U.S. patent application number 14/942903 was filed with the patent office on 2016-05-19 for digital control of on-chip magnetic particle assay.
This patent application is currently assigned to SILICON BIODEVICES, INC.. The applicant listed for this patent is Silicon BioDevices, Inc.. Invention is credited to Remy Cromer, Octavian Florescu, Tracie Martin, Daniel Wong, Duane Yamasaki.
Application Number | 20160139035 14/942903 |
Document ID | / |
Family ID | 51933949 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160139035 |
Kind Code |
A1 |
Florescu; Octavian ; et
al. |
May 19, 2016 |
DIGITAL CONTROL OF ON-CHIP MAGNETIC PARTICLE ASSAY
Abstract
An assay system and method for use in the field of chemical
testing is disclosed. The assay system can be used for filtering
whole blood for testing on an integrated circuit containing digital
control functionality.
Inventors: |
Florescu; Octavian;
(Berkeley, CA) ; Wong; Daniel; (Sunnyvale, CA)
; Martin; Tracie; (Oakland, CA) ; Yamasaki;
Duane; (El Cerrito, CA) ; Cromer; Remy;
(Saratoga, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Silicon BioDevices, Inc. |
Palo Alto |
CA |
US |
|
|
Assignee: |
SILICON BIODEVICES, INC.
Palo Alto
CA
|
Family ID: |
51933949 |
Appl. No.: |
14/942903 |
Filed: |
November 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2014/033607 |
Apr 10, 2014 |
|
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14942903 |
|
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61825464 |
May 20, 2013 |
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61891319 |
Oct 15, 2013 |
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Current U.S.
Class: |
506/40 |
Current CPC
Class: |
B01L 2200/0668 20130101;
C12Q 1/686 20130101; G06F 21/602 20130101; B01L 2400/0406 20130101;
G01N 33/54326 20130101; B01L 3/561 20130101; B01L 2300/0636
20130101; B01L 2300/023 20130101; B01L 3/502761 20130101; B01L
2200/148 20130101; B01L 2300/12 20130101; G01N 21/82 20130101; B01L
2200/16 20130101; B01L 2300/0838 20130101; Y02A 90/10 20180101;
B01L 2200/0605 20130101; G06K 19/06028 20130101; G01N 2201/068
20130101; B01L 2300/027 20130101; G16H 50/20 20180101; G01N
2201/061 20130101; G01N 21/255 20130101; G06K 19/06037 20130101;
B01L 2300/0874 20130101; B01L 2400/043 20130101; G01N 27/745
20130101 |
International
Class: |
G01N 21/25 20060101
G01N021/25; B01L 3/00 20060101 B01L003/00; G01N 33/543 20060101
G01N033/543 |
Claims
1. An assay system (10) for generating presentable assay
information from an aqueous sample (5), comprising: a filter (6)
capable of trapping or blocking particulate matter in an aqueous
sample (5); a delivery capillary (14) that fluidically connects the
filter (6) to a surface capillary (15); a first sedimentation
capillary (13) placed vertically above an integrated circuit 12 a
surface capillary (15) that fluidically connects the delivery
capillary (14) to the sedimentation capillary (13); a dry sphere
(3) placed at the top of the first sedimentation capillary (13),
wherein the dry sphere contains magnetic particles; magnetic
particle sensors embedded in the integrated circuit (12) at the
bottom of the first sedimentation capillary (13), wherein the
magnetic particle sensors are capable of detecting magnetic
particles specifically bound to the surface of the integrated
circuit; and a display (1) for displaying presentable assay
information.
2. The system of claim 1, configured to allow a user to take a
photograph of the display (1) to retrieve the presentable assay
information.
3. The system of claim 2, wherein the presentable assay information
retrieved by taking the photograph of the display (1) binds to a
patient ID in a secure manner.
4. The system of claim 1, wherein a magnetic particle sensor
detects a single specific molecular interaction between the surface
(12) of an IC (7) and a magnetic particle.
5. The system of claim 4, wherein an array of the magnetic particle
sensors is configured to count the number of specific molecular
interactions in the sensing area on the surface (12) of the IC
(7).
6. The system of claim 1, wherein a dry calibrant located on the
surface (7) of the integrated circuit (12) such that the dry
calibrant will be rehydrated by the aqueous sample and flow into a
second sedimentation capillary (63) but not into the first
sedimentation capillary (13).
7. The system of claim 6, wherein the signals from the first
sedimentation capillary (13) and the second sedimentation capillary
(63) is used to calibrate the native target signal.
8. The system of claim 1, wherein the third sedimentation capillary
(64) has a different height than the first sedimentation capillary
(13).
9. The system of claim 8, wherein the signal from the first
sedimentation capillary (13) and the third sedimentation capillary
(64) is used to measure the background signal.
10. The system in claim 1, wherein a passive unidirectional valve
eliminates and/or reduces the suck-back flow resulting from aqueous
sample evaporation through the filter (6).
11. The system in claim 1, wherein the magnetic particle sensors
comprise optical sensors, and wherein multiple light sources
producing illuminations of different color are used to identify
magnetic particles of different color.
12. The system of claim 11, wherein magnetic particles of different
color are coated with reagents that react with different target in
a multiplexed format.
13. The system of claim 1, wherein a cuvette (30) is placed at the
top of sedimentation capillary (13) to keep the dry sphere (3)
motionless.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of PCT
International Application No. PCT/US2014/033607, filed Apr. 10,
2014, which claims priority to U.S. Provisional Application No.
61/825,464, filed May 20, 2013; and U.S. Provisional Application
No. 61/891,319, filed Oct. 15, 2013, each of which are incorporated
herein by reference in their entireties.
BACKGROUND
[0002] 1. Technical Field
[0003] An assay system and method for use in the field of chemical
testing is disclosed. More particularly, the assay system can be
used for filtering whole blood for testing on an integrated circuit
containing digital control functionality.
[0004] 2. Summary of the Related Art
[0005] Point-of-Care (POC) diagnostic medical devices facilitate
early stage detection of diseases, enable more individually
tailored therapies, and allow doctors to follow up with patients
more easily to see if prescribed treatments are working. To ensure
widespread adoption, these tools must be accurate, easy to use by
untrained individuals, and inexpensive to produce and distribute.
Immuno-Assay (IA) applications are particularly well-suited for the
POC since a wide range of conditions, from cardiovascular disease
to cancer to communicable infections, can be identified from
soluble protein bio-markers. The detection and quantitation of
these bio-markers from raw samples such as whole blood often
involves labeling the target protein using fluorescent or
phosphorescent molecules, enzymes, quantum dots, metal particles or
magnetic particles. For high sensitivity applications, the labels
specifically bound to the target analytes must be distinguished
from the unbound ones that contribute to background noise. By
combining both label separation and detection in a low cost, easy
to use format, the Immuno-Chromatographic Test (ICT) achieves
stand-alone operation, i.e. the ability to perform an assay without
necessitating an electronic reader or an external sample
preparation system. Stand-alone operation is an often overlooked
attribute, but one that is key to the popularity of ICTs, achieved
despite other drawbacks such as low biochemical sensitivity, user
interpretation, inaccurate quantitation, timing requirements, and
awkward multiplexing.
[0006] The use of magnetic particle labeling is ideal for POC
applications; magnetic particles can be individually detected, so
sub-pico molar sensitivities can be achieved without signal
amplification steps that can take up to an hour as in case of
enzymatic labeling. Also, by micro-arraying the sensing areasensing
areas onto which the particles bind, multiplexed operation can be
achieved at low cost. The use of magnetic particles can reduce
incubation times, since they can bind to the target analytes with
solution-phase kinetics due to their high surface area to volume
ratio. Furthermore, the ability to pull the magnetic particles out
of solution magnetically and gravitationally overcomes the slow
diffusion processes that plague most high sensitivity protocols.
The signals from magnetic particles can be stable over time,
insensitive to changes in temperature or chemistries and detected
in opaque or translucent solutions like whole blood or plasma. The
biological magnetic background signal can he low, so high assay
sensitivity can be achieved with minimal sample preparation. Most
importantly, the use of magnetic particles as assay labels can
permit stand-alone device operation, since these particles can be
both manipulated and detected electromagnetically.
[0007] "Magnetic particles" are nano-meter or mico-meter sized
particles that display magnetic, diamagnetic, ferromagnetic,
ferrimagnetic, paramagnetic, super-paramagnetic or
antiferromagnetic behavior. "Magnetic particles" can refer to
individual particles or larger aggregates of particles such as
magnetic beads.
[0008] Magnetic particle sensors are sensor embedded in an
integrated circuit that can detect magnetic particles. Examples
include optical sensors, magnetic sensors, capacitive sensors,
inductive sensors, pressure sensors.
[0009] ICTs in which magnetic particles are used as the assay
labels are an improvement to conventional ICTs since the detection
of the particles is not limited to the surface of the strip, but
can be performed throughout the volume of the strip, resulting in
higher sensitivities and improved quantitative accuracy. However,
volumetric detection of magnetic particles cannot be readily
integrated in a stand-alone device, so these implementations
require an external device to measure the volume magnetization in
the strip.
[0010] One alternative for integration into a stand-alone assay
system is to use magnetic particles that bind to the target
analytes in solution before sedimenting via gravity or magnetic
force to sensing areas where the specifically bound particles can
be detected. A bio-functionalized IC can be used to detect the
specifically bound particles. However, most IC-based immuno-assay
implementations reported to date cannot operate stand-alone since
they require either off-chip components for particle detection, or
micro-fluidic actuation for particle manipulation and sample
preparation. Other implementations simply cannot reach the cost
structures necessary to compete in the current marketplace.
[0011] For POC application, it is desirable that the sample
preparation be rapid since the assay is limited to 10-15 minutes.
In addition, to obviate the need for refrigeration equipment and to
facilitate storage and distribution, a dry sample preparation
system is desired. It is also desirable to have a sample
preparation system that receives small unprocessed samples from
patients. The average hanging drop of blood from a finger stick
yields approximately 15 .mu.l of fluid. For more fluid, a
complicated venu-puncture can be necessary. Moreover, the sample
preparation system must be low-cost since biological contamination
concerns dictate that all material in contact with biological
samples be discarded. It is also desirable that the sample
preparation system be amenable to multiplexed operation.
BRIEF SUMMARY OF THE INVENTION
[0012] A sample preparation system that can fulfill the
requirements for speed, cost, and performance described above is
disclosed.
[0013] A porous material like a membrane filter can obviate the
need for centrifugation or complicated micro-fluidic sample
preparation. Since the membrane filters are compact and
inexpensive, system cost is reduced, enabling stand-alone POC
operation. Furthermore, the membranes can separate the plasma from
the whole blood cells without additional support in under 30
seconds. Incubation of the filtrate with functionalized magnetic
particles can achieve solution phase kinetics for rapid operation
with sub pico-molar sensitivities. The use of an IC to perform the
detection of the magnetic particles enables low cost, stand-alone
operation. Therefore, the combination of a filter, capillary,
magnetic particles and an IC can result in a stand-alone, accurate,
multiplexed platform with the form factor of a thumb-drive. The
size of the entire system excluding a battery and display can be
reduced to under 1 cm.sup.3.
[0014] The assay system can be used for immuno-assays. The assay
system can be used for nucleic acid, small molecule and inorganic
molecule testing, or combinations thereof.
[0015] A sample preparation system comprising a membrane filter and
a capillary channel configured to deliver magnetic particles to the
exposed surface of an integrated circuit (IC) that manipulates and
detects the particles is disclosed. The large particulate matter in
the sample, such as whole blood cells, can be trapped on top or in
the membrane, while the aqueous sample containing, the target
analytes traverses the membrane into the inlet of the capillary,
where the magnetic particles can re-suspend and bind to the target
analytes in the filtrate. The filtrate with the re-suspended
magnetic particles can flow through the capillary and onto the
sensing areas on the surface of the IC as a result of capillary
action.
[0016] Magnetic particles bound to a target analyte can bind
strongly through specific chemical interactions to the
functionalized sensing areas on the surface of the IC. The number
of magnetic particles specifically bound to the surface of the IC
is representative of the concentration of the target analyte in the
biological sample presented.
[0017] The surface of the IC can contain one or more sensing areas.
The sensing areas correspond to the areas on the surface of the
chip in which particle sensors can detect specifically bound
particles. The particle sensors can be embedded in the IC. Particle
sensors can be placed outside of the sensing areas to detect the
non-specifically bound particles removed from the sensing areas for
an accurate count of the total number of magnetic particles.
[0018] The IC can contain one or more magnetic force generators to
manipulate the non-specifically bound magnetic particles on the
sensing areas. These magnetic forces can be used to attract the
magnetic beads to the sensing areas and to remove the
non-specifically bound magnetic particles from the sensing areas.
The system can have two or more capillaries, for example where the
inlet of a delivery capillary is placed directly below the filter
and delivers the filtrate into a sedimentation capillary which is
placed vertically directly above the sensing area. The dried
magnetic particles can be placed at the top of the sedimentation
capillary. From the top of the sedimentary capillary, the dried
magnetic particles can sediment to the sensing area once the
filtrate reaches them. The length of time of the assay can be
determined by the height of the sedimentation capillary.
