance, or activating another enzyme that may activate the
enzyme of interest.
[0370] Variation 4:
[0371] The techniques described above with or without any
variations may be used in combinations with amplification using
materials and activation mechanism involving generation of high
local concentration.
Amplification Example 9
[0372] Amplification process: The reaction of the purple complex of
Co(III) and
2-(5-bromo-2-pyridylazo)-5[N-n-propyl-N-(3-sulfopropyl)amino]phenol
(5-Br-PAPS) and oxone has Co.sup.2+ (aq) as the autocatalyst.
(Endo, M.; Abe, S.; Deguchi, Y.; Yotsuyanagi, T., Kinetic
determination of trace cobalt(II) by visual autocatalytic
indication. Talanta 1998, 47, (2), 349-353; Endo, M.; Ishihara, M.;
Yotsuyanagi, T., Autocatalytic decomposition of cobalt complexes as
an indicator system for the determination of trace amounts of
cobalt and effectors. Analyst 1996, 121, (4), 391-394; Tsukada, S.;
Miki, H.; Lin, J. M.; Suzuki, T.; Yamada, M., Chemiluminescence
from fluorescent organic compounds induced by cobalt(II) catalyzed
decomposition of peroxomonosulfate. Analytica Chimica Acta 1998,
371, (2-3), 163-170.)
[0373] Inhibitory mechanism: A ligand that can be used to capture
Co.sup.2+. As a technical detail, the Co(III).(5-Br-PAPS) complex
may be separated from oxone by immobilizing them into two different
layers or keeping the mixture dry.
[0374] Activating mechanism: The input, which may be an enzyme or a
small molecule, may cleave or compete for the ligand that captures
Co.sup.2+ discussed in the inhibitory mechanism. Alternatively, the
input may also be a reducing agent that reduces Co.sup.3+ to
Co.sup.2+ rapidly but reduces oxone more slowly.
[0375] Readout process: The Co(III).(5-Br-PAPS) complex has a
purple color that is lost when the reaction gets activated because
of the conversion of Co(III) into Co(II) and the oxidation of the
organic ligand.
Amplification Example 10
[0376] Amplification process: The reduction of aqueous Ag(I) is
catalyzed by its product, Ag(0). Other metals (such as Pd, Au, and
Pt) may also be used for the autocatalytic reduction of metal
cations to lower oxidation states.
[0377] Inhibitory mechanism: The reductant may be chemically
protected. For example, the reductant may be based on hydroquinone,
with the hydroxyl groups protected by a group that is removed by
the process of interest. The metal cation may be captured by a
ligand. The autocatalyst metal may be coated with small molecules,
gel or polymer.
[0378] Activating mechanism: The input (enzyme or small molecule)
may deprotect the reductant by cleaving off the protective rings.
The input may also destroy or compete for the ligand. The input may
also remove the coating layer on the metal particle.
[0379] Readout process: Visual readout may be achieved via
aggregates (precipitates) of metal, specific indicator for
different oxidation states of the metals, or general
reduction-oxidation indicator.
Amplification Example 11
[0380] Amplification process: The reduction-oxidation reaction
between chlorite and iodide has iodine as the autocatalyst. (Dateo,
C. E.; Orban, M.; Dekepper, P.; Epstein, I. R., Systematic Design
of Chemical Oscillators 0.5. Bistability and Oscillations in the
Autocatalytic Chlorite Iodide Reaction in a Stirred-Flow Reactor.
Journal of the American Chemical Society 1982, 104, (2),
504-509.)
[0381] Inhibitory mechanism: A compound may be used to consume the
autocatalyst, iodine, thus slowing down the reaction effectively to
the degree that no significant output is visualized after a long
time.
[0382] Activating mechanism: The input may be iodine, or produce
iodine through one or multiple reactions. The input may also be a
compound that inhibits the consumption of iodine, or may produce
such compound after one or many steps.
[0383] Readout process: Starch may be used to give a strongly
colored blue complex with the product iodine.
Amplification Example 12
[0384] Amplification process: The reduction-oxidation reaction
between thiosulfate and chlorite is autocatalytic in both hydronium
ion and chloride ion, (Runyon, M. K.; Johnson-Kerner, B. L.;
Ismagilov, R. F., Minimal functional model of hemostasis in a
biomimetic microfluidic system. Angewandte Chemie-International
Edition 2004, 43, (12), 1531-1536.
[0385] Horvath, A. K.; Nagypal, I.; Epstein, I. R., Kinetics and
mechanism of the chlorine dioxide-tetrathionate reaction. Journal
of Physical Chemistry A 2003, 107, (47), 10063-10068; Horvath, A.