[0019] The assay system may be configured to take whole or
previously filtered blood, urine, tear, sputum, fecal, oral, nasal
samples or other biological or non-biological aqueous samples.
[0020] Chemicals, such as, but not limited to: aptamers,
oligonucloetides, proteins, agents to prevent clotting, target
analytes for internal calibration curves, bindive catalytic agents,
magnetic particles, or combinations thereof may be dried in the
membrane filter assembly along the shaft of the capillary or on the
surface of the IC and can be re-solubilized by the blood plasma but
remain bound to the surface upon which they were dried.
[0021] The assay system can contain user interface controls to
simplify user. The fully dry assay system can calibrate background
signal and the native target signal. The assay system may
invalidate the results if certain use-case conditions are not
met.
[0022] The assay system may transmit the results to a secondary
mobile device for storage and analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a cross sectional side view of a variation of the
assay system 10 that includes a sample preparation and delivery
module (SPDM) 8, a light source 2, an integrated circuit 12 (IC), a
printed circuit board (PCB) 9, a display 1, and a casing 11.
[0024] FIG. 2 is a cross sectional side view of a variation of the
assay system 10 as the aqueous sample 5 is filtered and wicked via
capillary action through a delivery capillary 14 into the surface
capillary 15 and onto a surface 7 of the integrated circuit 12.
[0025] FIG. 3 is a cross sectional side view of a variation of the
assay system 10 showing the aqueous sample 5 in the process of
wicking up the sedimentation capillary 13 due to capillary action.
Once the aqueous sample 5 reaches the top of the sedimentation
capillary 13, the aqueous sample 5 can re-hydrate a reagent sphere
3, releasing the particles 4 to sediment onto the surface 7 of the
IC 12.
[0026] FIG. 4A and 4B are top and cross sectional side views,
respectively, of a variation of the IC 12 containing a magnetic
separation field generator implemented with separation
conductors.
[0027] FIG. 4C is a cross-sectional view that presents the scenario
from FIG. 4A and 4B after non-specifically bound magnetic particle
25 is attracted to a separation conductor 23.
[0028] FIG. 5A is a cross sectional side view of cuvette 30 storing
the dry sphere 3.
[0029] FIG. 5B is a cross sectional side view of cuvette 30 after
the aqueous sample 5 dissolved the dried sphere 3 and released the
particles 4.
[0030] FIG. 6 is a cross sectional side view of a cuvette with
tapered side walls 40.
[0031] FIG. 7 is a cross sectional side view of a cuvette 30 with a
cover 50 to contain the dried sphere 3 in the cuvette.
[0032] FIG. 8 is a cross sectional side view of the surface
capillary 15 constructed from double sided tape 60.
[0033] FIG. 9 is a top view of the surface of the integrated
circuit 12 with the double sided tape 60 mounted on it. The sensing
area 21 can be situated under the sedimentation capillary, while
the active area 71 can be situated along the length of the surface
capillary.
[0034] FIG. 10 shows a cross of the system with a delivery
capillary 14 leading to two surface capillaries 15 and 62, which
lead to two sedimentation capillaries 13 and 63, respectively, for
controls or multiplexed operation.
[0035] FIG. 11 shows a cross section of the integrated circuit 12
mounted onto the PCB 9 and electrically connected via a wirebond
81. The wirebond 81 can be hermetically sealed by encapsulant
80.
[0036] FIG. 12 shows a cross section of the integrated circuit 12
mounted onto the PCB 9 and electrically connected by way of one or
more through-silicon vias 82.
[0037] FIG. 13 shows the top view of the integrated circuit surface
7 with one digitally addressable separation conductor 90 per
sensor.
[0038] FIG. 14 shows the top view of the integrated circuit surface
7 with one digitally addressable separation conductor 90 and one
digitally addressable concentration conductor 92 per sensor
[0039] FIG. 15 shows the cross section of the sedimentation
capillary 13 with a notch 100 to retain the dried sphere 3 in the
sedimentation capillary 3.
[0040] FIG. 16 shows the cross sectional view of the delivery
capillary 14, the surface capillary 15 and the sedimentation
capillary 13 and passive unidirectional valve that prevent the
suck-back of aqueous sample from the sedimentation capillary to the
filter.
[0041] FIG. 17A is a cross-sectional side view of a flow stop 120
placed above the dry sphere 3, before the aqueous sample 5 has
dissolved the dry sphere 3.
[0042] FIG. 17B is a cross sectional side view of a flow stop 120
that has hermetically sealed the top of the sedimentation capillary
13 after the aqueous sample 5 has dissolved the dry sphere 3 and
released the particles 4.
DETAILED DESCRIPTION
[0043] Biosensors that use non-magnetic or magnetic particle
labeling to perform assays are disclosed. A particle can serve as
an aid, or label, in detecting the presence or absence of a target
analyte if the particle is attached to a chemical entity that
reacts with the analyte, or analyte analogue, or analyte
by-product. The reaction can be immunological, nucleic acid based,
covalent, ionic, hydrogen bonding, van der Waals and other chemical
reaction phenomena capable of promoting or inhibiting the labeled
particle from binding to a surface.
[0044] Particles may be any spherical or arbitrarily shaped
localized objects, from several nanometers to tens of microns in
diameter, that modulate incoming light (e.g., reflect the light,
refract the light, block or absorb the light, increase or decrease
the intensity of the light, change the wavelength or spectral
composition of the light). Particles may also be magnetic. Magnetic
particles display diamagnetic, ferromagnetic, ferrimagnetic,
paramagnetic, superparamagnetic, or antiferromagnetic behavior.
Magnetic particles may include individual nanometer-sized particles
of magnetic material (often referred to as magnetic nanoparticles
or magnetizable nanoparticles) or larger aggregates of such
magnetic nanoparticles to form an essentially spherical bead (often
referred to as magnetic beads, magnetizable beads). Magnetic
particles may be covered with or encapsulated by a non-magnetic
material, such as a polymer, glass, ceramic, or any other
non-magnetic material, that may be coated with biological or
chemical molecules that react specifically to a target analyte. A
non-magnetic material refers to any material that displays no
magnetic properties or displays magnetic properties that are much
smaller in magnitude (e.g., less than 0.001%) than the
corresponding properties of the magnetic material in magnetic
particles. Magnetic particles may be from several nanometers to
tens of microns in diameter.
[0045] FIG. 1 shows an assay system 10 that includes a sample
preparation and delivery module (SPDM) 8, a light source 2, an
integrated circuit (IC) 12, a printed circuit board (PCB) 9, a
display 1, and a casing 11. The assay system 10 may be configured
to perform a biological and/or chemical assay on an aqueous sample
5 by introducing, detecting, and/or quantifying particles 4
specifically binding on the surface 7 of the IC 12. An assay may be
any procedure used to detect the presence of a target analyte or to
quantify the concentration or amount of the target analyte in the
aqueous sample 5. Target analytes may be enzymes, proteins, small
molecules, nucleic acids, and other biological, chemical, and
inorganic entities, or combinations thereof. The aqueous sample 5
may be whole blood, plasma, serum, diluted blood derivatives,
spinal fluid, sputum, pulmonary lavage, fecal samples, oral
samples, nasal samples, lachrymal fluid, other bodily fluids,
laboratory samples, environmental samples, any other fluids
potentially containing one or more target analytes, or combinations
thereof
[0046] Further, FIG. 1 shows a filter 6 that can be placed at the
top of the SPDM 8. The filter 6 may be any type of filter (e.g.,
membrane filter, microfilter, syringe filter) capable of blocking
or trapping particulate matter (e.g., red blood cells, white blood
cells, other cells and micron to millimeter size particulates) and
thus removing the particulate matter from the aqueous sample 5. The
filter 6 may also be adapted to remove certain biological or
chemical molecules from the aqueous sample 5 (e.g., a chemical
coating on the filter 6 may remove molecules that compete with the
target analyte or interfere, in any way, with the assay). Further,
the filter 6 may include chemicals, molecules, and other
dissolvable matter than may aid the assay protocol. For example,
the filter 6 may contain dry anticoagulation factors that prevent
blood samples from coagulating or dry assay additives to mitigate
the effects of interferers in the sample. Further still, the filter
6 may be coated with hydrophilic material to aid in aqueous sample
5 absorption. The filter can be attached onto the top surface of
the SPDM using double sided adhesive tape, transfer adhesive, hot
melt adhesive, an epoxy seal along the edge, or by heat sealing or
a similar bonding process. To minimize dead volume under the
filter, the height of the double sided tape, transfer adhesive or
epoxy seal can be less than 1 um, or less than 5 .mu.m, or less
than 10 .mu.m, or less than 20 .mu.m, or less than 50 .mu.m or less
than 100 .mu.m, or less than 250 um.
[0047] Further, FIG. 1 shows a surface capillary 15, a delivery
capillary 14 and a sedimentation capillary 13. The delivery
capillary 14 can fluidically connect the membrane filter 6 to a
surface capillary 15 allowing the aqueous sample 5 to flow from the
filter to the surface 7 of the IC 12. The surface capillary 15 can
fluidically connect the delivery capillary 14 to the sedimentation
capillary 13 thus allowing, the aqueous sample 5 to flow from the
delivery capillary 14 into the sedimentation capillary 13 and up
sedimentation capillary 13 to dry sphere 3. In one variation of the
assay system 10, the filter 6 may be placed inside the delivery
capillary 14 or surface capillary 15. The sedimentation capillary
13 may be placed vertically over the IC 12 and in contact with
reagents containing particles 4. The reagents may be configured in
a sphere (i.e., a reagent sphere 3) or any other shape. The reagent
sphere 3 or other shape may rest on top of the sedimentation
capillary 13 and may preferably be dry or lyophilized. The IC 12
may be mounted by any known method (e.g., wire-bonding, flip-chip
assembly, conductive epoxy, and combination thereof) to a PCB 9.
The assay system 10 can be encapsulated by a casing 11 with an
opening for a digital display 1 and an opening for the filter 6.
The display 1 may be driven by circuitry integrated on IC 12.
[0048] The SPDM 8 can be configured to accept an aqueous sample 5
from a sample source (e.g., a finger stick, a pipette, a syringe, a
capillary tube, or combinations thereof), filter the aqueous sample
5 using the filter 6, deliver the filtered aqueous sample 5 first
to the surface 7 of the IC 12 and subsequently to the reagent
sphere 3, re-hydrate dried particles 4 within the SPDM 8, mix and
incubate the particles 4 with the aqueous sample 5 and introduce
the particles 4 onto the surface 7 of the IC. The systems and
methods of use described herein can be applied to known SPDMs such
as those described in PCT Application No. WO 2011/059512, filed 16
Nov. 2010 (titled: FILTRATION DEVICE FOR ASSAYS) and in PCT
Application No. WO/2012/048288--MAGNETIC PARTICLE BASED BIOSENSOR,
which are incorporated by reference herein in their entirety. Other
variations, components, and functions of the SPDM 8 are further
described below.
[0049] FIG. 2 shows the aqueous sample 5 being wicked into the
filter 6 where particulate matter such as whole blood cells can be
blocked or discriminated by size. The aqueous sample 5 can then be
wicked from the outlet side of the filter 6 into the delivery
capillary 14 and delivered into the surface capillary 15 and onto
the surface 7 of the integrated circuit 12, as shown by an arrow.
The flow of the aqueous sample 5 can continue from the surface 7 of
the integrated circuit 12 up the sedimentation capillary 13 to the
dry sphere 3. The flow in the delivery capillary 14, surface
capillary 15 and the sedimentation capillary 13 can be maintained
by capillary action. Once the capillaries are filled and the dry
sphere 3 fully dissolved, the flow can cease. The amount of aqueous
sample 5 in the SPDM can be precisely controlled by the inner
volume of the capillaries to less than 0.5% variability, or to less
than 1% variability or to less than 2% variability or to less than
5% variability. The inner volume of the capillaries can be used to
precisely meter the amount of aqueous sample assayed. In cases
where the assay system 10 is placed vertically as shown in FIG. 2
and the delivery capillary 14 and surface capillary 15 are below
the filter 6, gravity can also assist the flow of the aqueous
sample 5. Pressure from vacuum or pumping can also be used to
facilitate the flow of the aqueous sample 5 through the delivery
capillary 14 and surface capillary 15. As discussed above, the
filter 6 may be a membrane filter and may have a surface area
between 0.1 mm.sup.2 and 100 cm.sup.2 and a thickness between 1
.mu.M and 10 mm. The membrane filter can be composed of
polyvinylpyrrolidone/polyethersulfone (PVP/PES). The membrane
filter can have a porosity gradient to effectively trap cells in
whole blood while allowing blood plasma and the analytes therein to
pass through the membrane. A preferable filter is a 0.26 mm thick
PVP/PES filter with a 35 .mu.m pore size on the top and a 2.5 .mu.m
pore size on the bottom. The membrane filter can be oriented in a
horizontal plane. The membrane filter can be oriented in a plane
parallel to the surface 7 of the IC 12. The delivery capillary 14
can be between 0.1 mm and 10 cm in length and between 10 .mu.m and
5 mm wide. A preferable delivery capillary 14 is 2 mm long, and
0.25 mm wide. The surface capillary 15 can be between 0.1 mm and 10
cm in length and 10 .mu.m and 5 mm wide. A preferable surface
capillary 15 is 5 mm long and 0.5 mm wide. The magnetic particles
may be dried on the bottom surface of the filter or inside the
filter.
[0050] FIG. 3 shows the aqueous sample 5 in the process of wicking
upwards inside the sedimentation capillary 13 due to capillary
forces. Gravity can also assist the flow of the aqueous sample 5 up
the sedimentation capillary 13 provided the sedimentation capillary
13 is placed below the bottom plane of the filter 6. Pressure from
vacuum or pumping can also be used to facilitate the flow. Once the
aqueous sample 5 reaches the top of the sedimentation capillary 13,
the aqueous sample 5 can dissolve the dry reagent sphere 3 placed
at the top of the sedimentation capillary 13. The particles 4 can
be released and sediment through the aqueous sample 5 to the
surface 7 of the integrated circuit 12, as shown by arrows. As the
particles 4 sediment, the particles 4 can react with the target
analytes in the aqueous sample 5 and bind specifically to the
surface 7 of the IC 12. The sedimentation capillary 13 can be
between 0.1 mm and 10 cm in length and 1 .mu.m and 5 mm wide. A
preferable sedimentation capillary 13 is 3 mm long and 1 mm
diameter. The dry reagent sphere 3 can be manufactured by
lyophilization and placed on the top of the sedimentation capillary
13 using an automated pick and place tool. Alternatively, the
magnetic particles can be placed in a cuvette 30 by air flowing
down the sedimentation capillary.
[0051] The surface 7 of the IC 12 can be illuminated by a light
source 2. The light source 2 can generate and/or direct light to
illuminate the surface 7 of the IC 12. The light source 2 may be or
include a luminescent light source such as a light emitting diode
(LED), laser emitting diode, incandescent light source such as a
light bulb, any other source of light internal or external to the
assay system 10, or combinations thereof. The light source 2 may be
any external light source (e.g., the sun, an external lamp, ambient
light in a room, and any other external light source that may be
used instead of or in combination with an internal light source to
illuminate the surface 7 of the IC). The light source 2 may be
positioned anywhere in the assay system 10 or external light may be
inputted anywhere into the assay system 10 and an optical module
may direct the light onto the surface 7 of the IC 12. The light
source 2 may be integrated on the IC 12 itself. For example, a
direct semiconductor may be used to fabricate light 2 in the IC 12
or a portion of a direct semiconductor may be added to the IC 12
(e.g., via wafer bonding, molecular beam epitaxy, and other
suitable fabrication processes). The light source 2 may be
configured to produce a light intensity anywhere from 1 mW/m.sup.2
to 10 kW/m.sup.2. The light source can be powered by a battery to
eliminate the AC tones prevalent with distributed power sources.
The light source can illuminate more than one sensing areas on one
IC or more than one sensing area on more than one ICs. The
activation of the light source and control of its intensity can be
done by circuitry embedded in the IC 12.
[0052] A shadow may refer to any type of light modulation caused by
a particle 4 in the direction of propagation of light that
increases or decreases intensity, changes the spectral composition,
blocks, changes the polarization, or otherwise modifies the
properties of said light. One or more light sources can be situated
or positioned directly above the integrated circuit 12 such that
particles 4 situated above the surface 7 of the IC 12 cast a shadow
that is projected downward onto the surface 7 of the IC. The shadow
then may be detected by one or more optical sensors 40 situated
below the surface 7 of the IC 12. However, the light source(s) may
be positioned and directed at any angle relative to the surface 7
of the integrated circuit 12 such that the light shines on least a
portion of IC 12 surface 7. Light at or near the red spectrum
(550-750 nm) may offer superior SNR since human whole blood and
plasma samples have an absorption minimum at that frequency.
[0053] In a variation of the assay system 10, multiple sources of
light may illuminate the surface 7 of the IC 12 indirectly and/or
at oblique angles. Multiple ICs can be illuminated by one source of
light or multiple sensing areas on the same IC can be illuminated
by one light source. Alternatively, one or more sensor regions on
one or more ICs can be illuminated by more than one light source.
The shadows or otherwise the modulated light due to the particles 4
can be projected at oblique angles (i.e., not straight
downward).
[0054] One or more reflectors, one or more lenses, one or more
optical fibers, one or more light pipes, and any other component or
combination of components may be used to direct light onto the
surface 7 of the IC 12. The light source 2 may be positioned on or
integrated into the PCB 9 and a reflector placed on the ceiling of
the casing 11 above the IC 12, or an optical fiber or light pipe
may direct the light originating from the light source 2 onto the
surface 7 of the IC 12. The light source 2 may be modulated (e.g.
turned on and turned off repeatedly) at a certain frequency, at
multiple frequencies, following a certain predetermined or random
sequence in time, or combinations thereof. For example, the light
source 2 may be turned on for a predetermined amount of time prior
to introducing the particles 4 in order to calibrate the optical
sensors 20 on the IC 12 (e.g., by measuring the sensitivity,
sensitivity distribution, saturation level, and other relevant
parameters of the optical sensors 20 and underlying electronic
circuits) and to calibrate the light source 2 (e.g., measure and
adjust the light intensity, light uniformity, and other relevant
parameters of the light source 2). Subsequently, the light source 2
may be turned on for a predetermined amount of time to allow for a
shadow or any other form of light modulation to be created by the
particles 4 and detected by the optical sensors 20.
[0055] The SPDM 8 can be opaque such that light from the light
source 2 can only be transmitted through the sedimentation
capillary 13. Multiple sedimentation capillaries above one or more
ICs can allow the propagation of light from one or more light
sources to one or more sensor regions. The SPDM can be partially
opaque or portions of the SPDM can be opaque to optimize the
quality of the light transmitted onto the surface of the chip. The
SPDM can be composed of two discrete portions, one opaque and one
translucent or transparent. The transparent region of the SPDM 8
useful when there is opaque material in the sedimentation capillary
13, like the dry sphere or lysed aqueous sample, that need to be
circumvented by the light in order to illuminate the particles 4 on
the surface 7 of the IC 12. For example, in the SPDM a thickness
along all or part of the length of the sedimentation capillary 13
can be transparent to allow the light to reach the surface 7 of the
IC 12, while the rest of the SPDM 8 can be opaque. For example, the
top portion of the SPDM 8 containing the cuvette 30 and to top of
the sedimentation capillary 13 can be transparent, while the bottom
portion of the SPDM 8 containing the rest of the sedimentation
capillary can be opaque. The SPDM can be created by laminating,
gluing, or otherwise binding two layers of material, one opaque and
one transparent or translucent. The transmission of the light onto
the surface of the chip can also be used to determine if aqueous
sample 5 has reached the top of the sedimentation capillary in the
SPDM and whether the dry sphere 3 has dissolved. Light pipes or
optical fibers and capillaries 13, 14 and 15 can be combined in a
single unit made of plastic (PMMA, PDMS or other silicon
derivatives, polycarbonate, polyacetate, polyurethanes,
polyvinylchloride, or other synthetic polymers).
[0056] Image processing filters can be used to eliminate
illumination artefacts for superior particle detection signal to
noise ratio. The image processing filter algorithm can be hardcoded
onto the IC 12, embedded in the memory on the IC 12 or described in
software stored on the IC 12 or on an external IC. Examples of the
image processing filters include spatial low pass filters, un-sharp
masks, convolution matrices and other algorithms that combine the
raw optical signals from multiple sensors in a logical algorithm to
reduce or eliminate the components of the raw optical signals that
are detrimental to particle shadow detection signal to noise ratio.
In this way, the image processing filter can be used for example to
estimate the background illumination, cross-talk between adjacent
or nearby optical sensors, global shadows, bead aggregates and
reflections on the surface 7 of the IC 12 due to constructive and
destructive optical interferences. The image processing filter can
eliminate or reduce the shadows resulting from debris or other
blemishes on the surface 7 of the IC 12 that does not correspond to
a magnetic particle.
[0057] To minimize stray light from the environment or external
light sources, the sample port where the sample is applied on the
device, for example the filter opening, can have an opaque lid that
closes after application of the sample. The opaque lid can be
limited to covering only the sample port or can large enough to
cover the entire device. The opaque lid can be hinged, screwed,
clipped or fastened on.
[0058] Illumination information from the optical sensors can be
interpreted by the IC 12, which can generate the commands to direct
the one or more light sources to alter the illumination
characteristics. The intensity, the color, the incident angle, the
position, the coherence and the number of light sources and their
constellation can all be directed according to the illumination
information from the optical sensors in order to improve detection
signal to noise ratio.
[0059] Multiple light sources producing illuminations of different
color can be used to identify magnetic particles of different
color. A first light source 200 can produce an illumination of a
first color 201 and a second light source 210 can produce an
illumination of a second color 211 onto the surface 7 of the IC 12.
A first particle 202 can be dyed or colored with color 201 and a
second particle 212 can be dyed or colored with color 211.
Particles 202 and 212 can bind specifically atop optical sensors
embedded in the IC 12. For equivalent illumination intensities from
light source 200 and light source 210, the intensity of the shadow
cast by particle 202 on the optical sensor resulting from the
illumination of light source 200 will be different from the
intensity of the shadow cast by particle 202 on the optical sensor
resulting from the illumination of light source 210. Similarly, for
equivalent illumination intensities from light source 200 and light
source 210, the intensity of the shadow cast by particle 212 on the
optical sensor resulting from the illumination of light source 200
will be different from the intensity of the shadow cast by particle
212 on the optical sensor resulting from the illumination of light
source 210. The color of a particle can be determined by measuring
the relative intensities of the shadows resulting from light
sources of different color. The lights of different color can be
turned on sequentially or at the same time. The optical sensors can
identify the color of a colored particle by measuring one or more
frequencies of the absorbance, reflectance, transmittance
phosphorescence, or fluorescence spectrum of the shadow cast by a
colored particle. The color of the shadow is the complementary of
the particle. More than one light source of more than one color can
be used to illuminate the colored particle with more than one
wavelength of light, thereby providing a spectral signature of the
colored particle. A broad spectrum light can be used for
calibration. Various intensities can be used for various
illumination colors and the relative intensities of the shadow, or
absorbance, reflectance, luminescence, fluorescence,
phosphorescence or transmittance of the colored beads can be used
to identify the color of the bead. Mono-chromatic, or
multi-chromatic lights can be used. Coherent, collimated or diffuse
lights can be used. Optical sensors sensitive to different
detection spectrums can detect the shadows from colored particles
and thereby can identify the color of the particle. A single
optical sensor that can measure illumination intensities at
different optical frequencies can be used to determine the color of
a particle casting a shadow on it.
[0060] Multiple optical sensors sensitive to different optical
spectra can be used to determine the color of a particle. The cross
sectional area of the optical sensors can be smaller than the cross
sectional area of the shadow of the colored particle so that they
can be laid out to measure the intensity of the shadow from the
same particle.
[0061] Optical sensors that are sensitive to different optical
spectra can be activated to detect the same shadow at the same
time, or they can be activated to detect the same shadow in an
alternating sequence.
[0062] Particles of the same color can be coated with one or more
reagents that react specifically to one or more targets such as
epitopes or one or more chemical groups or moieties on the same
target. Particles of different color can be coated with different
reagents that react specifically to different targets. Particles
202 can be coated with a first antibody 222 and particles 212 can
be coated with a second antibody 232. Particles 202 and 212 can be
dried in the same dry sphere 3 or can be dried in separate dried
spheres. Particle 202 and 212 can sediment in the same
sedimentation capillary 13 or sediment in separate sedimentation
capillaries. The surface of the chip can be coated with chemical
reagents that bind specifically in a capture format to particle 202
in the presence of a first specific target 242. The surface of the
chip can be coated with chemical reagents that bind specifically in
a capture format to particle 212 in the presence of a second
specific target 243. Particles of different colors can be
selectively identified and the concentration of their targets
measured at the same time or in sequence. Multiplexed assays can be
performed simultaneously on the same chip. The number of particles
of the same color can range from 1 to 100,000 to 1 trillion. The
number of different colors can range from 1 to 1000. Each target
can have a unique color, or multiple targets can share one color or
one color can identify multiple targets.
[0063] Examples of multiplexed embodiments:
1--entire chip is coated with a mixture of antibodies and all
particles of the same color are derivatized with a specific
antibody; 2--specific sites of the chip are derivatized with 1
specific antibody. Using specific colored particles (such as beads)
per assay allows determination of cross-reactive binding when wrong
color beads is at the wrong location; 3--when a certain bead is
derivatized with multiple antibodies, multiplexing is preferably
achieved by derivatizing specific locations of the chip with
specific antibodies.