K.; Nagypal, I.; Epstein, I. R., Oscillatory photochemical
decomposition of tetrathionate ion. Journal of the American
Chemical Society 2002, 124, (37), 10956-10957; Nagypal, I.;
Epstein, I. R., Systematic Design of Chemical Oscillators 0.37.
Fluctuations and Stirring Rate Effects in the Chlorite Thiosulfate
Reaction. Journal of Physical Chemistry 1986, 90, (23), 6285-6292.)
and may be activated by Ag.sup.+ which is predicted to oxidize
thiosulfate and release hydronium or chloride (preliminary
result).
[0386] Inhibitory mechanism: The amount of hydronium ion may be
kept in check by using a pH buffer. Chloride anion may be
sequestered by a cation that is a sufficiently weak oxidizer to not
react with thiosulfate. Ag.sup.+ may be sequestered by ligands.
Solids that produce an acidic environment or solids that are salts
of Cl.sup.- or Ag.sup.+ may be coated with materials such as gel or
polymer.
[0387] Activating mechanism: The input may be acidic or promote the
production of any acid through one or multiple steps. The input may
also be chloride anion or promote the production of chloride anion,
or compete with the cation that captures chloride anion, or react
with the cation to disable its ability to capture chloride anion.
The input may also be Ag.sup.+ or produce Ag.sup.+ by compete for
or inactivate the ligands for Ag.sup.+. The input may also break
the coating materials of the solid particles if such inhibitory
mechanism is used.
[0388] Readout process: A pH or reduction-oxidation indicator may
be used.
[0389] Using the techniques described in this patent and the
reaction conditions previously described, (Kastrup, C. J.; Runyon,
M. K.; Shen, F.; Ismagilov, R. F., Modular chemical mechanism
predicts spatiotemporal dynamics of initiation in the complex
network of hemostasis. Proceedings of the National Academy of
Sciences of the United States of America 2006, 103, (43),
15747-15752.) one can get response in 1-2 minutes.
Amplification Example 13
[0390] Amplification process: The organic reactions developed by
Ichimura and coworkers (Ichimura, K., Nonlinear organic reactions
to proliferate acidic and basic molecules and their applications.
Chemical Record 2002, 2, (1), 46-55.) have acids as
autocatalysts.
[0391] Inhibitory mechanism: The amount of hydronium ion may be
kept in check by using a pH buffer or a weak base.
[0392] Activating mechanism: The input may be acidic or promote the
production of any acid through one or multiple steps.
[0393] Readout process: A pH or reduction-oxidation indicator is
used.
[0394] The variations below can be applied individually or in
combinations with other variations to all of amplification examples
9-13 shown above
[0395] Variation 1:
[0396] Negative contrast is used instead of positive contrast.
[0397] Amplification processes: The processes used in examples 1-5
described above are used.
[0398] Inhibitory mechanism: There is no inhibitory mechanism.
[0399] Activating mechanism: Inhibitory mechanism used in examples
1-5 described above are used as input to see the contrast.
[0400] Readout process: The processes used in examples 1-5
described above are used here.
[0401] Variation 2:
[0402] The techniques described above with or without any variation
may be used in combinations amplification using materials and
activation mechanism involving generation of high local
concentration.
Amplification Example 14
[0403] Provided the analytes are particles that can be tagged with
some activating molecules through various methods (such as
antibodies, His-tag/Ni/NTA, biotin/avidin etc), a method can be
used to detect small concentration of the analytes in which the
amplification process may be any. The activating molecule is first
concentrated on particles. Then stochastic confinement is performed
to concentrate these particles into plugs. The concentrated
activating molecules act as input for the amplification process.
Because excess activating molecules are not concentrated on
particles, even after stochastic confinement, the concentration is
still not high enough to activate the amplification process.
[0404] For example, the amplification process may be chosen as the
one described in example 5 of section 1 and in FIG. 13, the
activating molecules may be chosen to be InhA1, as shown in FIG.
16. The particular example is predicted to be able to detect
particles of concentration of as low as 1fM, which may
hypothetically be amplified to 1 nM after stochastic confinement.
To total time of detection is predicted to be 1-10 minutes.
[0405] FIG. 16 illustrates combining amplification cascades with
stochastic confinement enables sensitive detection of single
particles or more. The general idea is described in the text. The
protease shown here represents an activating molecule in general.