[0064] The IC 12 can be a substrate that can incorporate one or
more optical sensors 20 and associated electronic circuits. At
least a portion of the surface 7 of the IC 12 is coated with
reactive molecules and the IC 12 is configured to accept particles
4 that may bind specifically (i.e., via the reactive molecules) or
non-specifically to the surface 7 of the IC 12, depending on the
concentration of the target analyte. The IC 12 may be used to
remove from atop the sensors any non-specifically bound particles
and quantify the number or concentration of remaining specifically
bound particles. The number of specifically bound particles may be
proportional to the concentration of the target analyte in the
sample. Generally speaking, specifically bound particles are
particles that are bound to a surface 7 via at least one specific
binding interaction (i.e., antibody-antigen binding). Generally
speaking, non-specifically bound particles are particles that are
bound to the surface 7 with weaker binding forces (e.g., van der
Waals forces). Specifically bound particles refer to particles that
are bound with one or more specific biochemical interactions such
as one or more antigen-antibody binding interactions and other
interactions discussed above and are not removed from the surface 7
by on-chip generated magnetic separation forces. Non-specifically
bound particles may be particles that are removed from the sensing
area 21 by on-chip generated magnetic separation forces.
Non-specifically bound particles may still contain one or more
specific binding, interactions but generally contain fewer specific
binding interactions than specifically bound magnetic particles.
For example, for large particles (e.g., those greater than 100
nanometers in diameter), multiple specific binding interactions may
be required for the particles to remain stationary in the presence
of separation forces (i.e., to be considered specifically-bound).
For example, particles with 2 or more antigen-antibody binding
interactions may be never removed with separation forces and thus
are always considered specifically bound, particles with fewer than
one antigen-antibody binding interactions may be always removed
with separation forces and thus are considered non-specifically
bound, whereas particles in between may be either
specifically-bound or non-specifically bound. The magnetic
separation forces can be tailored to select the desired number of
antigen-antibody interactions necessary to keep a magnetic particle
specifically bound to the surface. In so doing, the number of
magnetic particles remaining specifically bound to the surface can
also give an indication of the total number of antigen-antibody
interactions attaching the particles to the surface 7 of the IC
9.
[0065] The bio-chemical functionalization and the magnetic forces
can be tailored to ensure that only 1 specific molecular
interaction (such as one antigen-antibody interaction, one strand
of DNA, a complementary strand of DNA, a covalent bond, a hydrogen
bond, is sufficient to specifically bind a magnetic particle to the
surface 7 of the IC 12. Magnetic particles larger than 100 nm, such
as between 100 nm and 1 um, or between 1 um and 10 um, can also be
configured in the system to bind specifically to the surface 7 of
the IC 12 through one single specific molecular interaction. The
on-chip generated magnetic separation forces can be tailored to
pull away from the sensors the magnetic particles that have no
specific molecular interactions to the surface. The on-chip
generated magnetic separation forces can be tailored to leave
immobilized the magnetic particles that have exactly one specific
molecular interaction to the surface. A magnetic particle sensor
can detect a single magnetic particle and by extension, a magnetic
particle sensor can be used to detect a single specific molecular
interaction between the surface 7 of the IC 12 and a magnetic
particle. An array of individually addressable magnetic particle
sensors can be used to detect multiple magnetic particles
specifically bound to the surface 7 of the IC 12 through single
specific molecular interaction. The array of magnetic particle
sensors can be used to count the number of specific molecular
interactions in the sensing area on the surface 7 of the IC 12.
[0066] The assay system 10 can be handheld and portable. It can be
less than 1 L, 0.1 L, 0.01 L, or 1 mL in volume and weigh less than
1 kg, 100 g, 10 g or 1 g.
[0067] Particles may serve as light concentrators through internal
or external reflections. For example, the amount of light incident
on optical sensors 20 may be increased by over 1% and optical
sensors on the IC can be configured to detect this light intensity
increase. The particles may modulate the light (e.g., filter the
frequency spectrum of the light, luminesce with another frequency
of light, change the color or polarization of the light, fluoresce,
or phosphoresce). Likewise, the optical sensors 20 may be
configured to detect any of these color or polarization changes,
for example by using color or polarization filter arrays placed
over the optical sensors 20 or by using different optical sensor 20
types such as N-well diodes, N+ diodes, poly gate diodes, and P+
diodes, which are sensitive to different optical spectra or
frequencies. The electronic circuits may be any combination of
metal or semiconductor connections, resistors, capacitors,
inductors, transistors, diodes, amplifiers, digitizers, digital
logic, and other integrated electronic circuits used to obtain,
forward, process, and output a signal from the optical sensors 20.
The circuitry may be used to individually address any of the
optical sensors 20 in an array, either sequentially or in parallel.
The IC may be fabricated in any commercial integrated circuit
process (e.g., CMOS, CCD, BJT) or may be made in a custom
fabrication process. Other variations, components, and functions of
the IC 12 are further described below. Optical sensors 20 can
distinguish between different color beads by the relative amount of
light they transmit from the different color light sources. Optical
sensors 20 can distinguish different wavelengths of light that are
emitted by different particles such as beads.
[0068] The PCB 9 can be any rigid or flexible substrate that stores
the IC 12 and electrically and/or mechanically connects the IC 12
to any other components. The PCB 9 can contain one or more
batteries, one or more control modules, one or more voltage
regulators, one or more sensors, one or more actuators, one or more
displays, and combinations thereof As discussed above, the PCB 9
may also include the light source 2 that can provide light into the
IC 12 via an optical module. The PCB 9 may be placed on the bottom
of the housing or in any other position in the housing and may
contain connectors and daughter-boards or any other extensions that
may contain any of the components described above or described
below in any position and orientation inside or outside the housing
containing the SPDM 8. The PCB 9 components internal to the assay
system 10 and the circuitry and sensors of the IC 12 may be
controlled by a control module integrated on the IC 12 (e.g., a
control module core, a discrete control module mounted on the PCB
9, a central processing unit (CPU), a digital signal processing
(DSP) unit, a field-programmable gate array (FPGA), or any other
control module or combination of control modules. The terms control
module core and control module may be used interchangeably in this
specification and may be located on the PCB 9, in the IC, or in any
other part of the assay system 10. The control module may store
assay calibration parameters and assay protocol algorithms. Assay
calibration parameters may include a standard curve that relates
the number or concentration of particles 4 detected to the
concentration and/or amount of the target analyte in the aqueous
sample 5. Assay calibration parameters may also include an assay
time which may include any time intervals between different steps
in an assay (e.g., time from aqueous sample 5 detection to optical
sensor array readout, readout duration, magnetic separation force
duration, magnetic separation intervals and any other time
interval). Assay calibration parameters may also include magnetic
separation force and magnetic concentration force strength,
duration, frequency, pattern. Assay calibration parameters may
include any other parameters that may affect assay results. The
assay calibration parameters are adjusted in response to
measurements made by any sensors and components of the assay system
10. The battery or the battery unit can be removed in an easy
pop-out system or otherwise separated prior to discarding the
system. Portions of the circuitry or the entire electronic module
including display can be popped out or separated from the rest of
the device prior to discarding the system.
[0069] The assay system 10 can contain one or more inertial
sensors. The inertial sensors may include accelerometers,
gyroscopes, tilt sensors, and any other sensors capable of
detecting and quantifying position, velocity, acceleration,
orientation, and combinations thereof. The inertial sensors are
configured to sense the physical parameters discussed above and
output them to the control module. The control module may be
configured to read the output from the inertial sensors and
determine if any of the physical parameters are unusual and/or out
of the acceptable range. For example, the inertial sensors may send
an output to the control module indicating that the orientation of
the assay system 10 is incorrect (e.g., the IC 12 is at on a tilt
for a prolonged period of time) or the acceleration of the assay
system 10 is too high (e.g., a user is moving the assay system 10
beyond the recommended limits while the magnetic separation is
being performed). As a result, the control module may send a signal
to the user via the display 1 that an incorrect action took place
and that the results of the assay are invalid. The control module
may send a signal to the user via the display 1 that an incorrect
action took place and that the position of the device must be
adjusted in order for the assay to proceed normally. Alternatively,
the control module may attempt to compensate for any effects
resulting from incorrect orientation and/or applied acceleration.
The control module can modify and/or selects the assay calibration
parameters based on the measured values of relevant physical
parameters. The control module can perform more detailed
compensation on the sensor level, for example, by applying
different weights to signals from different optical sensors 20
positioned in different locations on the IC, or completely ignoring
the reading from certain optical sensors 20 altogether. The control
module may also modify the assay time based on the reading obtained
from the inertial sensors (e.g., the optical detection may be
turned on sooner/later, allowing particles 4 less/more time to
incubate with the target analyte, respectively). The inertial
sensors may be mounted in any component of the assay system 10
(e.g., mounted as a chip on the PCB 9, integrated into the IC,
mounted on any wall of the casing 11, or combinations thereof).
[0070] The optical sensors and other sensors can be used to
validate the manufacturing of the assay system 10. The assay can be
invalidated if too many or too few magnetic particles are detected
on the surface 7 of the IC 12. Too few magnetic particles can be an
indication of the assay system 10 being tilted during use, while
too many particles may be an indication of a manufacturing process
problem. The assay can also be invalidate the assay if the surface
density of the magnetic particles detected is not approximately
uniform. This can also be an indication that the assay system 10
was tilted during use. The assay system 10 can also detect aqueous
leaks in the double-sided tape by monitoring whether the magnetic
particles move across the surface 7 of the IC 12 when no strong
magnetic forces are applied.
[0071] The PCB 9 and/or the IC 12 may contain a read-only memory
module (ROM), a random access memory module (e.g., SRAM, DRAM), or
other module capable of storing data (PROM, EPROM, EEPROM, Flash,
and any other storage medium). The data module may be part of the
control module and may be used to store calibration data from the
various sensors, actuators, and modules in the assay system 10 that
is derived just prior, during, or after performing, an assay. The
data module may store calibration data generated during the design
process or after the IC 12 is manufactured and/or the assay system
10 is assembled. For example, the calibration data may compensate
for variations in manufacturing (e.g., ILD thickness, optical
sensor 40 sensitivity, and other parameters that may vary during
manufacturing). In another example, the calibration data may
compensate for variations in surface coating (e.g., surface
chemistry, reactive molecule density, reactive molecule type, and
other parameters that can vary during surface 7 coating). The
calibration data can include assay calibration parameters that are
derived from one or more chips in a particular batch (e.g., from
the same wafer, same surface coating batch, same assembly
batch).
[0072] The assay system 10 can include one or more temperature
sensors. The temperature sensors may include a thermistor, a
semiconductor sensor, a thermocouple, a temperature-dependent
resistor, or combinations thereof, and may be configured to measure
the temperature of the surroundings (e.g., the air temperature
outside the assay system 10, the temperature of the SPDM 8, and/or
the temperature in the vicinity of the IC 12) or the temperature of
the aqueous sample 5 directly (e.g., the SPDM 8 may be configured
to place the temperature sensor in contact with the aqueous sample
5, or the sensor may be located at or near the surface 7 of the IC
12). An assay may have distinct assay calibration parameters (e.g.,
standard curve, assay time) at each temperature level and as the
assay kinetics may be sped up or slowed down depending on the
temperature level. Accordingly, the temperature reading may be sent
to the control module and may be used to adjust the assay
calibration parameters to compensate for the changes in
temperature. Aside from being integrated or attached to the PCB 9,
the temperature sensor may be mounted in any other component of the
assay system 10 (e.g., integrated into the IC 12, mounted on any
wall of the casing 11, or combinations thereof). The IC 12 or SPDM
8 may contain one or more heating elements (e.g., resistors, coils,
wires) that can be used to keep the temperature of the surface 7 of
the IC 12, the aqueous sample 5, and/or the entire assay system 10
at a nearly constant, predetermined value. The information from the
temperature sensors can be read and the control module can control
the heating elements in order to keep the temperature constant in
the range of 20.degree. C. to 40.degree. C.
[0073] The assay system 10 can include one or more moisture
sensors. The moisture sensor(s) may be placed in contact with the
aqueous sample 5 and may be used to detect the presence of the
aqueous sample 5 (e.g., using electrodes to detect a change in
resistance or a change in capacitance between the electrodes as a
result of the presence of the aqueous sample 5). The moisture
sensor(s) may send a signal to the control module, either
continuously or upon detection of the aqueous sample 5, indicating
the moisture level reading, and the control module may enable other
components of the assay system 10 upon receiving a signal
indicating the presence of the aqueous sample 5. Aside from being
integrated or attached to the PCB 9, the moisture sensor may be
mounted in any other component of the assay system 10 (e.g.,
integrated into the IC 12, mounted on any wall of the casing 11, or
combinations thereof).
[0074] The PCB 9 can include one or more viscosity sensors. A
viscosity sensor can be placed in contact with the aqueous sample
5. The viscosity of blood plasma can vary. Higher fluid viscosities
may lead to longer assay kinetics and longer particle sedimentation
times. Accordingly, the viscosity sensor(s) may send a measurement
of the viscosity to the control module which in turn may modify the
assay time and other assay calibration parameters. Aside from being
integrated or attached to the PCB 9, the viscosity sensor(s) may be
mounted in any other component of the assay system 10 (e.g.,
integrated into the IC 12, mounted on any wall of the casing 11, or
combinations thereof). By including temperature, viscosity,
orientation, acceleration, and any other environmental factors into
account when performing an assay, the results of the assay may be
adjusted appropriately, via the assay calibration parameters, to
effectively cancel out these environmental effects, leading to
increased robustness, accuracy, and consistency of results in
diverse environments and settings. The moisture sensors placed at
different points along the path of the aqueous sample 5 can be used
to measure the viscosity of the fluid. Alternatively or in
combination, the optical sensors 20 can be used to measure the time
from the reagent sphere 3 dissolution to the time the particles 4
sediment onto the surface 7 of the IC 12. This time can also be
used to determine the viscosity and incubation time
information.
[0075] The assay system 10 can include a vibrator module. The
vibrator module may include an electric or piezoelectric motor with
an unbalanced mass, a piezoelectric or electromagnetic acoustic or
ultrasonic transducer, or any other module and method for
generating vibrations. The vibrator module may be turned on during
the sample delivery steps (i.e., between the time when the aqueous
sample 5 is introduced and the time the particles 4 finish
sedimenting on the surface 7 of the IC 12) in order to agitate the
aqueous sample 5 and/or the particles 4 and allow the particles 4
to more quickly disperse in the aqueous sample 5 and speed up assay
kinetics. The vibrator module may be enabled upon detection of the
aqueous sample 5 when the aqueous sample 5 comes in contact with
the SPDM 8 and/or the IC. The amplitude, frequency, and/or pattern
of the vibrations can be controlled by the control module and
adjusted based on various parameters obtained from the
environmental sensors discussed above and based on any assay
calibration parameters. For example, vibration amplitude may be
increased if the temperature is low and/or the viscosity of the
aqueous sample 5 is high in order to speed up assay kinetics.
[0076] A capacitive, inductive or resistive humidity sensor can be
used to detect the presence of the aqueous sample 5 on the surface
of the IC 12. The humidity sensor can be embedded in the IC 12
under the sedimentation capillary 13 or under the surface
capillary, or under the delivery capillary.
[0077] The PCB 9 can contain one or more external electromagnets or
permanent magnets generating fields from 0.1 .mu.T to 1 T at
excitation frequencies from DC to 100 MHz. Magnets can have an
appreciable effect on the particles 4 if the particles 4 are
magnetic particles. A permanent magnet may be placed below the IC
12 or in close proximity to the IC 12 in order to pull magnetic
particles more quickly towards the surface 7 of the IC 12 (i.e.,
increase the sedimentation velocity of the magnetic particles to as
high as 10 mm per second). The permanent magnet may be replaced
with an electromagnet (e.g., Helmholtz coil, current line, or
combinations thereof) mounted onto the PCB 9 and below the IC 12 to
selectively generate magnetic fields and magnetic forces. A second
electromagnet may be placed above the IC 12, near the ceiling of
the casing 11, in order to pull the magnetic particles up from the
sensor surface 7. This could be used to increase the incubation
time to over 10 minutes or to perform magnetic separation steps.
One or more electromagnets may be placed on the sides of the PCB 9
around the sedimentation capillary or extend into the assay system
10 and around the sample chamber in order to generate lateral
forces on the magnetic particles. Any of the electromagnets placed
above, below, or to the sides of the sample chamber may be used to
agitate the magnetic particles (e.g., move them side to side, make
them vibrate, make them change orientation) in order to create
convective forces in the aqueous sample 5 and/or to more quickly
sediment the magnetic particles to the IC 12 surface 7.
Electromagnets configured to generate lateral forces may be used to
compensate for any tilt in the assay system 10 (e.g., if the assay
system 10 is tilted to the left, an electromagnet on the right side
may turn on to ensure magnetic particles sediment evenly and do not
aggregate on the left side of the IC). The permanent magnets or
electromagnets may be mounted in any other component of the assay
system 10 (e.g., integrated into the IC, mounted on any wall of the
casing 11, or combinations thereof).
[0078] The assay system 10 can be run with the IC 12 at the top and
with its surface 7 facing down. A permanent or electromagnet can be
placed above the IC 12 to pull the magnetic particles 4 upward to
its surface. In this way, the system can run without the filter
directly on whole blood since the red blood cells will settle
downwards, away from the surface of the IC. Alternatively, the
system can be run directly with lysed samples. The whole blood
filter can be replaced by or stacked with a hydraulically permeable
solid membrane or matrix to ensure even and efficient mixing of the
cellular material with a lysing agent and other dry reagents.
[0079] The display 1 can be any display known in the prior art
(e.g., LCD display, LED display, OLED display and any other type of
display, touch-screen) that may display the presentable assay
information generated or stored by the chip 12 (e.g., concentration
of one or more target analytes, amount of one or more target
analytes, coefficient of variance, timestamp, timing of the assay,
validity of the results, patient and device identification number,
temperature, humidity, internet/telephone/physical locations,
help/counselling and information, angular information of the
device, device ID, patient ID and any other relevant results). The
presentable assay information may include status indicators that
can signal the device is ready, busy, testing, done, or in an error
state. The presentable assay information can include error messages
that indicate the device is tilted, additional sample is needed,
the on-board controls failed to fall within expected range,
temperature or humidity over/under the specified range or an
expiration date has been exceeded. The presentable assay
information may include information on the sample (e.g. whether it
is lysed, the viscosity, turbidity, lipemia, color of sample). The
presentable assay information may include written or visual
instructions to the user on how to use the assay system 10 to
perform a measurement. A speaker or earphone jack may also be
integrated into the assay system 10 to deliver the presentable
assay information in an audio format.
[0080] The presentable assay information can be displayed in an
encrypted format alone or alongside the presentable assay
information in a non-encrypted format. The display 1 can also
display a portion or all of the presentable assay information as a
one dimensional bar code or 2 dimensional QR code or other
machine-readable format. The results can appear as an encrypted
hexadecimal code or using other symbols or shapes. Users can take
one or more still photographs or one or more videos of the display
1 using a secondary mobile device to retrieve and decrypt a portion
or all of the presentable assay information. The user can have a
medical software application installed on a secondary mobile device
that processes the photograph or video of the display to retrieve
and decrypt the presentable assay information. The medical software
application can prompt the secondary mobile device's user for a
patient ID or can retrieve directly from the secondary mobile
device's login information. The presentable assay information
retrieved by taking the photograph or video of the display can be
bound to the patient ID in a secure manner, for example in a HIPAA
compliant manner. The display can be deactivated or the presentable
assay information can be removed from the display after a pre-set
time, or once a user presses a button or the a touch-screen
integrated into assay system 10 or from a prompt from the medical
software application. Tele-communication by taking still
photographs or videos of the display 1 does not require any
additional hardware either on the assay system 10 or on the
secondary mobile device and is therefore universally interoperable
with all modern consumer smart devices.
[0081] The medical software application can also store the
presentable assay information on the secondary mobile device and
can graph the presentable assay information. The medical software
application can combine the presentable assay information with
historical medical information from the patient. The medical
software application can connect the secondary mobile device
wirelessly or through a wire to a third storage device for
processing and storing the presentable assay information. The
medical software application can store or transmit all or portions
of the presentable assay information to a third device.
[0082] Presentable assay information can be transmitted to and
stored on the secondary mobile device without being displayed on
display 1. The medical software application can prompt the user to
get in contact with a doctor, counselor, insurance company
representative, drug company representative, clinical trial
representative, a reporting agency or other third party in order to
gain access to the presentable assay information. The medical
software application can automatically contact a third party and
direct transmission of all or part of the presentable assay
information to that third party. Example includes the CDC or other
healthcare professionals in the cases where public safety is at
risk. The medical software may omit the patient ID when sending
information third parties, but can include information like the
time and location of secondary wireless device . The medical
software application can combine the presentable assay information
with other information found on the secondary wireless device, such
as time of day, location, login information, contact to healthcare
professionals, emergency contacts, age and sex of patient or other
patient information stored on the secondary mobile device
[0083] The medical software application can be stored on the chip
12 or in other storage devices integrated in assay system 10 and
transmitted to the secondary mobile device prior to performing the
assay, after running the assay or during the assay. The web
location and/or routing information of the medical software
application can be stored on the chip 12 or in other storage
devices integrated in assay system 10 and can be included in the
presentable assay information. The assay system 10 can prompt the
secondary mobile device to download the medical software
application by transmitting the web location or routing
information.
[0084] The device can also provide multiple different sets of
results. A first set of displayed results can be displayed and
provided to the user and a second for example more detailed set of
comprehensive results can be sent to a third party.
[0085] A patient or user untrained as a caregiver, such as a family
member or home health aide, can perform the assay without help from
trained healthcare professionals. For special applications like
drug monitoring or emotionally difficult applications like HIV
testing, it may be undesirable for the patient to examine all or
part of the presentable assay information prior to their
examination by a third party. The device can encrypt and transmit
some or all the presentable assay information to a third party for
review without displaying them or granting access to them to the
patient. The third party can review the presentable assay
information and re-transmit reviewed assay information back to the
patient, or re-transmit access to the presentable assay
information, or re-transmit a different set of information or
additional information. The patient or user may be required by the
device to send the presentable assay information the secondary
mobile device to a third party in order to receive the reviewed
assay information or access to the latter. The presentable assay
information can be encrypted in a way that can only be decrypted by
the third party. The patient may or may not be the user of the
secondary mobile device or the assay system 10. A healthcare
professional, a family member or an untrained home health aide may
or may not be the user of the secondary mobile device or the assay
system 10. The secondary mobile device can be a tablet, a phone or
any wireless telecommunication device.
[0086] Presentable assay information and the medical software
application can be relayed to the secondary mobile device
wirelessly by assay system 10 (for example using Bluetooth, Zigbee
or Wifi protocols), visually on the screen, capacitively using
parallel plate, inductively or via optical links such as IR
communication or taking still photographs of the display 1. The
assay system 10 can contain full duplex communication with a
transceiver device. The assay system 10 can have an optical link or
a bar code reader integrated into it. The transceiver device can
send to the assay system 10 information regarding which assays were
ordered, and additional patient information like sex or age of
patient or other pertinent information to the assay. The assay
system 10 can modify the assay according to the received
information. In a multiplexed format, some assay may not be run, or
may be run but not reported if they weren't ordered.
[0087] The casing 11 can be an external shell that houses all the
other components of the assay system 10. The casing 11 may be made
in any standard or custom manufacturing process (e.g., injection
molding) and may be made from any standard material (e.g.,
plastic). The casing 11 may also include an outer flap over the
sample inlet or over the entire device to reduce the amount of
light that can shine through the seams of the casing 11.
[0088] The IC 12 can include one or more optical sensors 20
configured in an array. Each optical sensor 20 may be integrated
into the IC 12 and implemented in any technology (e.g., junction
photodiodes, avalanche photodiodes, PiN photodiodes, active pixel
sensors, charge-coupled devices, light-sensitive resistors, or
other solid-state optical sensors 40). Each optical sensor 20 may
be individually addressable and may output electrical signals that
may be amplified, digitized, stored and processed by circuitry on
the IC 12 and/or the PCB 9. Each optical sensor may be configured
to detect a shadow cast by a particle as a result of the particle
blocking the light rays from the light source. For example, an
optical sensor can detect a particle because the particle casts a
shadow over the sensor, decreasing the light intensity incident on
optical sensor from the light source. Consequently, as a result of
a particle blocking a portion of the light from the light source,
optical sensor generates a signal that is different from a baseline
signal without the particle, thus indicating the presence of a
particle over the sensor.
[0089] Magnetic particles in the sensing area 21 on the surface 7
of the IC 12 can be detected by magnetic sensors integrated in the
IC 12 as in WO/2009/091926--INTEGRATED MAGNETIC FIELD GENERATION
AND DETECTION PLATFORM, reference here in its entirety. Hall
sensor, GMR sensors, AMR sensors, variable inductance current lines
can all be used as magnetic sensors. If magnetic sensors are
employed, the light source 2 can he omitted.
[0090] FIGS. 4A, 4B, and 4C show a top and a cross sectional view,
respectively, of the light source 2 and the IC 12. One or more
magnetic separation field generators can be embedded in the
integrated circuit 12 at a lateral distance of 0.1 .mu.m to 100
.mu.m from the sensing area 21.
[0091] A portion of the magnetic particles 24 that sediment to the
surface 7 of the IC 12 and may bind strongly through specific
bio-chemical or inorganic interactions to the surface 7 of the IC
12. A portion of the magnetic particles 25 that sediment to the
surface 7 of the IC 12 may bind weakly to the surface 7 of the IC
12 through non-specific interactions.
[0092] The assay can be performed in various assay formats. In a
capture assay format, the presence of one or more target analytes
would promote specific binding of the particles 4 to the surface 7
of the IC 12. In a competitive assay format, the presence of one or
more target analytes would inhibit specific binding of the
particles 4 to the surface 7 of the IC 12. In a derivative capture
format, one or more by-products of one or more reactions with the
target analyte would promote specific binding of the particles 4 to
the surface 7 of the IC 12. In a derivative competitive format, one
or more byproducts of one or more reactions with the target analyte
would inhibit specific binding of the particles 4 to the surface 7
of the IC 12. Multiple assay formats can be performed concurrently
on the same chip 12, or on the same assay system 10 with multiple
chips. Electrodes and other bio-sensors can be integrated on the
same chip to detect ions, electrolytes and general chemistry
analytes.