(A and B) Particles are added to a container containing the
activating molecules. (C) The particles bind to the activating
molecules and are stochastically confined in plugs. (D) In plugs
loaded with particles, the concentration of activating molecules is
above the detection threshold and produces a threshold signal,
while plugs without particles do not. (E and F) In solutions with
excess but sub-threshold concentrations of activating molecules,
stochastic confinement of particles into plugs (G) results in a few
plugs containing a particle with multiple copies of the activating
molecules attached and many plugs with a few copies of activating
molecules. (H) Plugs containing a particle initiate the cascade,
because stochastic confinement of the particles has concentrated
the activating molecules in occupied plugs, whereas plugs without a
particle remain at the sub-threshold protease concentration of the
bulk solution.
Amplification Example 15
[0406] The analytes are particles which bind to activating
molecules. The reactions may be chosen from those described in
sections 1 and 2. For example, if the system with example 2 in
section 1 is chosen, the activating molecule is kallikrein.
[0407] Amplification process: The enzyme precursor is factor XII
that may be cleaved by factor XIIa to produce more factor XIIa in
the presence of dextran sulfate or negatively charge surface in
general.
[0408] Suppose a sample containing low concentration of type-B
particles (such as 100 CFU/mL) has a large excess of interfering
type-A particles (such as 10.sup.5 CFU/mL) that can bind much less
activating molecules per particle (such as 100 fold) (with a
specifically designed antibody or any other means), but much more
collectively. Under bulk detection mechanisms, either with
amplification or by classical methods for measuring enzyme
concentrations, the activity due to the excess of type-A particles
will dominate the response (FIGS. 17 A and B). When the tagged
particles are stochastically confined in plugs, only those
containing type-B particles will have enough activating molecules
to activate the amplification process. In general, any kind of
particles which can bind to any of activating molecules described
in sections 1 and 2 through antibodies or any other method may be
detected using this method. For example, this technique may be use
to detect B. anthracis from a sample containing a lot of
interfering bacteria such as B. circulans and other kinds.
[0409] FIG. 17 illustrates selective detection of particles by
using stochastic confinement. (A and B) In a bulk solution
containing both a high concentration of interfering type-A
particles (such as the bacteria B. circulans) (small gray particles
binding few activating molecules (such as kallikrein)) and a low
concentration of target type-B particles (B. anthracis) (large gray
particles binding many activating molecules), the activity of the
excess type-A particles dominates. Therefore, solutions with excess
type-A particles (A) and excess type-B particle with a low
concentration of type-A particles (B) both trigger a detection
response (dark gray) (C and D). When stochastically confined in
plugs, the amount of activating molecule in each plug made from
solution A remains below the detectible level, while the amount of
activating molecules of plugs containing type-B particles from
solution B results in readout of type-B particles (dark gray plug),
not from interfering type-A particles (lighter gray plugs).
Amplification Example 16
[0410] This technique is a variation of example 2 in section 2. Ag
particles are coated with tags to form Ag--(X--Y).sub.n. This
coated particle cannot bind with bacteria. However, the input
cleaves or produces some substance that cleaves X--Y, exposing X on
the surface of the particles. These particles now can be locally
concentrated using techniques shown in section 4ii.
Amplification Example 17
[0411] This technique is a variation of example 4 in section 2.
This system contains AgCl particles coated with an inert shell
(such as Ca.sub.3(PO.sub.4).sub.2) and protected
ethylenediaminetetraacetic acid (EDTA) ligands (in the solution or
in the particle), and thiosulfate and chlorite in the solution. The
input deprotects EDTA, allowing it to complex with Ca.sup.2+,
dissolving the inert shell, exposing AgCl to the
thiosulfate/chlorite mixture and activates the amplification
process. The threshold is set up by the thickness of the inert
shell. To detect different kinds of input, the method to protect
and deprotect EDTA may be customized. The major reaction in this
amplification process was described previously. (Runyon, M. K.;
Johnson-Kerner, B. L.; Ismagilov, R. F., Minimal functional model
of hemostasis in a biomimetic microfluidic system. Angewandte
Chemie-International Edition 2004, 43, (12), 1531-1536; Horvath, A.
K.; Nagypal, I.; Epstein, I. R., Kinetics and mechanism of the
chlorine dioxide-tetrathionate reaction. Journal of Physical
Chemistry A 2003, 107, (47), 10063-10068; Horvath, A. K.; Nagypal,
I.; Epstein, I. R., Oscillatory photochemical decomposition of
tetrathionate ion. Journal of the American Chemical Society 2002,
124, (37), 10956-10957; Nagypal, I.; Epstein, I. R., Systematic
Design of Chemical Oscillators 0.37. Fluctuations and Stirring Rate
Effects in the Chlorite Thiosulfate Reaction. Journal of Physical
Chemistry 1986, 90, (23), 6285-6292.)
* * * * *