[0093] The magnetic separation field generators can be used to
remove the non-specifically bound magnetic particles from the
sensing areas 21 so that the optical sensors 20 only detect
specifically bound magnetic particles. The magnetic separation
field generators can be implemented as electrical separation
conductors 23 embedded in the integrated circuit 12 and routed in
proximity to the sensing area 21. Current passing through the
separation conductors generates magnetic forces that act on the
magnetic particles inside the sensing area. The current can be from
0.01 mA to 200 mA depending on the separation force desired. A
value for the current passing though separation conductors to
separate 2.8 .mu.m magnetic particles can range from 1 mA to 100
mA. The separation conductors 23 on either side of the sensing area
21 can be activated at different times in order to pull the
magnetic particles. The current can be toggled between the two
separation conductors at a frequency from 0.001 Hz to 100 MHz. The
magnetic separation forces can be strong enough to displace
non-specifically bound magnetic particles from the sensing area
towards the separation conductor, but not strong enough to displace
specifically bound magnetic particles. A sequence of magnetic
separation forces is a series of magnetic forces resulting from
modulating one or more currents through one or more magnetic
separation conductors. The sequence of magnetic separation forces
can be controlled by an algorithm stored on the IC 12.
[0094] Non-specific binding forces may be on the order of 0.1 pN to
10 pN, while specific binding forces may be on the order of 20 pN
to 20 nN. For example, magnetic particle 24 may sediment over
optical sensor 26 and may specifically bind to the surface 7 of the
IC 12 over optical sensor 26. Thus, magnetic particle 24 may not be
removed by the separation force generated by a separation conductor
23 placed laterally to the sensing area 21 and may be detected by
optical sensor 26. On the other hand, magnetic particle 25 may
sediment over optical sensor 27 and may not bind specifically
(i.e., non-specifically bound) to the surface 7 of the IC 12 over
optical sensor 27. Thus, magnetic particle 25 may be removed by the
separation force generated by the conductors placed laterally to
the sensing area 21 and may not be detected by optical sensor 27.
The electric currents used to generate magnetic forces may be
pre-programmed onto the IC 12 during the design process or after
fabrication and may be adjusted at a later stage (e.g., before the
assay or dynamically during the operation of the assay) depending
on various parameters (e.g., temperature, viscosity of the aqueous
sample 5, magnetic content of the magnetic particles, size/shape of
the magnetic particles, and other factors). Magnetic forces can be
generated externally to the integrated circuit 12 using one or more
permanent magnets or external electromagnets (e.g., coils
integrated onto the PCB 9). In a variation of the assay system 10,
magnetic separation field generators may be omitted altogether from
the IC 12.
[0095] FIG. SA is a cross sectional side view of the dry sphere 3
placed in a cuvette 30 with vertical side walls 32. The cuvette can
be of any shape including square, rectangular, cylindrical and can
be smaller, greater or equal to the volume of the dried sphere 3.
The cuvette 30 can also be equal to or slightly narrower than the
diameter of the dried sphere 3 in order to hold it motionless in
place. The cuvette 30 can be wider than the dry sphere 3.
[0096] To promote the complete dissolution of the dried sphere 3,
the cuvette 30 can fill completely with the aqueous sample 5 as
shown in FIG. 5B. The fill stop structure 31 can be an enlarging of
the cuvette, a stop gap or a stop material.
[0097] To promote complete dried sphere 3 dissolution, the diameter
of the dried sphere 3 can be similar to the diameter of the
sedimentation capillary 13. For example the diameter of the dried
sphere 3 can be between 25% and 50%, between 50% and 75%, between
75% and 850%, between 85% and 100%, between 100% and 115%, between
115% and 125%, between 125% and 150% or between 150% and 200% of
the diameter of the sedimentation capillary 13.
[0098] To promote complete dry sphere 3 dissolution, the depth of
the cuvette can be similar to the diameter of the dried sphere. For
example the depth of the cuvette 30 can be between 10% and 25%,
between 25% and 50%, between 50% and 75%, between 75% and 100%,
between 100% and 125%, between 125% and 150% or between 150% and
200% of the diameter of the dried sphere 3. The cuvette 30 can be
partially filled with the aqueous sample 5 or the cuvette can
remain unfilled with the dry sphere 3 dissolving fully into the
sedimentation capillary 13 below without any fluid entering the
cuvette 30.
[0099] FIG. 5C shows the bottom of the cuvette acting as the fill
stop structure.
[0100] FIG. 6 is a cross sectional side view of a cuvette with
tapered side walls 40. The tapered sidewalls can hold the dried
sphere 3 in place firmly. The tapered sidewalls 40 can be designed
to wick the aqueous solution 5 via capillary force up the entire
length of the tapered sidewall 40, or up a portion of the tapered
sidewall 40. The tapered sidewalls 40 can be made to prohibit the
wicking of the aqueous solution 5.
[0101] FIG. 7 is a cross sectional side view of a cuvette 30 with a
cover 50 to hold the dried sphere 3 stationary. The cover 50 can be
manufactured of a breathable or porous material to let air pass, or
can be fully or partially hermetic to eliminate or reduce
evaporation of the aqueous sample through the top of the
sedimentation capillary 13. In the case that the cover 50 is
hermetic, an air opening 51 can be implemented to let the trapped
air evacuate as the aqueous solution 5 approaches. The air opening
can be designed into the SPDM 8 or into the cover 50. The cover 50
can also be transparent or partially transparent to let light
illuminate the dry sphere 3 and down the sedimentation capillary 13
after the dry sphere 3 dissolves. In this case, the optical sensors
embedded in the IC 9 can detect when the dry sphere 3 has
dissolved. The cover 50 can press on the dry sphere 3 in order to
keep it motionless in the cuvette. To eliminate adhesion of the dry
sphere 3 to the cover 50, the bottom of the cover 50 inside the
cuvette 30 can be made adhesion free. The cover can be glued,
taped, thermally bonded, or snap fit into location.
[0102] FIG. 8 is a cross sectional side view of the surface
capillary 15 constructed from double sided tape 60. A channel can
be cut, punched or milled into double sided tape 60 or transfer
adhesive or epoxy and that channel can form the sidewalls of the
surface capillary 15. The double sided tape 60 can provide a
hermetic seal on the surface of the chip 12. The surface 7 of the
IC 12 can be smaller than the bottom surface of the SPDM in such a
way that the bottom surface of the SPDM completely overlaps the
surface 7 of IC 12. Otherwise, any undesirable gaps in the double
sided tape 60 could result in persistent leaks that can pool on the
surface 7 of the IC 12.
[0103] The double sided tape 60 may be replaced by or used in
combination with other adhesives such as silicones, acrylates,
epoxies or others. A compression seal can be used instead of or in
combination with adhesives; in this case, the double sided tape 60
may be replaced by a flexible gasket, and mechanical pressure could
form the seal between the IC 10 and the SPDM 8. Alternatively, the
SPDM could be made out of flexible material such as rubber or
silicone, and the surface capillary 15 could be formed in the
bottom surface of the SPDM, without any gasket or tape. The height
of the double sided tape 60 or transfer adhesive can be less than
250 .mu.m, for example between 1 .mu.m and 10 .mu.m, or 10 .mu.m
and 25 .mu.m, or 25 .mu.m and 50 .mu.m, or 50 .mu.m and 100 .mu.m
or 100 .mu.m and 250 .mu.m. A thinner adhesive reduces the void
volume of the surface capillary 15 and bring the aqueous samples 5
in closer proximity to the surface 7 of the IC 12 for on-chip
pre-treatment of the aqueous sample 5. A thick tape can be used to
ensure a hermetic seal despite non uniformities on the surface 7 of
the IC 12 or on the bottom interface of the SPDM 8.
[0104] FIG. 9 is a top view of the surface of the integrated
circuit 12 with the double sided tape 60 mounted on it. The sensing
area 21 can be situated under the sedimentation capillary 13, while
the active area 71 can be situated along the length of the surface
capillary 15. Under the active area, a number of solid state
devices can be integrated for the pre-treatment of the aqueous
sample 5 as it flow by into the sedimentation capillary 13. One or
more temperature sensors and heating elements can be embedded under
the active area 71 to heat the aqueous sample 5 as it flows by and
measured the temperature of sample 5. The temperature of the SPDM
or the temperature of the aqueous sample in the SPDM can be
adjusted and kept at constant temperature for isothermal nucleic
amplification of oligonucleotides, or the temperature can be cycled
for PCR amplification of oligonucleotides. Similarly, one or more
pH sensors and hydrolysis electrodes or other pH adjusting elements
can be embedded under the active area 71 to respectively measure
and adjust the pH of the aqueous sample 5. The pH of the aqueous
sample in the SPDM can be adjusted kept constant for analyte
analysis, or the pH can be cycled to promote certain reactions.
Moisture sensors, blood cell counters and other solid state sensor
and actuators can be embedded under active area.
[0105] Heating elements can be placed under a portion of the active
area 71 or under a portion of sedimentation capillary 13. Heating
elements can be placed under the entire active area 71 or under the
entire sedimentation capillary 13 but only a portion can be
activated. Heating elements embedded under surface 7 of the IC 12
can be used to create eddy currents or convection currents to mix
the magnetic particles in solution. Heating elements under a
portion of the sedimentation capillary 13 can heat fluid in
proximity. The rising heated fluid from a portion of the
sedimentation capillary can generate eddy currents or convection
currents that keep the magnetic particles in suspension and
incubating with the target in the sedimentation capillary. The
heating elements can be enabled before the dry sphere 3 dissolves
or after the dry sphere dissolves.
[0106] FIG. 10 shows a cross section of the system with delivery
capillary 14 leading to a first surface capillary 15 and a second
surface capillary 62. Surface capillary 15 leads to a first
sedimentation capillary 13 and surface capillary 62 leads to a
second sedimentation capillary 63. Each sedimentation capillary can
have a different dried sphere at the top. The surface of the
sensing areas below each sedimentation capillary can have different
functional chemistry coatings. In this system, multiple assays can
be performed simultaneously without mixing between the
sedimentation capillaries. The heights of the different
sedimentation capillaries can be different and can be tailored to
the incubation time necessary for the assay being performed in the
sedimentation capillary. More than one assay can be performed in
one sedimentation capillary. More than two surface capillaries
leading to more than two sedimentation capillaries can be
integrated on the same system. The surface capillaries can be
connected in a star network, an H-network or any other network of
surface capillaries allowing the aqueous sample to flow from one or
more filters through one or more delivery capillaries to reach one
or more sedimentation capillaries.
[0107] To minimize the amount of sample and the number of
applications, all the surface capillaries can share the same
delivery capillary and the same filter. One or more sedimentation
capillaries can be reserved exclusively for performing assay
controls.
[0108] To ensuring proper functioning of all assay components,
conventional non-quantitative immunoassays rely on negative
controls using an irrelevant antibody of the same isotype to
determine the non-specific signal or background, and positive
controls using anti-species antibodies to generate a positive
signal.
[0109] Quantitative immunoassays rely on calibrators--known
quantities of analyte (calibrators) in a synthetic matrix--to
quantify unknowns.
[0110] This fully integrated assay system 10 can use a sample
specific internal assay calibration that relies on the sample
matrix itself to extrapolate the background signal and the native
target signal.
[0111] To calibrate the native target signal resulting from the
native target concentration in the aqueous sample 5, the assay
system can contain two sedimentation capillaries 13 and 63, that
may or may not be fluidically connected to the same delivery
capillary 14. A pre-determined quantity 1 of a dry calibrant
consisting of lyophilized synthetic target or target derivative, or
target analogue, can be added in the dry sphere at the top of
sedimentation capillary 63, along the sides of sedimentation
capillary 63, on the surface 7 of the chip 12 at the bottom of
sedimentation capillary 63, in the surface capillary 62 leading to
sedimentation capillary 63, or on the surface 7 of the chip 12 in
the surface capillary 62. The preferred location for the dry
calibrant is on the surface 7 of the chip 12 in the surface
capillary 62 since the lyophilized target can be deposited at the
same time as the sensing area is being coated, and since it enters
into the sedimentation capillary 62 in a dissolved state, akin to
the native target. The surface capillary 62 cannot flow into
sedimentation capillary 13 since the dry calibrant would corrupt
the detection of the native target signal. The dry calibrant will
be rehydrated by the sample and flow into the sedimentation
capillary 63. The quantity 1 of dry calibrant in sedimentation
capillary 63 can be between 1 zeptogram and 1 attogram, between 1
attogram and 1 femtogram, between 1 femtogram and 1 picogram,
between 1 picogram and 1 nanogram or between 1 nanogram and 1
microgram. A different quantity 2 of dry calibrant, or no dry
calibrant, can be loaded in the dry sphere at the top of
sedimentation capillary 13, along the sides of sedimentation
capillary 13, on the surface 7 of the chip 12 at the bottom of
sedimentation capillary 13, in the surface capillary 15 leading to
sedimentation capillary 13, or on the surface 7 of the chip 12 in
the surface capillary 15. The difference in signals in the two
sedimentation capillaries 13 and 63, i.e. the difference in the
number of specifically bound magnetic particles in sedimentation
capillaries 13 and 63 can be used to calibrate the native target
signal resulting from the native target concentration in the sample
by a signal calibration mathematical operation. The signal
calibration mathematical operation can include addition,
subtraction, multiplication, division, non-linear correlation
through look-up tables and can be performed digitally on the chip
12. An arithmetic logic unit can be integrated on the chip 12 to
perform the signal calibration mathematical operation. The
sedimentation capillaries 13 and 63 must be positioned to avoid the
dry calibrant intended to flow into sedimentation capillary 63 from
diffusion or traveling or flowing to sedimentation capillary 13 and
to avoid the dry calibrant intended to flow in sedimentation
capillary 13 from diffusion or traveling or flowing to
sedimentation capillary 63. The height of capillary 63 can be
shorter than capillary 13 to minimize the amount of sample volume
needed to calibrate the native target signal.
[0112] To calculate the background signal, i.e. the abnormally
strongly bound magnetic particles resulting from undesirable
non-specific interactions, the system can contain a first
sedimentation capillaries 13 and a third sedimentation capillary
64. The sedimentation capillary 64 can have a different height than
sedimentation capillary 13 resulting in a different sedimentation
times and by extension incubation time. In so doing, the number of
specifically bound magnetic particles resulting from the native
target concentration in the sample will be different in
sedimentation capillary 13 versus sedimentation capillary 64 due to
the different incubation times. Meanwhile, the background signal,
or the number of non-specifically bound magnetic particles will
remain approximately equal. The difference in the number of bound
magnetic particles, both specifically and background
non-specifically, in sedimentation capillaries 13 and 64 can be
used to determine the number of background non-specifically bound
magnetic particles by a background calculation mathematical
operation. That background calculation mathematical operation can
include addition, subtraction, multiplication, division, non-linear
correlation through look-up tables and can be performed digitally
on the chip 12. An arithmetic logic unit can be integrated on the
chip 12 to perform the background calculation mathematical
operation.
[0113] The native target signal is correlated to the magnetic bead
settling time, i.e. the incubation time, while the background
signal remains approximately constant with incubation time. To
measure the background signal, 2 concurrent assays, Assay 1 and
Assay 2, can be run in two different sedimentation capillaries with
different heights, corresponding to for example 12 and 2 minutes
incubation times respectively. Assay 1 bead count B1 consists of a
background signal Bkg and a native target signal component Sig1:
B.sub.1=Bkg+Sig.sub.1. Similarly, Assay 2 bead count B2 consists of
the same background signal Bkg but with a different native target
signal Sig1: B.sub.2=Bkg+Sig.sub.2, where Sig.sub.1 equals
approximately 6 Sig.sub.2 according to the ratio of the incubation
times. The background signal and the native target signals can
easily be extracted arithmetically:
Sig.sub.1=(B.sub.1-B.sub.2)*(6/5), Bkg=B.sub.1-Sig.sub.1, and
Sig.sub.2=B.sub.2-Bkg. Note that the two incubation times depend on
the heights h.sub.1 and h.sub.2 of the chambers and their ratio can
be controlled tightly by design, irrespective of sample viscosity.
The ratio of h1 to h2 can be increased as much as possible for more
precise measurement of the background signal. The ratio can be
between 1:1 and 1.5:1, between 1.5:1 to 2:1, between 2: 1 and 4:1,
between 4:1 and 8:1, between 8:1 and 16:1, between 16:1 and
100:1.
[0114] More than 2 sedimentation capillaries can be implemented on
the same assay system 10 to perform native target signal
calibration and background non-specifically bound magnetic
particles calculation from the same aqueous sample 5. Three
sedimentation capillaries can be used per analyte. A first
sedimentation capillary can be used to perform the standard assay,
a second sedimentation capillary can be used to measure the
background signal, while a predetermined amount of a dry calibrant
can be spiked into the surface capillary leading to a third
sedimentation capillary, which can be used to calibrate the native
target signal.
[0115] In a multiplexed format, the sedimentation capillaries can
be grouped to minimize the total amount of volume of aqueous sample
5 needed. Multiple background signal measurements for multiple
analytes can be performed using the same sedimentation capillaries,
while multiple native signal calibrations for multiple analytes can
be performed using the same sedimentation capillaries. Native
signal calibration for a first analyte and background measurement
for a second analyte can be performed using the same sedimentation
capillary.
[0116] The background signal measurement and native target signal
measurement can also be used qualitatively to invalidate the test
for example should they fall outside expected ranges. The
background signal measurement and native target measurement can be
performed on more than one targets concurrently or in series on the
same system or moreover on the same chip.
[0117] For qualitative yes/no measurements, the background signal
measurement and the native target signal calibration can be
performed using a single sedimentation capillary 63 to minimize the
volume of aqueous sample 5 needed. In this case, the height of
sedimentation capillary 63 can be a ratio r of the height of
sedimentation capillary 13. Seimentation capillary 63 can contain
the dissolved dry calibrant. The bead counts in the sedimentation
capillaries 13 and 63 is given respective by B.sub.1=Sig.sub.1+Bkg
and B.sub.2=Sig.sub.2+Bkg +Cal. The difference between the bead
counts is given by B.sub.1-B.sub.2=Sig.sub.1+Bkg-Sig.sub.2-Bkg-Cal.
Sig.sub.1 and Sig.sub.2 are ratioed according to the ratio r of the
heights of the sedimentation capillaries 13 and 63 and Cal is the
solubilized dry calibrant concentration in sedimentation capillary
63. B.sub.1-B.sub.2=Sig.sub.1*(1-r)-Cal. The amount of dry
calibrant can chosen to be so that the concentration of
resolubilized dry calibrant in sedimentation capillary 63 is equal
to a qualitative concentration threshold multiplied by (1-r).
B1-B2=Sig*(1-r)-Threshold*(1-r). As a results B1>B2 when
Sig>Threshold and B2>B1 when Sig<Threshold.
[0118] The bead count to target concentration relationship is
non-linear so additional calculation can be performed when assay
system operates in the non-linear slope of the bead count to
concentration curve. The IC 12 can perform the non-linear
calculation or store a look-up table with the relationship to
convert between bead count and target concentration and back.
Target concentration refers to native target and calibrant
concentration.
[0119] The on-chip generated magnetic separation forces can be
adjusted such that the background signal is zero beads. In this
case, no measurement of the background signal is needed.
[0120] FIG. 11 shows a cross section of the integrated circuit 12
mounted onto the PCB 9 and electrically connected via a wirebond
81. The wirebond 81 can be hermetically sealed by encapsulant 80.
The SPDM 8 can be placed directly on the exposed surface 7 of the
IC 12. The encapsulant can be an epoxy, acylate, urethanes,
silicones or other adhesives.
[0121] FIG. 12 shows a cross section of the integrated circuit 12
mounted onto the PCB 9 and electrically connected by way of one or
more through silicon vias 82. The use of through silicon vias which
can be placed under the active sensing area can minimize the area
of the IC 12 devoted to pads or input/output functions and by
extension minimize the cost of IC 12. The SPDM 8 can be placed
directly on the exposed surface 7 of the IC 12.
[0122] FIG. 13 shows the top view of the integrated circuit surface
7 with one dedicated magnetic separation conductors 90 for each
sensor 20. Each dedicated magnetic separation conductor can be
individually addressed and activated. The current through each
dedicated magnetic separation conductor can be precisely set to
achieve the desired force on a magnetic particle 24 atop a sensor
26. When a magnetic particle lands on a sensor, it can be detected
and the dedicated magnetic separation conductor can be used to
remove it if it is non-specifically bound without disturbing other
magnetic particles more than one sensor length away. The
non-specifically bound magnetic particles can be removed one by one
off their corresponding sensors by dedicated separation conductors
for superior assay control and precision.
[0123] Each sensor can have more than one dedicated magnetic
separation conductors. For example, each sensor can have two
dedicated magnetic separation conductors, one on each side for
hi-lateral magnetic separation. A dedicated magnetic separation
conductor can be shared with one or more neighboring sensor.
[0124] FIG. 14 shows the top view of the integrated circuit surface
7 with one dedicated magnetic concentration conductor 92 for each
sensor 20. Each dedicated magnetic concentration conductor 92 can
be individually addressed and activated. The current through each
dedicated magnetic concentration conductor can be precisely set to
achieve the desired concentration force on a magnetic particle
sedimenting atop a sensor 20. Magnetic particles can be pulled atop
sensors by dedicated magnetic concentration conductor 92, and once
the magnetic particle it atop the sensor, the dedicated magnetic
concentration conductor can be switched off.
[0125] Each sensor can have more than one dedicated magnetic
concentration conductors for rastering the magnetic particles. A
dedicated magnetic concentration conductor can be shared with one
or more neighboring sensor. Rastering is the process of moving or
rolling a particle on the surface 7 of the IC 12 to promote
specific binding.
[0126] The magnetic particles can be rastered on the surface by the
dedicated concentration conductors and the dedicated separation
conductors applying one or more magnetic rastering forces. On the
flat X-Y planar surface 7 of the IC 12, the dedicated separation
conductors can be arrayed in rows in the x-direction and the
dedicated concentration conductors can be arrayed in columns in the
y-direction. In so doing, non-specifically bound magnetic particles
can be rastered 2-dimensionally in the positive and negative X and
Y directions by a rastering algorithm that controls the movement of
each particle individually, or one or more ensembles of particles.
The particles can be rastered by on-chip generated magnetic
rastering forces until they form a binding interaction on the
surface 7 of the IC 12, preventing the particle from rastering
further. A detection algorithm can detect when a particle is no
longer rastering on the surface of the IC, and activate the
magnetic separation forces. The magnetic separation forces can be
applied and if the particle is non-specifically bound, the particle
can be removed atop the sensor and continue to be rastered across
the array. The magnetic separation force can be higher than the
magnetic rastering force. The magnetic rastering force can be
different from the magnetic concentration force. However, if a
particle is specifically bound atop the sensor, then the sensor can
detect the particle and the dedicated concentration and dedicated
separation conductors for that sensor can be de-activated such that
other particles are not pulled atop that sensor. The magnetic
concentration forces can be used to pull the magnetic particles
atop the sensors, to raster the magnetic particles across the
sensor or to raster the magnetic particle on top of the sensor. In
the latter case, the particle is detectable throughout the entire
raster process. One or more magnetic concentration conductors can
be placed directly atop a sensor or laterally spaced to a sensor.
The optical sensors can be arrayed densely to ensure a 100% fill
factor so that all the particles or ensembles of particles on the
surface of the chip are detected.
[0127] When the particles 4 are released from dissolution of the
dry sphere 3, they can disperse throughout the sedimentation
capillary 13 and land on the surface 7 of the IC 12 at different
times. In this scenario, the incubation time on the surface 7 of
the IC 12 for a given particle 4 can vary depending on when it
landed on the surface 7 of the IC 12. The magnitude of non-specific
interactions can depend on the time the particle 4 lies on the
surface 7 of the IC 12, leading to variability in the assay. To
overcome this variability, a sequence of magnetic separation forces
can be initiated at different magnetic separation times. The
magnetic separation times can be determined dynamically at run-time
or pre-determined and stored in memory. At each magnetic separation
time, a sequence of magnetic separation forces can be applied to
remove the non-specifically bound particles away from the sensors.
Once the magnetic separation is complete, the sequence of magnetic
separation forces can be deactivated until the next magnetic
separation time to allow more particles to settle. The assay
protocol can consist of more than one magnetic separation times
with different or variable intervals between them. The intervals
between magnetic separation times can vary from 5 seconds to 15
minutes, or from 30 seconds to 10 minutes, or from 1 to 5 minutes.
The shorter the interval between magnetic separation times, the
shorter the opportunity for the particles to land on the surface
and bind specifically. The longer the interval between magnetic
separation times, the more chance for abnormally strong
non-specific interactions to form. The sequences of magnetic
separation forces applied at each magnetic separation time can be
different, for example in magnetic force magnitude, conductors
activated, frequency of forces, length of application of forces,
magnetic force profile and algorithm of activation of
conductors.
[0128] The number of particles can be detected before and after a
sequence of magnetic separation forces is applied. The specific
particle binding ratio is the ratio of the pre-separation number of
magnetic particles detected by the sensors before the magnetic
separation forces are applied to the post-separation number of
magnetic particles detected by the sensors after the magnetic
separation forces are applied. The specific particle binding ratio
is a useful indicator of binding since it eliminates or mitigates
the dependence to the absolute number of particles that sedimented
on the surface 7 of the IC 12.
[0129] The sequences of magnetic separation force can be tailored
such that when applied, few magnetic particles--i.e. less than 1,
or less than 10, or less than 100, or less than 1000, or less than
10000, or less than 1000000--can land on the sensor but rather the
sedimenting particles can be pulled toward the separation conductor
before they have the opportunity to land on the surface 7 of the IC
12. This way, the post-separation number can avoid being corrupted
by non-specifically bound particles landing on the sensors during
the application of the magnetic separation force.
[0130] When a sequence of magnetic separation forces is
deactivated, the current through the respective separation
conductors can be switched off and the magnetic particles can
settle on the sensor indiscriminately, while during the magnetic
separation sequence, few magnetic particles--i.e. less than 1, or
less than 10, or less than 100, or less than 1000, or less than
10000, or less than 1000000--may be able to sediment on a
sensor.
[0131] More than one sequence of magnetic separation forces
generating separation forces on different areas of the IC 12 can be
applied at the same magnetic separation times, or at different
magnetic separation times. Multiple areas of the ICs can perform
multiple assays independently.
[0132] An assay can consist of multiple magnetic sequences of
magnetic separation forces initiated at multiple magnetic
separation times. A final sequence of magnetic separation forces
can be using stronger magnetic separation forces or magnetic
separation forces for longer duration. A specific particle binding
ratio can be calculated for each magnetic separation sequence, and
the multiple specific binding ratios can be combined to give a
final particle binding ration, which can correspond to the target
analyte concentration in the aqueous sample 5.
[0133] A final particle binding ratio can be calculated by dividing
the final number of particles detected by the total number of
particles that sedimented onto the sensors. The total number of
particles that sedimented onto the sensor is not straight-forward
to calculate if prior magnetic separation sequences were applied
before the final magnetic separation sequence. The total number of
particles is equal to the sum of all the pre-separation particles
counts minus the sum of all the post-separation particle counts
plus the final post-separation particle count.
[0134] The sequence of magnetic separation forces can include a
magnetic force chirp, where the magnetic forces applied can be
toggled between the left and right side of a sensor at increasing
frequency. At the beginning of the magnetic chirp, strong
separation forces strongly remove the non-specifically bound beads,
while at the end of the magnetic chirp, the toggle frequency is too
high for the particles to move away from the separation conductors.
This prevents distant separation conductors from rastering
non-specifically bound particles across sensors prior to
detection.
[0135] The assay system 10 can provide different assay results at
different times. Intermediate results can be provided ahead of
final results. The intermediate results can include assay progress
information and qualitative assay results before the final
quantitative results are complete and displayed or transmitted. The
intermediate results can provide expedited qualitative
information.
[0136] The intermediate results of the assay after each
intermediate sequence of magnetic separation forces can be
displayed or transmitted for real-time updates. In this case, after
each intermediate sequence of magnetic separation forces, the
particle binding ratio can be arithmetically processed is if it
were a final sequence of magnetic forces.
[0137] To expedite access to assay information, expedited
qualitative results of the assay can be displayed or transmitted
before the full assay is complete and before precise quantitative
information is available. The relative particle binding ratio at
the end of the first magnetic separation interval or any subsequent
magnetic separation interval can be used to provide the expedited
qualitative information.
[0138] An example of an on-chip assay protocol is given below:
1. IC 12 in standby until detection of aqueous sample on surface of
IC under surface capillary. Once detected, IC 12 activates the
heating elements to heat the aqueous sample to 37C and proceed with
protocol. 2. IC 12 waits until detect of aqueous sample on surface
of IC 12 under sedimentation capillary 13. IC 12 proceeds with
protocol when detected or send error message if timed out. 3. IC 12
detects dissolution of dry sphere by changes in light on sensing
area 21. 4. IC 12 activates magnetic concentration conductors. 5.
IC 12 waits 2 minutes. 6. IC 12 reads out each sensor and counts
the pre-separation number of particles to give Count 1. 7. IC 12
de-activates magnetic concentration conductors. 8. IC 12 activates
the first sequence of magnetic separation forces to remove all the
non-specifically bound particles from the sensor surfaces. 9. IC 12
reads out each sensor and counts the post-separation number of
particles to give Count 2. 10. IC 12 calculated ratio of Count
2/Count 1 and correlates ratio to a concentration. 11. IC 12
displays the concentration or the qualitative information.
[0139] Protocol elements 4-10 can be repeated until all the
particles have settled onto the surface of the chip. In each repeat
of protocol elements 4-10, the magnetic concentration forces and
separation forces can be varied. A Cumulative Pre-Counts and a
Cumulative Post-Count can be the sum of all the pre-separation
counts and all the post-separation counts, respectively. The
different pre-separation counts and post-separation counts from
each sequence of magnetic separation forces can be combined
arithmetically to give a final particle count and a final binding
ratio. In protocol element 11, the correlation function that
translates the final particle count or final binding ratio to a
concentration of target analyte can be stored on chip from values
obtained during manufacturing. The correlation function can also
include the results from the background measurement and the native
target signal calibration. The assay can be initiated by the
humidity or moisture sensors, or the assay can be initiated by a
button or a touch screen integrated in the assay system 10.
[0140] When detecting a particle, the optical sensors can
internally perform correlated double sampling to a calibration
value acquired during manufacturing, or to a value obtained in real
time running the assay. The optical sensor can measure the optical
signal before and after a particle lands on the sensor. The
difference or the ratio can be compared to a threshold to determine
whether a particle is present. The sensor can detect the optical
signal before and after magnetic separation to detect the removal
of a particle.
[0141] FIG. 15 shows the cross section of the sedimentation
capillary 13 with a notch 100 to prevent the dried sphere 3 falling
into the sedimentation capillary 13. The dried sphere 3 can have
smaller diameter than the sedimentation capillary 13 but can be
large enough to be prevented by the notch 100 from falling into the
sedimentation capillary.
[0142] FIG. 16 shows a cross section of the delivery capillary 14,
the surface capillary 15 and the sedimentation capillary 13 atop
the IC 12. When a small volume of aqueous sample 5 is applied (i.e.
less than 200 ul, or less than 100 ul, or less than 50 ul or less
than 30 ul or less than 20 ul, or less than 10 ul, or less than 5
ul, or less than 2 ul or less than 1 ul), the evaporation of the
aqueous sample 5 can results in small fluidic flows that can
disturb the assay on the surface 7 of the chip 12. To reduce or
eliminate evaporation through the top of the sedimentation
capillary 13, the cover 50 can be made or a material that reduces
or eliminates evaporation. Moreover, the air opening 51 can be
small enough in cross section to limit by diffusion or other
effects the amount of aqueous sample 5 that can evaporate through
it.
[0143] A second source of evaporation can occur through the filter
6. In this case a "suck-back" pressure (vacuum) can be generated as
the aqueous sample evaporates from the filter surface. The filter
has a large surface area and can evaporate fluid at a high rate.
When a small amount of sample is applied, the aqueous sample 5 can
traverse the filter into the delivery capillary 14, into the
surface capillary 15 and into the sedimentation capillary 13, but
can be sucked back through the filter due to evaporation before the
full assay can be performed. This can be overcome by a lid that can
close over the sample port on assay system 10 after the aqueous
sample 5 has been applied and eliminate or reduce the amount of
aqueous sample 5 that evaporates into the surrounding
environment.
[0144] Another way of mitigating the evaporation through the top of
the filter 6 is to implement a passive unidirectional valve in the
filter 6, or in the delivery capillary 14, or in the surface
capillary 15, or in the sedimentation capillary 13 or at the top of
the sedimentation capillary 13 or at the top of the cuvette 30. The
passive unidirectional valve can allow the fluid to flow from the
filter 6 to the delivery capillary 14, or to the surface capillary
15, or to the sedimentation capillary 13, or to the top of the
sedimentation capillary 13 or to the top of the cuvette 30 but not
in the reverse direction. The passive unidirectional valve can
eliminate or reduce the "suck-back" flow resulting from aqueous
sample evaporation through the filter 6.
[0145] For ease of use of the assay system 10, a passive
unidirectional valve rather than an actuated unidirectional valve
is desirable. A Martin vent 110 is a passive unidirectional valve
that can relieve the "suck-back" pressure with air. The Martin vent
110 provides a low impedance path for air to be sucked back towards
the filter without passing through the sedimentation capillary 13,
thereby leaving the fluid in the sedimentation capillary 13 intact
and the assay able to complete unmolested. To prevent the aqueous
sample 5 from leaking out of the Martin vent, a microfluidic stop
gap 112 or fluid trap can be implemented at the terminus of the
Martin vent 110. The design of this microfluidic stop gap can be
such that its surface tension in the direction of the filter is
less than the surface tension in the sedimentation capillary or in
the cuvette in the direction of the filter such that air will
preferentially flow from the Martin vent as opposed to the cuvette
or sedimentation capillary.
[0146] The Martin vent can be placed anywhere along the length of
the delivery capillary or surface capillary that prevents a
"suck-back" pressure being generated by the filter when the fluid
in the filter begins to evaporate from pulling or sucking back the
fluid in the SPDM. Alternatively, a microfluidic check valve may be
placed anywhere between the outlet of the filter and the
sedimentation capillary.
[0147] FIG. 17A and B present cross sectional side views of a
passive unidirectional valve that can seal the sedimentation
capillary 13 once the aqueous sample dissolves the dry sphere 3 by
blocking the flow of air or fluid through the top of the
sedimentation capillary 13. A flow stop 120 can be placed above the
dry sphere 3. While the dry sphere 3 remains dry, the flow stop 120
cannot seal the sedimentation capillary 3 and can allow air and
fluid to move through the top of the sedimentation capillary 13
(FIG. 17A). Once the dry sphere 3 dissolves, the flow stop 120 can
drop down vertically or through other mechanism seal the top of the
sedimentation capillary (FIG. 17B) and prevent or impede air or
other fluid flowing through the top of the sedimentation capillary
13.
[0148] The flow stop 120 can be any shape that creates a hermetic
seal or high impedance seal with the top of the sedimentation
capillary 13 or to create a hermetic seal or high impedance inside
the sedimentation capillary 13. For example, the flow stop 120 can
fit flush with the top or inner sidewall of the sedimentation
capillary 13. The flow stop 120 can be shaped to allow a small
amount of air or fluid through. The flow stop 120 can use the
surface tension from a vapor seal to seal the top of the
sedimentation capillary 13.
[0149] The flow stop 120 can be sized such that is cannot tilt or
move inside the cuvette or the sedimentation capillary 13 before
the dry sphere dissolves. It can be light-weight such that the
weight of flow stop 120 does not crush the dry sphere 3 during use,
in manufacturing or transportation. The flow stop 120 can be
transparent or translucent to allow light to pass through it into
the sedimentation capillary 13.
[0150] Another example of a passive unidirectional valve is an air
opening 51. The air opening 51 can be a small diameter capillary or
small diameter opening (i.e. less than 1 mm, or less than 0.1 mm,
or less than 0.01 mm, or less than 1 um, or less than 1 nm) and can
be placed at the top of the sedimentation capillary 13 or at the
top of the cuvette 30 or in the cover 50. The air opening 51 can
allow the air or aqueous fluid through but will not allow the
aqueous fluid back out. The air opening 51 can block the fluid from
exiting by capillary force if the effective diameter of the air
opening 51 is small enough. The air opening 51 can be coated with a
material that reacts with the aqueous sample to constrict the air
opening 51 or seal it altogether
[0151] The surface 7 of the chip 9 can be coated with a thin
optically transparent reagent adhesion layer. The protein adhesion
layer can consist of gold, silver, chrome, polymer, silicon
dioxide, polyimide or silicon nitride. The reagent adhesion layer
can be thermally deposited, chemically deposited or spun on, or
other method. The reagent adhesion layer can be less than 50 nm or
less than 25 nm or less than 20 nm or less than 15 nm or less than
10 nm or less than 5 nm or less than 3 nm or less than 1 nm. For
proper adhesion to silicon or silicon dioxide of the reagent
adhesion layer, an additional adhesion layer of chromium or
titanium can be used. The additional adhesion layer can be
optically transparent and can be less than 50 nm or less than 25 nm
or less than 20 nm or less than 15 nm or less than 10 nm or less
than 5 nm or less than 3 nm or less than 1 nm. The reagent adhesion
layer can be coated with streptavidin by passive adsorption.
Biotinylated anti-bodies can be bound to the streptavidin. The
reagent adhesion layer can be deposited over the entire chip, or
the sensing area or localized on the individual sensors. The
reagent adhesion layer can be deposited after the IC has been
assembled onto the PCB to eliminate any contamination that occurred
during the manufacturing process.
[0152] To minimize power dissipation and heat generation, the
separation conductors can be implemented in thick top metal (top
metallization having a deposition thickness greater than 1 um, or
greater than tum or greater than 3 um). To eliminate the topology
from the thick top metal can affect the assay performance, the
surface can be chemically mechanically polished (CMPed). Openings
in the top metal for illuminating the optical sensors below can be
used to collimate the light for improved detection SNR. The
increased thickness of the top metal could increase the SNR despite
the increased distance from the particle to the optical sensor.
[0153] The platform described herein can be used for applications
including, but not limited to, diagnostics such as simplex assays,
parallel or multiplexed assays, DNA micro-array assays, glucose,
cholesterol, metabolites, and small molecules detection;
environmental assays such as for food contamination, and water
and/or soil contamination; proteomics such as protein-protein
binding force measurements, protein-protein binding resonant
frequencies, protein kinetics research; genomics such as DNA
methylation profile, and DNA force measurements; magnetic particle
4 atomic force microscopy (AFM) such as low l/f noise AFM, AFM with
digitally controlled force and frequency, and multiplexed AFM;
Magnetic Particle Characterization such as exploration of magnetic
properties of particles of different sizes and characteristics; Low
Cost Bio-sensor Networks such as integrated and direct wireless
transmission of assay results, and real-time outbreak and/or
contamination monitoring; and any combinations thereof.
[0154] Variations of the systems, devices and methods have been
shown and described herein by way of example only. Variations,
changes, and substitutions can occur. For example, the methods can
be performed with any one or more elements of the methods absent,
and any one or more element of the devices can be omitted. Various
alternatives and combinations of elements between the variations
described herein may be employed. All publications, patents, and
patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *