U.S. patent application number 10/338787 was filed with the patent office on 2003-08-21 for nucleic acid biosensor diagnostics.
Invention is credited to Damha, Masad, Hudson, Robert H.E., Krull, Ulrich J., Piunno, Paul A., Uddin, Andre H..
Application Number | 20030157538 10/338787 |
Document ID | / |
Family ID | 25679432 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157538 |
Kind Code |
A1 |
Krull, Ulrich J. ; et
al. |
August 21, 2003 |
Nucleic acid biosensor diagnostics
Abstract
A biosensor for direct analysis of nucleic acid hybridization by
use of an optical fiber functionalized with nucleic acid molecules
and fluorescence transduction is disclosed. Nucleic acid probes are
immobilized onto the surface of optical fibers and undergo
hybridization with complementary nucleic acids introduced into the
local environment of the sensor. Hybridization events are detected
by the use of fluorescent compounds which bind into nucleic acid
hybrids. The invention finds uses in detection and screening of
genetic disorders, viruses, and pathogenic microorganisms.
Biotechnology applications include monitoring of gene cultures and
gene expression and the effectiveness (e.g. dose-response) of gene
therapy pharmaceuticals. The invention includes biosensor systems
in which fluorescent molecules are connected to the immobilized
nucleic acid molecules. The preferred method for immobilization of
nucleic acids is by in-situ solid phase nucleic acid synthesis.
Control of the refractive index of the immobilized nucleic acid is
achieved by the support derivatization chemistry and the nucleic
acid synthesis. The preferred optical fiber derivation yields a DNA
coating of higher refractive index than the fiber core onto the
fiber surface.
Inventors: |
Krull, Ulrich J.;
(Mississauga, CA) ; Piunno, Paul A.; (Mississauga,
CA) ; Hudson, Robert H.E.; (London, CA) ;
Damha, Masad; (St. Hubert, CA) ; Uddin, Andre H.;
(Georgetown, CA) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
5370 MANHATTAN CIRCLE
SUITE 201
BOULDER
CO
80303
US
|
Family ID: |
25679432 |
Appl. No.: |
10/338787 |
Filed: |
January 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10338787 |
Jan 7, 2003 |
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09446222 |
Feb 16, 2000 |
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6503711 |
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09446222 |
Feb 16, 2000 |
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PCT/CA98/00402 |
Apr 30, 1998 |
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60050970 |
Jun 19, 1997 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
C12Q 1/6825 20130101;
C12Q 1/6839 20130101; G01N 21/7703 20130101; Y10S 977/924 20130101;
G01N 21/648 20130101; G01N 21/6428 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 1997 |
CA |
2,208165 |
Claims
We claim:
1. A biosensor for detecting a target nucleic acid in the presence
of a fluorophore by detection of fluorescence from the fluorophore
which comprises: (a) an optical element having an index of
refraction and which comprises an interaction surface; (b) an
immobilized layer having an index of refraction which comprises a
nucleic acid or nucleic acid analogue covalently attached to the
interaction surface of the optical element, the nucleic acid or
nucleic acid analogue capable of hybridizing to the target nucleic
acid to form a hybridized nucleic acid complex; (c) a light source
for introducing light capable of stimulating fluorescence of the
fluorophore into the optical element in contact with the
interaction surface; and (d) a detector for detecting fluorescence
emitted by the fluorophore on binding to a hybridized nucleic acid
complex; wherein the index of refraction of the immobilized layer
is equal to or greater than the refractive index of the interaction
surface of the optical element such that direct excitation of the
fluorophore in the immobilization layer to emit fluorescence
results in the detection of the target nucleic acid, wherein the
nucleic acid or nucleic acid analogue is attached to the
interaction surface through a linker molecule that is at least
about 25 .ANG. in length and the index of refraction of the
immobilized layer is controlled by changing the density of linker
molecules immobilized on the interaction surface of the optical
element and wherein the linker contains ethylene glycol
subunits.
2. The biosensor of claim 1 wherein the linker is hexaethylene
glycol.
3. The biosensor of claim 1 wherein the nucleic acid or nucleic
acid analogue is attached to the linker by in situ solid-phase
oligonucleotide synthesis.
4. The biosensor of claim 3 wherein the nucleic acid is attached to
the linker by in situ oligonucleotide synthesis using the
phosphoramidite method.
5. The biosensor of claim 4 wherein after synthesis of a protected
oligonucleotide on the linker the oligonucleotide protecting groups
are removed without removing the oligonucleotides from the
linker.
6. The biosensor of claim 5 wherein the protecting groups are
phenoxyacetyl protecting groups.
7. The biosensor of claim 5 wherein the oligonucleotide protecting
groups are removed using a triethylamine/acetonitrile solution.
8. The biosensor of claim 7 wherein the oligonucleotide protecting
groups are removed using a solution of 2:3
triethylamine/acetonitrile at room temperature for 1.5 hours.
9. The biosensor of claim 1 wherein the interaction surface is a
silica surface and the immobilized layer is prepared by treatment
with glycidoxypropyltrimethoxysilane (GOPS), followed by extension
via treatment with hexaethylene glycol (HEG) under acidic
conditions.
10. The biosensor of claim 1 wherein the fluorophore is covalently
tethered to the nucleic acid or nucleic acid analogue in the
immobilization layer.
11. The biosensor of claim 1 wherein the index of refraction of the
immobilized layer is measured using ellipsometry.
12. The biosensor of claim 1 wherein the optical element is an
optical fiber.
13. A method for detecting one or more target nucleic acids in a
sample which comprises the steps: (a) contacting the immobilized
layer of the biosensor of claim 1 with the sample such that target
nucleic acids in the sample can hybridize to the nucleic acids or
nucleic acid analogues of the immobilization layer; (b) contacting
the immobilization layer of the biosensor with a fluorophore and
allowing the fluorophore to bind to hybridization complexes of the
nucleic acids or nucleic acid analogues with the one or more target
nucleic acid in the immobilization layer; (c) introducing light
into the optical element of the biosensor in contact with the
interaction surface of the optical element to stimulate emission
from bound fluorophore; and (d) detecting the fluorescence emitted
by bound fluorophore; whereby the target nucleic acid is
detected.
14. The method of claim 13 wherein the immobilization layer is
contacted with the fluorophore by covalently tethering the
fluorophore to nucleic acids or nucleic acid analogues of the
immobilization layer.
15. The method of claim 13 wherein the one or more target nucleic
acids are those of bacteria, viruses, fungi, unicellular or
multicellular organisms.
16. The method of claim 13 wherein the one or more target nucleic
acids are those of a cell, a cellular homogenate, a tissue or an
organ.
17. The method of claim 13 wherein the biosensor is internally
calibrated by comparing fluorescence emission from the fluorophore
in the immobilization layer before and after the biosensor is
contacted with the sample.
18. The method of claim 13 wherein the biosensor is internally
calibrated using time-resolved fluorescence measurements.
19. The method of claim 13 wherein the optical element is an
optical fiber.
20. A method for making a biosensor having an optical element
comprising an immobilized layer covalently attached to an
interaction surface of the optical element which comprises the
steps of: (a) activating the interaction surface of the optical
element of the biosensor; (b) covalently attaching nucleic acids or
nucleic acid analogues, which can hybridize to a target nucleic
acid, to the activated interaction surface; (c) measuring the
refractive index of the immobilized layer by ellipsometry; and (d)
adjusting the conditions of step b, if necessary, to obtain an
immobilized layer having an index of refraction equal to or greater
than the index of refraction ofthe interactive surface of the
optical element.
21. The method of claim 20 wherein the interaction surface of the
optical element is activated with spacer molecules of at least 25
.ANG. in length which have either an amine or a hydroxyl
functionality at the terminus of the spacer molecule.
22. The method of claim 20 wherein activation is performed using a
bifunctional amphiphilic support derivatization agent created by
condensing .gamma.-aminopropyltriethoxysilane (APTES) with
12-nitrododecanoic acid.
23. The method of claim 20 wherein the interaction surface is
cleaned prior to activation.
24. The method of claim 23 wherein the interaction surface is
cleaned by treatment with a 1:1:5 (v/v) solution of 30% ammonium
hydroxide/30% hydrogen peroxide/water at 80.degree. C. for five
minutes, followed by treatment with a solution of 1:1:5 (v/v) conc.
HCl/30% hydrogen peroxide/water for 80.degree. C. for five minutes
and followed by sequential washing with methanol, chloroform and
diethyl ether.
25. A method for cleaning an optical element of a biosensor for
detecting a target nucleic acid in the presence of a fluorophore by
detection of fluorescence from said fluorophore wherein said
optical element comprises an interaction surface having an index of
refraction to which an immobilized layer also having an index of
refraction is attached, the immobilized layer comprising a nucleic
acid or nucleic acid analogue covalently attached to said
interaction surface of said optical element, said nucleic acid or
nucleic acid analogue capable of hybridizing to said target nucleic
acid to form a hybridized nucleic acid complex and wherein said
index of refraction of said immobilized layer is equal to or
greater than the refractive index of said interaction surface of
said optical element wherein said method comprises one or more
steps selected from the group consisting of: sonicating said
optical element in ethanol; heating and flushing said optical
element; incubating said optical element in highly polarized salts
and flushing; and autoclaving said optical element; wherein after
cleaning, biosensor function is at least maintained compared to
biosensor function before cleaning.
26. The method of claim 25 wherein said sonicating is performed for
15 minutes in 1:1 ethanol/water.
27. The method of claim 25 wherein said sonicating is performed for
15 minutes in 100% ethanol.
28. The method of claim 25 wherein said heating and flushing
comprises: (a) flushing said optical element with 30 ml of
85.degree. C. buffer solution for 30 seconds; (b) waiting 5
minutes; and (c) flushing said optical element with 30 ml of
85.degree. C. buffer solution; wherein complementary nucleic acid
is dissociated and washed away.
29. The method of claim 28 wherein said buffer is sterile and
comprises 1.0 M NaCl and 50 mM sodium phosphate at pH 7.4 in
water.
30. The method of claim 29 wherein said autoclaving is performed
for 20 minutes, at 120.degree. C., and at 4 atm pressure.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 09/446,222 filed Feb. 16, 2000, which is a 35
USC Section 371 national stage application of PCT/CA98/00402, filed
on Apr. 30, 1998, now WO 98/58079, which claims priority to U.S.
Provisional Application No. 60/050970, filed Jun. 19, 1997, and to
Canadian Application No. 2,208165, filed Jun. 18, 1997, all of
which applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is directed generally to biosensors
that are useful in the identification and analysis of biologically
significant nucleic acids. The biosensors of the present invention
and their applied methods provide a means for the direct analysis
of nucleic acid hybridization and, therefore, have application to a
myriad of biological fields including clinical diagnostics.
BACKGROUND OF THE INVENTION
[0003] The detection and identification of microorganisms is a
problem common to many areas of human and veterinary health. For
example, the detection of pathogenic species such as Salmonella
typhimurium, Listeria monocytogenes, and Escherichia coli, which
are causative agents of major food borne epidemics, is a great
concern within the food industry with respect to the quality and
safety of the food supply. In other areas of human and veterinary
health care, detection and identification of infectious diseases
caused by pathogenic microorganisms and viruses is a first step in
diagnosis and treatment. For example, it is estimated that 10-15
million office visits per year are for the detection and treatment
of three major pathogens--Chlamydia ssp., Trichomonas vaginalis and
Gradenerella vaginitis. Infections of these organisms annually
affect 3.75 million, 0.75 million and 1.5 million patients,
respectively.
[0004] Classical techniques routinely used for the detection and
identification of microorganisms are often labor intensive,
involving plating procedures which require lengthy analysis times.
To illustrate, the method currently employed for the detection of
Listeria monocytogenes in food and feed commodities involves a
three stage analysis. The analysis begins with enrichment of the
sample to be analyzed in a nutrient broth for 2 to 4 days. After
the enrichment period, plating of the sample onto selective agar
media is done and the sample is allowed to incubate for 2 days in
order to obtain colonies for biotyping and serotyping, which may
take as long as 20 days to complete (McLauchlin et al., 1988,
Microbiology Review, 55: 578).
[0005] Detection processes based on culturing require analysis
times which are too lengthy for effective monitoring and timely
intervention to prevent the spread of biohazardous materials or
treat disease. In addition, although these methods have been
improved over the last decade, the chance of obtaining false
negative results is still considerable, and many microorganisms are
difficult to culture. Thus, plating/culture methods are limited
with respect to their sensitivity, specificity, and lengthy
analysis times that are required.
[0006] In order to shorten the time required to detect and identify
pathogenic bacteria, viruses and genetic diseases, rapid tests such
as enzyme immunoassays (EIA) have been developed (Olapedo et al.,
1992). Although immunoassay techniques can be very sensitive and
effective, there are practical drawbacks which have restricted the
use of these methods. Such drawbacks include the need for highly
skilled personnel, lengthy analysis and preparation times, and the
large quantities of costly reagents that are required to do such
analysis.
[0007] With the advent of nucleic acid amplification techniques
(the polymerase chain reaction), the in-vitro amplification of
specific sequences from a portion of DNA or RNA is now possible.
Detection of very low numbers of microorganisms has been
demonstrated (Rossen et al., 1991; Golsteyn et al., 1991; Wernars,
K., et al., 1991). The polymerase chain reaction technique is
sensitive and specific but involves complex manipulations in
carrying out the tests and is not particularly well-suited for
large numbers of samples. Due to the sensitivity of Polymerase
Chain Reaction (PCR) technology, special rooms or areas for sample
preparation and analysis are required to prevent contamination. In
many tests PCR results must be confirmed by additional
hybridization analysis. RNAs are difficult to assay by PCR but are
very important for human viral detection. In general, PCR needs to
be automated for acceptance as a practical diagnostic tool.
Hybridization methods require as much as three or four days to
complete results. Although the actual hybridization step can be as
short as 18 hours, the entire detection process of a DNA/DNA hybrid
can take as long as three days with a radioisotope marker.
[0008] Thus, there is a great need for simpler, faster and more
cost-effective means for detecting specific biologically important
RNA and DNA sequences in the fields of human and veterinary
in-vitro diagnostics, food microbiology, and forensic
applications.
[0009] Biosensors developed to date begin to overcome drawbacks
associated with the current state of the art in detecting and
identifying microorganisms. A biosensor is a device which consists
of a biologically active material connected to a transducer that
converts a selective biochemical reaction into a measurable
analytical signal (Thompson et al., 1984. Trends in Analytical
Chemistry, 3: 173; Guilbault, 1991, Current Opinion in
Biotechnology, 2: 3). The advantages offered by biosensors over
other forms of analysis include the ease of use (by non-expert
personnel), low cost, ease of fabrication, small size, ruggedness,
facile interfacing with computers, low detection limits, high
sensitivity, high selectivity, rapid response, and reusability of
the devices.
[0010] Biosensors have been used to selectively detect cells,
viruses, other biologically significant materials, biochemical
reactions and immunological reactions by using detection strategies
that involve immobilization of enzymes, antibodies or other
selective proteins onto solid substrates such as quartz and fused
silica (for piezoelectric and optical sensors) or metal (for
electrochemical sensors) (Andrade et al., 1990, Biosensor
Technology: Fundamentals and Applications, R. P. Buck, W. E.
Hatfield, M. Umana, E. F. Bowden, Eds., Marcel Dekker Inc., New
York, pp. 219; Wise, 1990, Bioinstrumentation: Research,
Developments and Applications, Butterworth Publishers, Stoneham,
Mass.). However, such sensors are not widely available from
commercial sources due to problems associated with the long-term
stability of the selective recognition elements when immobilized
onto solid surfaces (Kallury et al, 1992, Analytical Chemistry, 64:
1062; Krull et al, 1991, Journal of Electron Microscopy Techniques,
18: 212).
[0011] An alternative approach is to create biosensors with
long-term chemical stability. One such approach takes advantage of
the stability of DNA. With the recent advent of DNA probe
technology, a number of selective oligomers which interact with the
DNA of important biological species, for instance salmonella, have
been identified (Symons, 1989, Nucleic Acid Probes, CRC Press, Boca
Raton, Fla.; Bock et al., 1992, Nature, 355: 564; Tay et al., 1992,
Oral Microbiology and Immunology, 7: 344; Sherman et al., 1993,
Bioorganic & Medicinal Chemistry Letters, 3: 469). These have
been used to provide a new type of biorecognition element which is
highly selective, stable, and can be easily synthesized in the
laboratory (Letsinger et al., 1976, Journal of the American
Chemical Society, 98: 3655; Beaucage et al., 1981, Tetrahedron
Letters, 22: 1859; Alvarado-Urbina et al., 1981, Science, 214:
270).
[0012] Until recently, the only other research group in existence
which has published work done on the fluorimetric detection of
nucleic acid hybridization immobilized onto optical substrates is
that of Squirrell et al. (C. R. Graham, D. Leslie, and D. J.
Squirrel, Biosensors and Bioelectronics 7 (1992) 487-493.) In this
work, single-stranded nucleic acid sequences ranging in length from
16-mer oligonucleotides to 204-base oligomers functionalized with
an aminohexyl linker at the 5' terminus were covalently attached to
optical fiber sections functionalized with 3-aminopropyl
triethoxysilane via a gluteraldehyde linkage. All investigations of
nucleic acid hybridization were done by monitoring fluorescence
intensity in an intrinsic mode configuration using complementary
strands which had been previously labeled with a fluorescein
moiety. This yielded a reusable assay system in which signal
generation was observed to occur within minutes and nanomolar
detection was achieved. However, this optical sensor technology
developed by Squirrell et al. does not contain a transduction
element which can transduce the binding event in a reagentless
manner. For this assay to function, the target strands must be
labeled prior to doing the assay in order for detection, making
this technique unsuitable for practical applications.
[0013] Abel and co-workers (Abel, A. P.; Weller, M. G.; Duveneck,
G. L.; Ehrat, M. and Widmer, H. M. Anal. Chem. 1996 68, 2905-2912)
of Norvartis Ltd. (formerly Ciba-Geigy Ltd.) have recently reported
an automated optical biosensor system. Their device utilizes
5'-biotinylated-16-mer oligonucleotide probes bound to an optical
fiber functionalized with avidin to detect complementary
oligonucleotides pre-labeled with fluorescein moieties in a total
internal reflection fluorescence (TIRF) evanescent wave motif
similar to that of Squirrell. Each assay consisted of a 3 minute
pre-equilibration, 15 minute hybridization time, 10 minute washing
procedure followed by a 5 minute regeneration cycle (chemical or
thermal). A chemical denaturation scheme was observed to be the
preferred embodiment for sensor regeneration as exposure of the
oligonucleotide functionalized optical sensor to temperatures
exceeding 52.degree. C. caused irreversible damage to the device,
owing to denaturation of the avidin used for immobilization. This
limitation renders the device function labile against sterilization
techniques, such as autoclaving, and also indicates that rigorous
cleaning of the sensor surface, such as by sonication, would also
compromise the integrity of the sensor via denaturation of the
affinity pair used to anchor the probe oligonucleotide. In order to
detect nucleic acids not pre-labeled with fluorescein, and to
overcome the limitation of Squirrell, a competitive binding assay
was employed by Abel and co-workers. Detection of the unlabelled
analyte was done by pre-treatment of the sensor with fluorescein
labeled "tracer-DNA" followed by monitoring decreases in the
fluorescence intensity of the sensor upon exposure to and
subsequent displacement of the tracer-DNA by complement analyte
nucleic acid. The dose-response curves reported by Abel et al. show
a detection limit of 132 pmol (8.times.10.sup.13 molecules) for
this detection strategy. However, in addition to high detection
limits and the inability of the device to withstand sterilization,
this device cannot be classified as a biosensor technology due to
the necessity for external treatment with tracer-DNA in order to
achieve transduction.
[0014] The prior art with respect to patent literature contains
many examples of "sensor" devices which are based on nucleic acid
molecules immobilized on waveguide supports and transduction
strategies based on evanescent excitation. The technology of Gerdt
and Herr (David. W. Gerdt, John. C. Herr "Fiber Optic Evanescent
Wave Sensor for Immunoassay", U.S. Pat. No. 5,494,798) describes
detection of nucleic acid hybridization based on alterations in the
quantity of light transmitted from one optical fiber in a coupled
fiber system (similar to that of a Mach-Zehnder interferometer) to
the second fiber of the waveguide system. The quantity of light
transferred is a function of the refractive index of the media on
or surrounding the waveguides. Refractive index alterations affect
the penetration depth of the evanescent wave emitted from the first
waveguide into which optical radiation is launched. This standing
wave of electromagnetic radiation subsequently propagates into (and
thus transfers optical radiation to) the second waveguide.
Therefore, the device is sensitive to refractive index alterations
occurring within a volume surrounding the first waveguide with a
thickness of ca. one wavelength of the light propagating within
that waveguide. One of the arms of the waveguide may be
functionalized with immobilized nucleic acid molecules which serves
to provide selective binding moieties. The change in refractive
index of the thin film of nucleic acids on the first waveguide upon
the occurrence of hybridization with target nucleic acid sequences
alters the quantity of light transferred to the second waveguide,
thereby providing a means of signal transduction. Hybridization
events may then be identified based on changes in the output ratios
of the two waveguide arms in the coupled fiber system. One
limitation of this technology lies in the fact that any alterations
in refractive index near the surface of the waveguides will provide
alterations in the output ratios of the two fibers. Therefore,
non-specific binding events (such as protein adsorption) will
provide false positive results.
[0015] In order to avoid the problem of interferents providing
false positive results, a transduction strategy which is sensitive
to the structure of the binding pair (i.e. recognition element and
target) is required. The technologies of Fodor, Squirrell (David
James Squirrell "Gene Probe Biosensor Method" International
Application Number PCT/GB92/01698, International Publication Number
WO 93/06241, International Publication Date: 1 Apr. 1993.),
Sutherland etal. (Ranald Macdonald Sutherland, Peter Bromley and
Bernanrd Gentile "Analytical Method for Detecting and Measuring
Specifically Sequenced Nucleic Acid." European Patent Application
Number 87810274.8, Publication Number 0 245 206 A1, Date of Filing:
30 Apr. 1987.), Hirschfeld (Tomas B. Hirschfeld, "Nucleic Acid
Assay Method" U.S. Pat. No. 5,242,797, Date of Patent: 7 Sep.
1993.), and Abel et al. (Andreas P. Abel, Michael G. Weller, Gert
L. Deveneck, Markus Ehrat, and H. Michael Widmer, Analytical
Chemistry, 1996, 68, 2905-2912.) overcome this limitation by using
fluorescent probes which associate with the binding pair or are
attached to selective binding moieties capable of binding to a
portion of the binding pair. These inventions provide methods to
measure nucleic acid hybridization on waveguide surfaces based on
evanescent excitation and TIRF. In each embodiment, an
oligonucleotide probe capable of selective binding to a target
sequence is covalently immobilized on a waveguide surface. For the
cases of Squirrell and Abel et al, each define two preferred
embodiments for the detection of hybridization events. The first
embodiment of Squirrell and Abel et al. are essentially identical
wherein the target nucleic acid is functionalized with a
fluorescently detectable agent (by chemical or enzymatic methods)
as a first step prior to detection. Upon hybridization between the
labeled target and immobilized nucleic acid, the fluorescent agent
is then bound in close proximity to the waveguide surface where it
may be excited by evanescent wave formation and emission from the
fluorophore collected and quantitatively measured. In the second
preferred embodiment of Squirrell, hybridization between the
immobilized oligonucleotide and the target sequence is first done.
Subsequent to the first hybridization event, a fluorescently
labeled oligonucleotide present in the system may then undergo
hybridization with all or a portion of the remainder of the target
sequence not hybridized with the immobilized sequence. The binding
of the third (labeled) oligonucleotide provides a fluorescent
species bound in close proximity to the waveguide which may furnish
transduction via evanescent excitation and collection of the
emitted radiation. In the second embodiment of Abel et al., a
method for the detection of nucleic acids not pre-labeled with a
fluorescent moiety via a competitive binding assay is described.
Detection of the unlabelled analyte was done by first pre-treating
the optical sensor with immobilized probe nucleic acid with
fluorescein labeled "tracer-DNA". The quantity of tracer-DNA may be
monitored via the evanescent excitation and collection motif.
Binding of the analyte could be followed by monitoring decreases in
the fluorescence intensity from the sensor as a function of the
displacement of the tracer-DNA via competitive binding with
non-fluorescent analyte nucleic acid in a dose-response
convention.
[0016] In the methods of Sutherland et al. and Hirschfeld,
transduction of hybridization events is provided by fluorescent
intercalating dyes (e.g. ethidium bromide). Following hybridization
between the single-stranded target and immobilized probe nucleic
acids, intercalant fluorescent dye molecules from solution insert
into the base stacking regions of the immobilized double-stranded
nucleic acid. An increase in the fluorescence quantum efficiency,
fluorescence lifetime, stokes shift of the fluorescent intercalant
probes often occurs upon association with double-stranded nucleic
acid. It is claimed by the inventors that these enhanced features
may be monitored by evanescent excitation and collection of
fluorescence emission.
[0017] Fodor et al. have employed light-directed chemical synthesis
to generate miniaturized, high density arrays of oligonucleotide
probes. DNA oligonucleotide arrays have been fabricated using
high-resolution photolithography in combination with solid-phase
oligonucleotide synthesis. This form of DNA chip technology may be
used for parallel DNA hybridization analysis, directly yielding
sequence information from genomic DNA segments. Prior to sequence
identification, the nucleic acid targets must be fluorescently
labeled, either prior to or after hybridization to the
oligonucleotide array, via direct chemical modification of the
target strand or by use of an intercalant dye subsequent to
hybridization on the DNA chip. The hybridization pattern, as
determined by fluorescence microscopy, is then deconvolved by
appropriate chemometric processing to reveal the sequence of the
target nucleic acid. Rather than focusing on selective detection of
trace quantities of a particular nucleic acid sequence, this
technology has focused on sequence analysis of nucleic acids in
suitably high copy number so as to sufficiently occupy the
oligonucleotide array.
[0018] Notwithstanding the indubitable accomplishments of the
aforementioned prior art, there yet exists limitations in these
technologies for which further improvements are most desirous.
Although the strategies employed by Sutherland et al. and
Hirschfeld overcome the limitations of Gerdt and Herr with regard
to signal origin and the generation of false positive results,
these assay methods are limited by the amount of signal which can
be generated by evanescent excitation. For multimode waveguides,
less than 0.01% of the optical radiation carried within the
waveguide is exposed to the outer medium in the form of an
evanescent wave (R. B. Thompson and F. S. Ligler, "Chemistry and
Technology of Evanescent Wave Biosensors" in Biosensors with
Fiberoptics, Eds.: Wise and Wingard, Humana Press Inc., New Jersey,
1991, pp.111-138.). In the case where monomodal waveguides are
used, ca.10% of the radiation carried by the waveguide is exposed
to the outer medium in the form of an evanescent wave (David. W.
Gerdt, John. C. Herr "Fiber Optic Evanescent Wave Sensor for
Immunoassay", U.S. Pat. No. 5,494,798). In the classic total
internal reflection fluorescence (TIRF) evanescent wave
configuration, the critical angle (.theta..sub.c) for the
waveguide/solution interface (.theta..sub.c.sup.W/S) is larger than
.theta..sub.c for the waveguide/biological film interface
(.theta..sub.c.sup.W/B), only the evanescent component of the
propagated radiation will enter the biological film. The principle
of optical reciprocity states that light coupled back into a
waveguide as a plane wave will be in the same way as the primary
process when a plane wave generates an evanescent wave (Ranald
Macdonald Sutherland, Peter Bromley and Bernanrd Gentile
"Analytical Method for Detecting and Measuring Specifically
Sequenced Nucleic Acid" European Patent Application Number
87810274.8, Publication Number 0 245 206 A1, Date of Filing: 30
Apr. 1987, p.13.). Thus, for the fluorophores excited by evanescent
waves created from modes propagating at or near
.theta..sub.c.sup.W/S, none of the fluorescence emission can be
coupled back into the waveguide in the same propagation mode as
.theta..sub.c.sup.W/S would be >90.degree. (U. J. Krull, R. S.
Brown and E. T. Vandenberg, "Fiber Optic Chemoreception" in Fiber
Optic Chemical Sensors and Biosensors, vol.2, Ed. O. S. Wolfbeis,
CRC Press, Boca Raton, 1991, pp.315-340.). Hence a large portion of
the signal would be lost to the surroundings for systems in which
fluorescence emission originates from thin films of a lower
refractive index than that of the waveguide onto which they are
immobilized. It has been shown by Love et al. that under optimal
conditions, only 2% of the light emitted by the fluorophore in the
medium of lower refractive index may be captured and guided by the
fiber (W. F. Love, L. J. Button and R. E. Slovacek, "Optical
Characteristics of Fiberoptic Evanescent Wave Sensors: Theory and
Experiment" in Biosensors with Fiberoptics Eds.: Wise and Wingard,
Humana Press Inc., New Jersey, 1991, pp.139-180.).
SUMMARY OF THE INVENTION
[0019] The present invention concerns biosensors for direct
detection of nucleic acids and nucleic acid analogs. The device
comprises a light source, a detector, and an optical element for
receiving light from the source and conveying it to an interaction
surface of the optical element. A nucleic acid or nucleic acid
analog for a particular nucleic acid sequence or structure (i.e.
which is complementary to the target nucleic acid(s)), is
immobilized onto the interaction surface of the optical element.
Fluorescent ligands are provided that will bind into or onto the
hybridized nucleic acid complex and fluoresce when stimulated by
the light source. Subsequent to excitation by electromagnetic
radiation of suitable wavelength bound within the optical element,
the resultant fluorescence is collected within the optical element
and guided to the detector to signal that the target nucleic
acid(s) has complexed with the immobilized probe and thus indicate
the presence of the target in the sample. An interaction surface is
defined to mean a surface of the optical element on which nucleic
acid is immobilized, and at which the fluorescent molecules
interact with the light.
[0020] This invention provides biosensors in which the interaction
surface is functionalized with nucleic acid probe sequences such
that the index of refraction of the immobilized layer (Substrate
Linker/Nucleic Acid/Fluorescent Ligand) is equal to or greater than
the refractive index at the surface of the waveguide such that the
organic coating becomes an extension of the waveguide. The index of
refraction of the immobilized layer is dependent, at least in part,
on the loading of immobilized molecules and linkers on the surface
and the chemical nature of the immobilized molecules and any
linkers.
[0021] Preferred biosensors which offer high-sensitivity and
low-detection limits may be realized by activating the interaction
surface of an optical element with substrate linker molecules of at
least about 25 .ANG. (Angstrom) in length followed by attachment of
a selected probe nucleic acid sequence to that linker. (A probe
nucleic acid is, at least in part, complementary to a target
nucleic acid.) The preferred method for attachment of the probe
nucleic acid to the substrate linker is by in-situ synthesis of the
nucleic acid sequence onto the linker terminus using solid-phase
nucleic acid synthesis methods or routine modifications of thereof.
Such methods of in-situ synthesis are particularly useful for
immobilization of nucleic acids of 50 or fewer bases and more
particularly useful for nucleic acids of 30 or fewer bases.
[0022] The fluorophore may be tethered to the immobilized DNA, for
example, by use of a hydrocarbon tether. The use of tethered probes
can significantly reduce biosensor response time as the response
mechanism is not diffusionally controlled. The associated
fluorophore provides for internal calibration of optical source
intensity and detector drift. It also provides for calibration of
photobleaching, and provides for internal calibration by monitoring
bound against free dye by use of, for example, time-resolved
fluorescence measurements.
[0023] The optical element preferably comprises an optical
waveguide which also conveys the fluorescent light to the detector.
The optical waveguide preferably conveys the emitted light by total
internal reflection to the detector. The optical waveguide can
comprise an optical fiber, a channel waveguide, or a substrate that
confines light by total internal reflection. The fluorescent
molecules preferably provide sufficient Stokes shift such that the
wavelength of the light source and the wavelength of the
fluorescent light are easily separated. The fluorescent molecules
can be provided in a solution in which the optical element is
immersed, or by a tether to the nucleic acid that is immobilized to
the linker.
[0024] In the practice of the present invention, the light source
can be any suitable source such as a gas laser, solid state laser,
semiconductor laser, a light emitting diode, or white light source.
The detector can be any suitable detector such as a photomultiplier
tube, an avalanche photodiode, an image intensifier, multi-channel
plate, or semiconductor detector. The biosensor system can be a
multi-wavelength, multi-fluorescent system. The light coupling of
the system can also be modified to allow a multitude of disposable
biosensors to be analyzed either sequentially or in parallel.
[0025] The biosensor system of the present invention can be
constructed and used to detect each of a mixture of target nucleic
acids (for example, Chlamydia and Gonorrhea in urogenital
infections or E. coli and Salmonella during food processing). This
may be done by using a plurality of fluorophores (which, for
example, fluoresce at different wavelengths), each of which is
tethered to an immobilized nucleic acid probe that is
characteristic of or specific for detection of a given species or
strain. In this example, the observed wavelength(s) of fluorescence
emission will then be specific for hybridization of a given target
nucleic acid to its complementary immobilized probe.
[0026] The biosensors of the present invention have an improved
detection limit and sensitivity with respect to the prior art and
are shown to be stable over prolonged storage and severe washing
and sterilization conditions. Sensors stored over 1 year in vacuo,
in 1:1 ethanol/water solutions, absolute ethanol, or dry at
-20.degree. C. provide identical response characteristics to those
freshly prepared. Adsorbed fluorescent contaminants accumulated
through storage can be removed (as confirmed through fluorescence
microscopy investigations) by sonicating the biosensors in 1:1
ethanol/water where the sensitivity of the device has consistently
been observed to increase by a factor of c.a. 2.5 from this
pre-treatment with respect to that of freshly prepared biosensors
not cleaned before use. Unlike those of the prior art (e.g. Abel et
al.), the optical biosensors of the present invention have also
shown to be thermally stable wherein device function is maintained
after sterilization by autoclaving (20 minutes, 120.degree. C., 4
atmospheres over-pressure). The ability to clean and sterilize a
biosensor device so that it may be usable in an on-line
configuration and/or in clinical applications is a significant
advantage yet realized only by the technology reported herein.
Biosensors of this invention also allow for more rapid sample
analysis with improved response time for signal generation.
[0027] The present invention also provides a recyclable or
disposable biosensor for detecting a target nucleic acid, which
biosensor includes an optical element for receiving and conveying
light to an interaction surface of the optical element and nucleic
acid, for a particular nucleic acid sequence which is complementary
to the target nucleic acid, immobilized onto the interaction
surface of the optical element. The recyclable or disposable
biosensor preferably comprises an optical waveguide, which
preferably conveys the light by total internal reflection to the
interaction surface of the optical waveguide when the organic
coating is of equal or higher refractive index in comparison to the
surface of the waveguide. The optical waveguide preferably
comprises an optical fiber. Fluorescent molecules are provided in a
solution in which the recyclable or disposable biosensor is
immersed that will bind upon hybridization of the immobilized
nucleic acid with complementary target nucleic acid and fluoresce
when stimulated by light. Alternatively, the fluorescent molecules
are provided bound by a tether to the immobilized nucleic acid.
[0028] The present invention provides biosensors for direct
analysis of nucleic acid hybridization by use of an optical
substrate such as an optical wafer or an optical fiber, and nucleic
acids or nucleic acid analogs which have been immobilized onto the
optical substrate. Generation of a fluorescence signal upon
hybridization to complementary nucleic acids and nucleic acid
analogs in a sample may be achieved in a number of different ways.
Biosensors of this invention are sufficiently sensitive to directly
detect very small quantities of target nucleic acids in a sample
without the need to employ nucleic acid amplification methods such
as PCR techniques. Biosensors of this invention can have detection
limits for target nucleic acids below 10.sup.6 molecules.
[0029] The optical biosensor comprises nucleic acid strands or
nucleic acid analogs of a specific selected sequence immobilized
onto activated optical supports. The selected immobilized sequences
are capable of binding to target sequences, including sequences
characteristic of and selective for viruses, bacteria, or other
microorganisms as well as of genetic disorders or other conditions.
Biosensors having such characteristic or selective immobilized
sequences are useful for the rapid screening of genetic disorders,
viruses, pathogenic bacteria and in biotechnology applications such
as the monitoring of cell cultures and gene expression. One
important avenue which has been widely ignored by the nucleic acid
biosensor community is the investigation of multi-stranded
(.gtoreq.3) nucleic acid formation. For example, triple-helical
oligonucleotides have been reported to offer potential use as:
sequence-specific artificial nucleases ({a} Moser, H. E.; Dervan,
P. B. Science, 1987, 238, 645. {b} Strobel, S. A.; Doucettestamm,
L. A.; Riba, L.; Housman, D. E.; Dervan, P. B. Science, 1991, 254,
1639.), DNA-binding protein modulators/gene expression regulators
({a} Cooney, M.; Czernuszewicz, Postel, E. H.; Flint, S. J.; Hogan,
M. E. Science, 1988, 241, 456. {b} Durland, R. H.; Kessler, D. J.,
Gunnel, S., Duvic, M.; Pettit, B. M.; Hogan, M. E.; Biochem., 1991,
30, 9246. {c} Maher, L. J.; Dervan, P. B.; Wold, B.; Biochemistry,
1992, 31, 70. {d} Maher, L. J. BioEssays, 1992, 14, 807. {e} Maher,
L. J. Biochemistry, 1992, 31, 7587. {f} Duvalvalentin, G.; Thoung,
N. T.; Hlne, C. Proc. Nat. Acad. Sci. USA, 1992, 89, 504. {g} Lu,
G.; Ferl, R. J. Int. J. Biochem., 1993, 25, 1529.), materials for
genomic mapping ({a} Ito, T., Smith, C. L.; Cantor, C. R. Proc.
Natl. Acad. Sci. U.S.A., 1992, 89, 495. {b} Ito, T., Smith, C. L.;
Cantor, C. R. Nucleic Acids Res. 1992, 20, 3524.), and highly
selective screening reagents to detect mutations within duplex DNA
(Wang, S. H., Friedman, A. E., Kool, E. T. (1995) Biochemistry 34,
9774-9784.). The present invention can also be used to detect the
formation of multi-stranded nucleic acid hybrids (for example,
formation triple-helical nucleic acids), and therefore could, for
example, operate to monitor the effectiveness, dose dependence and
intracellular concentration of nucleic acid pharmaceuticals used in
gene therapy applications or as an assay to identify multi-strand
formation associated with any of the aforementioned potential
applications associated with triple-helical oligonucleotides.
[0030] The invention is a biosensor system for detecting a target
nucleic acid, which consists of at least three layers, two of which
are a waveguide, wherein one layer includes a nucleic acid or
nucleic acid analog capable of hybridizing to the target nucleic
acid, and wherein a fluorophore is tethered to the nucleic acid or
nucleic acid analog and wherein the biosensor functions according
to direct excitation. The invention also relates to a biosensor for
detecting a target nucleic acid, which comprises an inner layer, a
middle layer and an outer layer, wherein
[0031] the inner layer has refractive index n.sub.1,
[0032] the middle layer includes a nucleic acid or nucleic acid
analog capable of hybridizing to the target nucleic acid and has
refractive index n.sub.2, which is greater than or equal to
refractive index n.sub.1, and
[0033] the outer layer has refractive index n.sub.3, which is less
than refractive index n.sub.2.
[0034] and wherein a fluorophore is tethered to the nucleic acid or
nucleic acid analog of the middle layer and wherein the biosensor
functions according to direct excitation.
[0035] In a preferred embodiment, the inner layer is an optical
fiber or optical wafer and the outer layer is an ambient. The outer
layer is an aqueous based solution. The biosensor is useful for
detection of triplex formation or multi-stranded nucleic acid
formation. The triplex formation preferably involves a branched
antisense nucleic acid which inhibits expression of a target
nucleic acid sequence by triplex formation with the sequence.
[0036] The biosensor is useful for detection of nucleic acids of
bacteria, viruses, fungi, unicellular or multicellular organisms or
for the screening of nucleic acids of cells, cellular homogenates,
tissues or organs.
[0037] Preferably, a fluorophore is tethered to a nucleic acid or
nucleic acid analog which is one of the layers of a biosensor
having at least three layers and the biosensor functions according
to direct excitation. The invention also includes the use of a
fluorophore for detecting a target nucleic acid.
[0038] The invention also relates to a method of detecting a target
nucleic acid, comprising:
[0039] pre-treating a sample so that target nucleic acids
characteristic of or selective for said sample are available for
hybridization;
[0040] contacting the sample with the middle layer of the biosensor
of claim 2, such that the target nucleic acids can hybridize to the
nucleic acids or nucleic acid analogs of the middle layer;
[0041] allowing the fluorophore tethered to the nucleic acids of
the middle layer to bind upon hybridization of the target nucleic
acids with the nucleic acids or nucleic analogs of the second
layer;
[0042] illuminating the fluorescent molecules with light such that
fluorescence is stimulated; and
[0043] detecting the emitted fluorescence,
[0044] whereby the presence of the target nucleic acid is
detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1(a). Synthetic scheme of Arnold et al. used to
activate the glass or fused silica surfaces with long chain
aliphatic spacer molecules terminated with
5'-O-dimethoxytrityl-2'-deoxythymidine.
[0046] FIG. 1(b). Synthetic scheme of Brennan et al. used to create
alkylamine substrate linker molecules on hydroxylated fused silica
surfaces.
[0047] FIG. 1(c). Synthetic scheme of Maskos and Southern used to
functionalize hydroxylated fused silica surfaces with GOPS followed
by extension with HEG.
[0048] FIG. 1(d). Possible closed loop structure formation as a
consequence of the synthesis scheme used in FIG. 1(c).
[0049] FIG. 1(e). Synthetic scheme used to extend GOPS
functionalized substrates with DMT-HEG via a base catalyzed
mechanism.
[0050] FIG. 1(f). Synthetic scheme used to covalently link DMT-HEG
onto hydroxylated fused silica surfaces via activation with
methanesulfonyl chloride.
[0051] FIG. 2. The phenoxyacetyl protecting group used for
exocyclic amine (R) protection on nucleoside phosphoramidite
synthons.
[0052] FIG. 3(a). Synthetic scheme used to create a
hydrocarbon-tethered analogue of Ethidium Bromide.
[0053] FIG. 3(b). Synthetic scheme used to create a
polyether-tethered phosphoramidite analogue of Ethidium
Bromide.
[0054] FIG. 3(b). Synthetic scheme used to create a
polyether-tethered analogue of the bis-intercalative fluorescent
probe YOYO-1. Removal of the DMT protecting group followed by
treatment with .beta.-cyanoethyl-N,N-diisopropyl phosphityl
chloride will yield the tethered YOYO-1 phosphoramidite
synthon.
[0055] FIG. 4(a). Schematic diagram of one embodiment of an
apparatus used to measure fluorescence intensity from optical
fibers coated with immobilized DNA.
[0056] FIG. 4(b). Schematic diagram an example of a dedicated
instrument for analysis of nucleic acid samples by the fiber-optic
nucleic acid biosensor of the present invention.
[0057] FIG. 4(c). Schematic representation of a biosensor system in
which light from a suitable source is directed through a dichroic
mirror beam splitter and focused onto a fiber or waveguide coupler
and then into an optical fiber having single-stranded nucleic acid
bound to the surface thereof, and in which any resultant
fluorescent light travels back through the coupler, and passes
through the beam splitter and is directed to a photomultiplier
detector.
[0058] FIG. 5. Illustration of the operating principles of the
fiber-optic nucleic acid biosensor. Hybridization of complement
single-stranded oligonucleotide from solution with immobilized
nucleic acid probe on biosensor is followed by intercalation of the
tethered fluorescent ligand which provides transduction of the
selective binding process into a measurable analytical signal.
[0059] FIG. 6. Fluorescent intensity as a function of temperature
for the mixed base sequence icosanucleotide functionalized fibers.
Upper Curve: response of the optical sensor to 20 pmol of linear
complement icosanucleotide in the presence of 2.5.times.10.sup.-8M
ethidium bromide. Lower Curve: response of the optical sensor to
2.5.times.10.sup.-8 M ethidium bromide.
[0060] FIG. 7.(a) A Model of parallel (T*AT) triplex formation
using dT.sub.10 and an optical biosensor functionalized with
immobilized dA.sub.10. dT.sub.10:dA.sub.10 duplex is first formed
upon cooling the system below the duplex T.sub.m followed by
formation of the triple-stranded complex with further cooling below
the T.sub.m for triplex formation. (b) The dA.sub.10 of the optical
sensor capturing the branched "V" compound 1 (see FIG. 15). Note
how the fluorescent probe is excluded from the triplex as the
temperature is cooled.
[0061] FIG. 8. Quantity of trityl cation released during each
detritylation step of the automated phosphoramidite synthesis of
dT.sub.20 onto fused silica optical fibers functionalized by the
protocols of examples 1 and 5.
[0062] FIG. 9(a). Response characteristics of an optical biosensor
to complement and non-complement DNA.
[0063] FIG. 9(b). Response characteristics of an optical biosensor
to 570 ng.multidot.ml.sup.-1 of complement RNA.
[0064] FIG. 10. Response time of the optical sensor constructed as
per the protocols in examples 1 and 5 and effect of ethidium
bromide incubation time.
[0065] FIG. 11. Response of a DNA optical biosensor (a) after
storage for one month used without cleaning and (b) after storage
for eleven months and cleaned by sonication in ethanol for 10
minutes. Note: A 1-month-old sensor which had been cleaned by
sonication (data not shown) provided a response similar to (b).
[0066] FIG. 12. Thermal denaturation profiles of aqueous
dA.sub.20+dT.sub.20 and immobilized dT.sub.20 with aqueous
dA.sub.20.
[0067] FIG. 13. Response of the optical sensor with immobilized
nucleic acid probe for Candida albicans to complement DNA.
[0068] FIG. 14. Response of a reagentless biosensor as described in
Example 14. The graph measures fluorescence from the tethered dye
on the terminus of the immobilized nucleic acid as a function of
time after exposure to a sample of 720 ng of cDNA.
[0069] FIG. 15. The structures of dT.sub.10 and compound 1, a
branched oligonucleotide with identical oligo(thymidine) chains
linked to the 2'- and 3'-positions of a ribose branch-point
nucleoside i.e., rA.sub.3'.fwdarw.5'-dT10.sup.2'.fwdarw.5'-dT10
binds to dA.sub.10 to yield a triple-stranded complex containing
only T.circle-solid.AT (reverse Hoogsteen-Watson/Crick) base
triplets.
[0070] FIG. 16(a). Response (.circle-solid.) of the optical sensor
with a 5'-end terminated recognition sequence to 40 pmol of linear
dT.sub.10 in the presence of 2.5.times.10.sup.-8M ethidium bromide.
Response (X) of the optical sensor to 2.5.times.10.sup.-8 M
ethidium bromide and no dT.sub.10. . . . Melting profile of the
same nucleic acid system in bulk solution by measurement of
absorbance (260 nm) in 10 mM TRIS and 50 mM MgCl.sub.2 at pH
7.3.
[0071] FIG. 16(b). Response (.circle-solid.) of the optical sensor
with a 3'-end terminated recognition sequence to 40 pmol of linear
dT.sub.10 in the presence of 2.5.times.10.sup.-8M ethidium
bromide.
[0072] Response (X) of the optical sensor to 2.5.times.10.sup.-8 M
ethidium bromide and no dT.sub.10. . . . Melting profile of the
same nucleic acid system in bulk solution by measurement of
absorbance (260 nm) in 10 mM TRIS and 50 mM MgCl.sub.2 at pH
7.3.
[0073] FIG. 16(c). Response (.circle-solid.) of the optical sensor
with a 3'-end terminated Recognition Sequence to 40 pmol of 1 (see
FIG. 15) in the presence of 2.5.times.10.sup.-8M ethidium bromide.
Response (.circle-solid.) of the optical sensor to
2.5.times.10.sup.-8 M ethidium bromide with no 1. . . . Melting
profile of the same nucleic acid system in bulk solution by
measurement of absorbance (260 nm) in 10 mM TRIS and 50 mM
MgCl.sub.2 at pH 7.3.
[0074] FIG. 17(a). Photograph of a UV-shadowed native
polyacrylamide gel containing single strands, duplex and triple
helical complexes of branched and linear controls. DNA samples were
loaded in 50 mM MgCl.sub.2, and 30% sucrose. Lanes 4-10 are
dT.sub.10, dT.sub.10:dA.sub.10 (1:1), dT.sub.10:dA.sub.10 (2.5:1),
dT.sub.10:dA.sub.10(4:1), dA.sub.10, 1+dA.sub.10, and 1,
respectively. As can be noted the dT.sub.10:dA.sub.10 triplex (lane
7) showed a greater retardation in the mobility relative to the
corresponding duplex (lanes 5 and 6). The slowest mobility was
observed in lane 9 for 1:dA.sub.10. Note: See FIG. 15 for the
structure of 1.
[0075] FIG. 17(b). Photograph of an ethidium bromide stained native
polyacrylamide gel (same gel as FIG. 17{a}) containing single
strands, duplex and triple helical complexes of branched and linear
controls. DNA samples were loaded in 50 mM MgCl.sub.2, and 30%
sucrose. Lanes 4-10 are dT.sub.10, dT.sub.10:dA.sub.10 (1:1),
dT.sub.10:dA.sub.10 (2.5:1), dT.sub.10:dA.sub.10(4:1), dA.sub.10,
1+dA.sub.10, and 1, respectively. As can be noted the
dT.sub.10:dA.sub.10 triplex (lane 7) showed a slight retardation in
the mobility relative to the corresponding duplex (lanes 5 and 6).
The slowest mobility was observed in lane 9 for 1:dA.sub.10. Notice
that only the duplexes and triplexes showed ethidium bromide
fluorescence. Note: See FIG. 15 for the structure of 1.
[0076] FIG. 18. Schematic diagram illustrating the experimental
concept for light scattering investigations of a two-layer system
with n.sub.Fused Silica>n.sub.Film.
[0077] FIG. 19. Schematic diagram illustrating the experimental
concept for light scattering investigations of a three-layer system
with n.sub.Fused Silica>n.sub.film>n.sub.Ambient.
[0078] FIG. 20. Schematic diagram illustrating the experimental
concept for light scattering investigations of a three-layer system
with n.sub.Fused Silica<n.sub.Film>n.sub.Ambient.
[0079] FIG. 21. Schematic diagram of the instrument used for
investigations of angularly dependent light scatter.
[0080] FIG. 22. Control experiments for the Angularly Dependent
Light Scattering Technique Using Substances of Known Refractive
Index.
[0081] FIG. 23. Results of the light scattering experiments done
with substrates coated with a thin organic films.
[0082] FIG. 24. Results of the light scattering experiments done
with substrates coated with covalently immobilized
oligonucleotides.
DETAILED DESCRIPTION OF THE INVENTION
[0083] The invention relates to a biosensor which functions
according to an intrinsic mode of operation. Using the chemistries
as disclosed in this patent application) for attaching linker
molecules onto optical waveguide supports (preferably optical
fibers) and an automated DNA synthesizer, control over the
orientation and a wide range of oligonucleotide packing densities
on the waveguide is afforded. In this way, immobilized films of
oligonucleotides of desired refractive index may be constructed on
waveguide supports so that the oligonucleotide film is made to be
an extension of the waveguide. This intrinsic mode of operation
provides a highly efficient means of signal generation and
collection where fluorescence excitation and emission occur within
the waveguide itself, providing an expected enhancement in
sensitivity and lowering of detection limits by six orders of
magnitude.
[0084] The second major improvement provided by our technology is
the use of fluorescent dyes tethered to or otherwise associated
with the immobilized oligonucleotide. Thompson and Krull ({a} M.
Thompson and U. J. Krull, Trends in Analytical Chemistry, 3 (1984)
173-178. {b} M. Thompson and U. J. Krull, Analytical Chemistry, 63
(1991) 393A-405A.) teach that biosensors may be defined as devices
which consists of a biorecognition element and a transduction
element. The biorecognition element may be a biological material
capable of participating in highly selective binding to a target,
usually a biologically significant molecule. The transduction
element converts the selective binding reaction into a measurable
analytical signal. The transduction strategy of Gerdt is too
non-selective for the technology to be classified as a biosensor
whereas the devices of Fodor, Squirrell, Abel et al., Sutherland et
al. and Hirschfeld do not contain a transduction element at all. In
addition to the requirement for external reagent treatment, in the
cases of Fodor, Sutherland et al., and Hirschfeld, there also
exists the extra shortcoming that all intercalant dyes are known or
suspected mutagens. Therefore, the troublesome issues of collection
and disposal of hazardous chemical waste exists subsequent to each
analysis. By associating the transduction element with the
biorecognition element, the device may function without the need
for external reagent treatment and obviated the need to collect and
dispose of hazardous waste. Such a technology then readily lends
itself to automated and in-line analysis and precludes the need for
skilled technicians to partake in the analysis procedure or
disposal of waste (provided the sample itself is not
biohazardous).
[0085] The other advantage provided by the incorporated dye is
internal calibration. More specifically, three key advantages may
be realized: 1) the associated dye provides a means to determine
the quantity of fluorophore and immobilized nucleic acid on the
waveguide; 2) the fluorophore in the presence of single-stranded
nucleic acid provides a baseline signal to which all signals can be
referenced, hence providing meaningful analytical data; and 3) the
useful lifetime of the device can be determined from alterations in
the background fluorescence signal from the incorporated
fluorophore over time. Therefore, by including the associated
fluorescence transduction unit, an internal reference marker and
diagnostic tool for the device status is included as an integral
part of the optical biosensor.
[0086] Nucleic acid oligomers are covalently immobilized onto
optical fibers by first activating the surface of the optical fiber
with a long chain spacer arm terminated by a chemically protected
terminus, normally a dimethoxytrityl (DMT) moiety, followed by
automated solid-phase DNA synthesis. Detection of nucleic acids or
nucleic acid analogs at the fiber surface after hybridization
between immobilized nucleic acid and its complementary nucleic acid
is achieved by measuring enhanced fluorescence emission of the
fluorophore.
[0087] The optical fiber may be activated with a number of
different compounds. The method of Arnold and co-workers (Arnold et
al., 1989, Collect. Czech. Chem. Commun., 54: 523) may be used for
the activation of the fused silica wafers, optical waveguides, and
optical fibers whereby 25 atom-long spacer molecules terminated by
a dimethoxytrityl protected nucleoside are immobilized onto the
cleaned optical fiber substrate, as illustrated in FIG. 1(a). In
this method, the length of the spacer between the substrate and the
first nucleoside is sufficiently long so that the environment of
the terminal nucleoside is fluid enough to permit efficient
coupling with successive nucleotide monomers during automated
phosphoramidite synthesis of the immobilized nucleic acid probe.
This is in accord with the report of Beaucage et aL (1992,
Tetrahedron, 48: 2223-2311) wherein it was stated that substrate
linkers of lengths of at least 25 atoms are required to achieve
high (.gtoreq.99.5%) synthon coupling yields. The synthetic scheme
of Arnold et al. requires inexpensive chemicals, is facile to
perform, and is done as a one pot procedure wherein product
isolation and purification is obviated. Because the linker is
terminated by a protected nucleoside, any reactive sites on the
support which would lead to the production of unwanted side
products during automated synthesis can be eliminated by treating
the derivatized supports with acetic anhydride prior to synthesis.
Lastly, the coverage of linker on the support is easily determined
by determining the amount of trityl cation released during the
first trichloroacetic (TCA) deprotection step of the automated
synthesis. This methodology does however place limits on the types
of nucleobase protecting chemistries can be used as treatment with
strong base will cleave the succinate bond between the substrate
linker and the oligonucleotide probe.
[0088] An amine-terminated solid support suitable for automated
oligonucleotide synthesis may be prepared according to the method
of Brennan et al. (1993, Sensors and Actuators B, 11: 109). A
bifunctional amphiphilic support derivatization agent is created by
condensing .gamma.-aminopropyltriethoxysilane (APTES) with
12-nitrododecanoic acid. The resulting long chain spacer molecule
is covalently immobilized onto the surface of the optical fibers by
an S.sub.n2 reaction between the hydroxyl groups present at the
surface of the fiber and the silane moiety of the amphiphile. With
the terminus of the substrate linker in the non-reactive
nitro-form, the support may then be capped using standard methods
employed during automated synthesis (acetic anhydride), or with
chlorotrimethylsilane (R. T. Pon Methods in Molecular Biology,
Vol.20: Protocols for Oligonucleotides and Analogs, S. Agrawa, Ed,
1993, Humana Press, Inc. Totowa N.J.), thereby masking other sites
of reaction which may produce unwanted side products during
oligonucleotide synthesis. Reduction of the terminal
nitro-functionalities is then achieved by treatment of the
derivatized support with an acidic zinc solution. The resulting
amine headgroups may then be used directly for automated synthesis
wherein an ammonolysis/base resistant phosphoramidate linkage is
made between the activated support and the first nucleotide. An
outline of a synthetic procedure used to immobilize alkyl amine
monolayers covalently onto fused silica substrates is depicted in
FIG. 1(b).
[0089] The hydrolysis resistant linkage of Maskos and Southern may
also be employed to provide waveguides functionalized with
substrate linkers. Analogous to the natural internucleotidic
linkage, a phosphodiester linkage between the substrate linker and
first nucleotide is completely resistant to ammonolysis under the
conditions which remove standard base-protecting groups. This
linkage is produced by derivatization of optical fibers with the
bifunctional silylating reagent 3-glycidoxypropyltrimethoxy silane
via silyl-ether bond formation with the hydroxylated waveguide
surface. This yields a substrate derivatized with short spacer
molecules with terminal epoxide moieties. The length of the spacer
arm is then extended by nucleophilic attack of a polyether, such as
hexaethylene glycol (HEG), in an acid catalyzed expoxide
ring-opening reaction, yielding a stable ether linkage (U. Maskos
and E. M. Southern, 1992 Nucl. Acids Res., 20(7). 1679), as shown
in FIG. 1(c). Polyether chains provide for hydration, flexibility
for molecular motion, and improved biocompatibility in terms of
minimization of non-selective binding to biological compounds. By
extending the spacer molecule ensemble to one composed of at least
25 atoms, optimal phosphoramidite synthon coupling efficiencies are
realized (Beaucage et al., 1992 Tetrahedron, 1992 48, 2223). This
support, terminated with a hydroxyl functionality, is then used
directly for automated oligonucleotide synthesis, obviating the
need for tedious nucleotide functionalization of the support.
[0090] Since polyethylene glycols are bifunctional, there exists
the possibility of creating non-reactive closed-loop structures
which may significantly decrease the amount of loading of
oligonucleotides on the surface of an optical fiber, as shown in
FIG. 1(d). To eliminate any such problem and improve upon the prior
art, one terminus of the polyether is protected with a suitable
blocking group, for example, with a DMT functionality, prior to
extension of the glycidoxypropyltrimethyl silane. In the case where
a chromophoric protecting group is used (such as DMT), an
additional advantage is provided wherein facile determination of
the amount of support linkers may be determined by monitoring the
absorbance of the deprotection solution (e.g. 504 nm for
DMT.sup.+). Mono-dimethoxytrityl protected polyethylene glycols may
be introduced onto the surface of fused silica waveguides by a
number of methods. Waveguides first functionalized with GOPS, as in
the method of Maskos and Southern, may then be treated with a
solution of mono-dimethoxytritylated polyethylene glycol over
sodium hydride to afford linkage of the polyether to the terminal
epoxide moiety of the immobilized GOPS via a base catalyzed epoxide
ring-opening reaction as shown in FIG. 1(e).
Mono-dimethoxytritylated polyethylene glycols (such as DMT-HEG) can
also be directly linked to the surface of fused silica waveguides
by activation of the terminal hydroxyl moiety of the polyether with
methane sulfonyl chloride or .beta.-cyanoethyl N,N-diisopropyl
phosphityl chloride, as shown in FIGS. 1(e) and 1(f), respectively.
In the later case, the polyether substrate linker is attached as a
phosphoramidite synthon which can be done as part of the automated
oligonucleotide synthesis procedure; thereby making the entire
biosensor fabrication protocol completely automated after cleaned
waveguide pieces are introduced into the synthesis column of the
automated synthesizer.
[0091] The biorecognition element to be bound onto the terminus of
the substrate linker in configuration of the described biosensor
can include immobilized nucleic acids (DNA and RNA), modified
nucleic acids, and nucleic acid analogs prepared by well-known
methods or by straight-forward extension or modification of those
methods. The term nucleic acid includes polynucleotides, oligomers,
relatively short polynucleotides (up to about 50 bases), longer
polynucleotides ranging up to several hundred bases, and
doubled-stranded polynucleotides. There is no specific size limit
on nucleic acids used for immobilization in this invention.
However, problems due to self-hybridization and reduced selectivity
may occur with longer nucleic acids. As used herein, the term
"nucleic acid analogs" includes modified nucleic acids. As used
herein, the term "nucleotide analog" includes nucleic acids where
the internucleotide phosphodiester bond of DNA or RNA is modified
to enhance bio-stability of the oligomer and "tune" the
selectivity/specificity for target molecules (Ulhmann, et al, 1990,
Angew. Chem. Int. Ed. Eng., 90: 543; Goodchild, 1990, J.
Bioconjugate Chem., I: 165; Englisch et al, 1991, Angew, Chem. Int.
Ed. Eng., 30: 613). Such modifications may include and are not
limited to phosphorothioates, phosphorodithioates,
phosphotriesters, phosphoramidates or methylphosphonates.
[0092] In the present invention, nucleic acid sequences are
covalently attached to the surface of the optical fiber. In a
preferred embodiment, an automated DNA synthesizer is used to grow
nucleotide oligomers onto the surface of activated optical fibers
via the well established .beta.-cyanoethylphosphoramidite method.
Any commercially available automated DNA synthesizer can be used.
The use of an automated synthesizer to grow nucleic acids or
nucleic acid analogs on the optical fiber substrates provides many
advantages over conventional techniques of DNA immobilization.
Conventionally, nucleic acid strands are adsorbed onto a suitable
support (usually nitrocellulose) with little known about strand
orientation. The use of an automated oligonucleotide synthesizer
provides full control of the oligomer sequence, strand orientation,
and packing density in association with activation of the optical
fiber substrates. Control over these parameters is critical to the
development of a nucleic acid detection method based on
hybridization as the alignment of the immobilized strands with
respect to the availability of target nucleotides for hybridization
and intermolecular interactions (electrostatic and steric) between
oligomers will have direct ramifications on the kinetics and
thermodynamics of hybrid formation and dissociation. The use of a
gene machine, in addition to the chemistry used to activate the
surface of the optical fibers, allows for the creation of membranes
of desired density and structural order to permit rapid and
reversible hybridization, and to control refractive index.
[0093] The use of the phosphoramidite method of oligonucleotide
synthesis has been widely reviewed and has become the synthetic
method of choice owing to the high coupling efficiencies and
robustness of the reagents, in addition to the fact that the
necessity of numerous product isolation and purification steps
(which are required for liquid phase methods) are avoided. There
are two readily available types of phosphoramidites which may be
used to synthetically grow oligonucleotides, namely,
methylphosphoramidites and cyanoethylphosphoramidites. The method
utilizing .beta.-cyanoethyl phosphoramidites is preferable as
complete deprotection of the oligonucleotides can be done using
aqueous ammonia (as opposed to thiophenol) for the case where
oligonucleotides were grown onto controlled pore glass (CPG).
Triethylamine is used to deprotect the .beta.-cyanoethyl protected
oligonucleotides grown onto fused silica wafers or optical fibers
without liberating the oligonucleotides from the support. An
overview of the .beta.-cyanoethylphosphoramidite synthesis is as
follows:
[0094] The first step in each cycle of solid phase automated
phosphoramidite synthesis involves the removal of the
dimethoxytrityl protecting group on the immobilized nucleotide.
Detritylation is done by introducing a solution of 3%
trichloroacetic acid (TCA) in 1,2 dichloroethane (DCE) onto the
synthesis column in order to yield a 5'-hydroxyl functionality onto
which the next nucleotide monomer may be coupled. TCA is the
reagent of choice for detritylation due to its rapid reaction rate
so that the oligonucleotide is only exposed to the acid for short
periods of time, thereby avoiding the acid catalyzed removal of the
adenine and guanine moieties from the nucleotide sugar groups by
the process of depurination. Once the reaction has been completed,
the acid is removed by flushing the column with acetonitrile. The
eluent containing the released trityl cation is sent to a fraction
collector so that the coupling efficiency of the synthesis may be
monitored by absorption spectroscopy.
[0095] Coupling is the next stage of the synthesis cycle. The
contents of the synthesis column are dried by alternatively washing
with acetonitrile and flushing with dry argon. This ensures that
the support is anhydrous and free of nucleophiles. The desired
phosphoramidite and tetrazole are then sent into the synthesis
column. Tetrazole is a weak acid (pK.sub.a=4.8) which is used to
activate the phosphoramidite. Nucleophilic attack by the
5'-hydroxyl group on the activated phosphoramidite moiety forms an
internucleotide linkage. A ten-fold molar excess of phosphoramidite
in an excess of tetrazole is added to the synthesis column to
ensure that high coupling yields are achieved.
[0096] The next step of the synthesis is the capping step. This is
done to eliminate further growth of sequences onto which coupling
did not occur. The failed sequences are rendered unreactive by
introducing acetic anhydride in the presence of
dimethylaminopyridine in order to acetylate any remaining
unprotected 5'-hydroxyl moieties.
[0097] Because the trivalent internucleotide phosphite moieties are
labile to both acidic and basic conditions, a solution of aqueous
iodine is added after flushing the capping reagents from the
column. This is done in order to oxidize the trivalent
internucleotide phosphite moieties to the more stable pentavalent
phosphate moieties found in naturally occurring nucleic acids. This
procedure is termed the oxidation step.
[0098] Following the oxidation step, one cycle of nucleotide
addition is complete. The process may be repeated many times until
oligonucleotides of desired length and base sequence have been
constructed. After addition of the last nucleotide, a final
detritylation step is usually done in order to yield a 5'-hydroxyl
group on the completed sequence.
[0099] Triethylamine is used for the removal of .beta.-cyanoethyl
protecting groups on the internucleotidic phosphotriester moieties
of oligonucleotides grown onto optical substrates. This procedure
is known to cause quantitative loss of the phosphate protecting
groups via a .beta.-elimination mechanism while not cleaving the
single-stranded nucleic acids from the optical fibers. Ammonia
treatment of the immobilized oligonucleotides is avoided by
choosing an all-thymine base sequence. Thymine does not contain a
primary amine functionality which would require protection during
oligonucleotide synthesis. This approach is not limited to the use
of phosphoramidite synthons, but is compatible with all
commercially available solid-phase synthesis such as the
H-phosphonate chemistry (Froehler, B. C., 1986, Tetrahedron
Letters, 27: 5575; Stein et al, 1990, Analytical Biochemistry, 188:
11).
[0100] Contrary to the conventional preparation of oligonucleotides
by solid-phase synthesis, post-synthesis removal of the product
from the support is not desired. In order to prevent cleavage of
the oligonucleotide from the support (optical fiber) while removing
the protecting groups of the nucleobases, two modifications to the
usual synthetic protocol can be made. The approach involves the
combination of a hydrolysis resistant linkage between the oligomer
and support along with the use of labile base protecting groups.
Thus, an oligomer of any sequence can be prepared and deprotected
yet remain attached to the support, available for
hybridization.
[0101] The phenoxyacetyl (PAC) family of protecting groups
represents a convenient method for blocking the exocyclic amino
functions of guanine, adenine and cytosine residues (thymine or
uracil require no nucleobase protection). The half-time of
deprotection with concentrated ammonium hydroxide at 20.degree. C.
is 8 min, 7 min and 2 min, respectively (Wu et al, 1989). Under
these conditions, the cyanoethyl phosphate protecting groups are
removed within seconds (Letsinger and Ogilvie, 1969), whereas the
linkage which joins the oligomer to the surface of the fused silica
fiber (e.g., a phosphodiester or phosphoramidate) is completely
stable under these conditions. Alternative labile protecting groups
are derivatized phenoxyacetyl groups including alkyl substituted
PAC groups, more specifically t-butylphenoxyacetyl groups. The
t-butylphenoxyacetyl group can be quickly removed compared to
hydrolysis of the linkage to the spacer thereby reducing the
possibility of cleavage of the immobilized sequence from the
surface. N-phenoxyacetyl deoxynucleoside
3'-cyanoethylphosphoramidites and the analogous
t-butylphenoxyacetyl phosphoramidites are commercially available.
It has been reported by Polushin and Cohen (N. N. Polushin and J.
S. Cohen, Nucleic Acids Research, 1994, 22, 5492-5496) that the
t-butylphenoxyacetyl nucleobase protecting groups can be
quantitatively be removed by treatment with ethanolamine for 10
minutes at room temperature or by treatment with a mixture of
hydrazine/ethanolamine/MeOH (1:1:5 v/v/v) for 3 minutes. Beaucage
and co-workers (J. H. Boal, A. Wilk, N. Harindranath, E. E. Max, T.
Kempe and S. L. Beaucage, Nucleic Acids Research, 1996, 24,
3115-3117.) also report the rapid and quantitative removal of
t-butylphenoxyacetyl protecting groups by treatment of the
support-bound protected nucleic acid with gaseous amines ({a}
anhydrous ammonia gas, 10 bar, 25.degree. C., 35 min. or, {b}
methylamine, 2.5 bar, 25.degree. C., 2 min.)
[0102] Other possible labile protecting groups could include the
"FOD" (fast oligonucleotide deprotection available from Applied
Biosystems Inc.) based on N,N-dialkylformamidines (Vinayak et al,
1992, Nucleic Acids Research, 20: 1265-1269). Kuijpers et al
(Tetrahedron Lett., 1990, 31 6729-6732 and Nucleic Acids Res.,
1993, 21, 3493-3500) have described a method of nucleobase
protection using 2-(acetoxy-methyl) benzoyl (AMB) moieties which
can be removed by treatment with anhydrous potassium carbonate in
methanol for 90 minutes at room temperature. Use of protecting
groups that can be selectively removed under conditions that will
not cleave the oligomer from the support, such as the levulinyl
group (removed by hydrazine treatment) (Letsinger et al, 1968,
Tetrahedron Letters, 22: 2621-2624; Hassner et al, 1975, J. Amer.
Chem. Soc., 97: 1614-1615) are also contemplated by the present
invention. Even synthesis without nucleobase protecting groups is
possible for nucleic acid oligomers of up to 20 nucleobases in
length using the phosphoramidite approach (Gryaznov et al, 1991, J.
Amer. Chem. Soc., 113: 5876) or H-phosphonate chemistry (Kung et
al, 1992, Tetrahedron Letters, 33: 5869). Any of these approaches
circumvents difficulties in removing nucleobase protecting groups
while leaving the oligomer attached to the support.
[0103] Free short strands of nucleic acids can also be covalently
attached to the optical fiber directly or via linker molecules.
This approach allows the use of DNA or RNA isolated from natural
sources, amplified nucleic acids or their analogs, or synthetic
samples provided in the fully deprotected form. Protocols provide
end-attached oligomers of a well defined orientation. Chemically
stable linkages between the support and oligonucleotide may be
employed to enhance the robustness of the biosensor.
[0104] Quartz (or interchangeably fused silica) optical fibers
derivatized with linker molecules terminated with either hydroxyl
or amino groups can serve as substrates for carbodiimide-mediated
coupling with terminally phosphorylated single-stranded nucleic
acids. Coupling to the hydroxyl fiber produces a phosphodiester
bond while coupling to an amine fiber yields a phosphoramidate
bond. Oligonucleotides can be phosphorylated, in solution, either
chemically via a modification of Ouchi's method (Sowa et al Bull.
Chem. Soc., Japan 1975, 48 2084) or enzymatically.
[0105] Covalent attachment of free short strands of single-stranded
nucleic acid to the optical fibers can be achieved by a slight
modification of the method Ghosh and Musso (Ghosh and Musso, 1987,
Nucl. Acids Res. 15: 5353). Coupling of a 5'-aminohexyl derivatized
oligomer with activated carboxyl fibers affords end-attached
oligomers. This method is known to minimize reaction at the amino
groups of the DNA bases (which would potentially compromise the
hybridization event) and affords surfaces with excellent nucleic
acid coverage. The synthesis of the 5'- or 3'-terminally modified
oligomers can be achieved readily by standard methods (Ghosh and
Musso, 1987; Beaucage and Iyer, 1993).
[0106] RNA may be assembled on the support or prepared separately
and linked to the support post-synthesis. RNA monomers are
commercially available, as are some 2'-O-modified synthons. The
2'-O-methyl, allyl and 2'-deoxy-2'-fluoro RNA analogs, when
incorporated into an oligomer show increased biostability and
stabilization of the RNA/DNA duplex (Lesnik et al, 1993,
Biochemistry, 32: 7832).
[0107] As used herein, the term "nucleic acid analogs" also include
alpha anomers (.alpha.-DNA), L-DNA (mirror image DNA), 2'-5' linked
RNA, branched DNA/RNA or chimeras of natural DNA or RNA and the
above-modified nucleic acids. Back-bone replaced nucleic acid
analogs can also be adapted to use in the biosensor of the present
invention.
[0108] For purposes of the present invention, the peptide nucleic
acids (PNAs) (Nielsen et al, 1993, Anti-Cancer Drug Design, 8: 53;
Engels et a., 1992, Angew, Chem. Int. Ed. Eng., 31: 1008) and
carbamate-bridged morpholino-type oligonucleotide analogs (Burger,
D. R., 1993, J. Clinical Immunoassay, 16: 224; Uhlmann, et al.,
1993, Methods in Molecular Biology, 20,. "Protocols for
Oligonucleotides and Analogs," ed. Sudhir Agarwal, Humana Press,
New Jersey, U.S.A., pp. 335-389) are also embraced by the term
"nucleic acid analog." Both exhibit sequence-specific binding to
DNA with the resulting duplexes being more thermally stable than
the natural DNA/DNA duplex. Other back-bone replaced nucleic acids
are well-known to those skilled in the art and may also be used in
the present invention (See e.g., Uhlmann et al 1993, Methods in
Molecular Biology, 20, "Protocols for Oligonucleotides and
Analogs," ed. Sudhir Agrawal, Humana Press, New Jersey, U.S.A., pp.
335).
[0109] Optical substrates such as planar wafers and optical fibers
may be used in the present invention. A preferred embodiment
utilizes optical fibers. Optical fibers are particularly
advantageous as membrane supports due to their small size, high
light transmission capability, and ability to allow total internal
reflection (TIR) of light. Optical fibers also provide a compact an
rugged sensing device, and offer the ability to do remote
spectroscopic measurements (Love et al, 1991, Biosensors with
Fiberoptics, D. L. Wise and L. B. Wingard (Eds.), Humana, N.J., pp.
139-180).
[0110] There are two fundamental configurations in which
alterations in fluorescence parameters from fluorescently doped
membranes on optical fibers may be monitored, namely, extrinsic
mode and intrinsic mode. Extrinsic mode configurations are those in
which the waveguide is simply used as a light pipe or conduit.
End-on extrinsic mode investigations are usually done using optical
fibers. In a biosensor which uses end-on extrinsic mode
configurations, the fluorescent dyes and selective chemistry are
located on or near the distal end of the fiber. The fiber is used
as a light-pipe or conduit, where the excitation or emission
radiation is simply guided from the sampling region to the
detector. Fluorescence is stimulated by coupling excitation
radiation into the near end of a fiber, and emission can be
monitored by placing light sensing equipment directly opposite the
distal end of the fiber.
[0111] Alternatively, the detector is placed at the near end of the
fiber as some of the fluorescence may be coupled back into the
fiber and totally internally reflected back to the near end. The
side-on extrinsic mode approach is typically used for
investigations carried out on planar supports, but may also be used
for fibers. The immobilized single-strand nucleic acid and
fluorophore are placed along the length of the optical fiber
waveguide/wafer. The fiber is illuminated by a light source located
normal to the length of the fiber and fluorescence emission is also
monitored by equipment placed normal to the fiber. Extrinsic
configurations provide the advantage that simple and inexpensive
equipment, including conventional light sources and detectors, are
used (Krull et al, 1991, Fiber Optic Chemical Sensors and
Biosensors, Vol. II, O. S. Wolfbeis, Ed., CRC Press, Boca Raton,
pp. 315). However, the extrinsic sampling configuration provides
poorer sensitivity owing to the short path length and sensitivity
to interferents present in the surrounding media. In a preferred
embodiment, an intrinsic mode arrangement, based on careful control
of refractive index is used to monitor fluorescence emission from
the surface of optical fibers.
[0112] Fluorophores present at either the surface or just below the
surface of the fiber may be excited through the formation of a
standing wave electric field which propagates normal to the surface
of the fiber upon total internal reflection of radiation in the
fiber. The process of TIR occurs when the angle of incidence,
.theta., at the interface between a fiber of high refractive index,
n.sub.1, and the external medium of lower refractive index,
n.sub.2, is larger than a critical angle, .theta..sub.c, defined
as: 1 Sin c = n 2 n 1 ( 1 )
[0113] The amplitude of the electric field of the reflecting
radiation decreases exponentially as a standing wave into the
medium having the lower refractive index. This decaying radiation
is referred to as an evanescent wave and can be used to excite
fluorophores located near the boundary for TIR. The propagation
intensity, I, of the evanescent wave depends on the reflection
angle, .theta., the wavelength of the transmitted radiation,
.lambda., and a Fresnel transmission factor, T: 2 I = T ( ) exp ( -
2 x d p ) ( 2 )
[0114] where x represents distance normal to the boundary for TIR,
and d.sub.p is the penetration depth which is given by (Krull et
al, 1990, Talanta, 37: 801-807): 3 d p = 4 n 1 2 sin 2 ( ) - n 2 2
( 3 )
[0115] The penetration depth is defined as the distance at which
the intensity of the evanescent field has decayed to 1/e of the
intensity at the reflection boundary. Typically, the evanescent
wave propagates into a thin zone beyond the surface of a fiber with
a penetration depth ranging from about 200 nm to 400 nm for visible
light.
[0116] Fluorophores within the evanescent wave propagation zone are
excited by that evanescent wave to emit fluorescence. Fluorophores
further from the interface with the optical fiber will experience
lower intensity of light at the excitation frequency and a
resultant concomitant decrease in intensity of emitted
fluorescence.
[0117] A major limitation of the evanescent wave excitation is that
less than 0.01% of all of the excitation radiation on a fiber
actually leaks beyond the fiber as an evanescent wave, and less
that 2% of the fluorescence caused by the evanescent wave is
actually recovered back into the fiber for transmission to the
detector by total internal reflection (Love et al, 1991, Biosensors
with Fiberoptics, D. L. Wise and L. B. Wingard (Eds.), Humana,
N.J., pp. 139-180). As such, the evanescent wave mode of excitation
and fluorescence signal recovery is very inefficient and not the
preferred mode of operation for optical sensor devices.
[0118] For the case where the refractive index of the immobilized
layer is effectively the same or greater than the index of
refraction of the substrate for immobilization (e.g., the silica
surface of the optical element) the boundary for TIR effectively
becomes the interface between the immobilized layer and the
solution. Fluorophores bound to nucleic acid in the immobilized
layer are directly exposed to the electromagnetic radiation bound
within the waveguide thereby providing a vastly improved excitation
efficiency and, as a consequence, emit increased intensity
fluorescence. For example, the index of refraction of a monolayer
of organic media (n.sub.monolayer=1.46 to 1.5; Ducharme et al,
1990, J. Phys. Chem. 94: 1925) is very similar to that of fused
silica or fused silica (n.sub.quartz=1.46; O'Hanian, H. C. 1985,
Physics, W. W Norton & Co. New York. p. 835). Fluorophores in
the immobilized layer then emit fluorescence radiation within the
waveguide itself to provide a much improved probability for
transmission of the fluorescence signal by total internal
reflection to the detector, yeilding increased sensitivity and
lower target nucleic acid detection limits.
[0119] Fluorescence is the analytical method chosen for the
transduction of hybridization events into a measurable analytical
signal, since fluorescence techniques have long been known to
provide high sensitivity (comparable to radioisotopic methods) and
detailed information about structure at the molecular level
(Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum
Press, New York). Changes in the polarity, pH, temperature,
microviscosity, or orientation of molecules in the local
environment of a fluorophore may result in alteration of the
electronic structure or collisional probabilities of the
fluorophore. Such environmental changes may be detected by
monitoring fluorescent signal parameters such as intensity,
wavelength, lifetime, or polarization. For example, it is not
uncommon for the efficiency of fluorescence emission (quantum
yield) and fluorescence lifetime of an intercalant fluorophore to
increase by an order of magnitude or more when inserted into the
rigid and hydrophobic base stacking region of a double-stranded
nucleic acids with respect to that of the unbound dye in
solution.
[0120] The present invention utilizes, and is not limited to, the
fluorescence intensity response of the bound fluorophore via
monitoring in a total internal reflection configuration along the
optical fiber substrate to quantify the presence of hybridized
nucleic acids at the surface of the fiber. The fluorescence
intensity is directly proportional to the amount of target nucleic
acid or nucleic acid analog initially present in solution. It is
also possible to use the time dependence of the rate of change of
the fluorescence intensity increase upon hybridization to determine
the concentration of target nucleic acid.
[0121] The fluorophore of the present invention can be for example
ethidium bromide (EB). The ethidium cation
(3,8-diamino-6-phenyl-5-ethyl-- phenanthridium) is a fluorescent
compound which strongly associates with double stranded nucleic
acids by intercalation into the base-stacking region and, in some
cases, the major groove of the double helical structure (Monaco et
al., 1993, Journal of Bimolecular Structure and Dynamics, 10: 675).
The ethidium cation is particularly well suited for investigations
of nucleic acid hybridization for a number of reasons. Firstly, the
quantum yield of the dye is known to increase as much as 100-fold
when intercalated into the base stacking region with respect to
that of the unbound dye in aqueous solution (Bauer et al, 1989,
Proceedings of the National Academy of Science USA, 56: 7937).
Secondly, the binding affinity and the fluorescence enhancement of
the dye are independent of base composition (Cuniberti et al, 1990,
Biophysical Chemistry, 38: 11). Thirdly, intercalation of the
ethidium cation is known to increase duplex stability as the two
3,8-amino substituents hydrogen bond with the internucleotide
phosphate groups on each of the DNA strands (whereas other
intercalators are known to significantly decrease duplex stability)
(Cuniberti et al, 1990, Biophysical Chemistry, 38: 11). The
absorption maximum of ethidium bromide is 510 nm, which is
sufficiently close to the output wavelength of 488 nm of an
Ar.sup.+ laser which may be used to excite the fluorophore. The dye
has an emission maximum of 595 nm when bound to DNA which is a
sufficiently large Stoke's shift to make separation of the emission
radiation from the excitation radiation straight forward, and to
prevent inner filter effects, by the use of a dichroic mirror or
other standard optical components (Haugland, 1992, Molecular
Probes: Handbook of Fluorescent Probes and Research Chemicals, 5th
Ed:, USA: Molecular Probes Inc.). Due to the above mentioned
reasons, the use of EB has been shown to provide a sensitive means
to detect the presence of nucleic acid duplexes for this
application.
[0122] A specific example of a tethered fluorophore is illustrated
in the synthetic schemes of FIGS. 3a, b, and c. In this case a
modified ethidium-type dye with tether, here C.sub.13 acid moiety,
is synthesized as shown (FIG. 3a). The ethidium analogue with acid
tether is attached to 5'-hexylamine functionalized oligonucleotides
immobilized on the surface of an optical fiber to generate the
biosensor with the tethered fluorophore probe. For the case where
the nucleotides are grown on the support via solid phase
phosphoramidite synthesis, the 5'-hexylamine functionalization can
readily be achieved through the use of the commercially available
reagent Aminolink 2.RTM..
[0123] The fluorophore or reporter group may be attached to the 5'-
or 3'-end of the oligomer by not only a hydrocarbon tether but
other types of tethers such as polyether, mixed aliphatic/aromatic,
or peptidic. The tether need not be restricted to the 3'- or
5'-ends of the oligomer but may be attached to a terminal or
internal ribo-residue via the 2'-hydroxyl (Yamana et al, 1991,
Tetrahedron Letters, 32: 6347). Similarly, a tether can be attached
to a terminal or internal nucleobase using pyrimidines (Pieles et
al, 1990, Nucleic Acids Research, 18: 4355) or purines (Roduit et
al, 1987, Nucleosides and Nucleotides, 6: 349). Furthermore, the
internucleotidic linkage can be a site for tether attachment
(Agrawal et al, 1990, Nucleic Acids Research, 18: 5419). Obviously,
any combination of these methods could be used to site specifically
incorporate multiple reporter groups.
[0124] The choice of fluorophores which may be tethered to the
oligonucleotide include organic intercalating complexes, such as
the commonly used nucleic acid stain ethidium bromide, thiazole
orange and analogs thereof as prepared by L. G. Lee et al (1986,
Cytometry 7: 508) and the YOYO, BOBO, and TOTO series of cyanine
based intercalant fluorophores which are commercially available
from Molecular Probes Inc. (Eugene, Oreg.). Inorganic coordination
complexes, such as the "molecular light switch" Ru(phen').sub.2
dppz PF.sub.6 developed by Jenkins et al. (1992, J. Amer. Chem.
Soc. 114: 8736) may also be used as well as groove binding dyes,
such as Hoechst 33258 and Hoechst 33342, which are commercially
available from Aldrich Chemical Co. (Milwaukee, Wis.). These
fluorophores are chosen such that the fluorescent probe is quenched
(non-emissive) when in the presence of single-stranded nucleic
acids and provides intense luminescence when in the presence of
double stranded nucleic acids. This change in observed luminescence
occurs via changes in the relative rates of radiative and
non-radiative relaxation processes of the probe when the external
environment changes from aqueous solution to a hydrophobic and
highly structured one in the base stacking region of
double-stranded nucleic acids.
[0125] Other examples of classes of fluorophores which can be used
in the present invention include acridine dyes, phenanthrides,
phenazines, phenothiazines, quinolines, alfatoxin, polycyclic
hydrocarbons, oxirane derivatives, actinomyces, anthracyclinones,
thiaxanthenones, anthramycin, mitomycin, platinum complexes,
polyintercalators, norphilin-A, fluorenes and fluorenones,
furocoumarins, benzodipyrones and monostral fast blue. Preferred
dyes are also those that provide large Stoke's shifts, can be
excited at long wavelengths and have large differences in
fluorescence lifetime, quantum efficiency, and/or wavelength of
excitation and emission when in solution as compared to when bound
to hybridized nucleic acids.
[0126] Light emitted from fluorophores (after direct excitation) at
the surface of the fiber is preferentially coupled back into the
fiber and can be monitored by a photomultiplier tube (PMT) or any
other suitable light detection equipment. Increasing the length of
coated fiber results in a greater optical path length and better
sensitivity (Krull et al, 1991, Fiber Optic Chemical Sensors and
Biosensors, Vol. II, O. S. Wolfbeis, Ed. , CRC Press, Boca Raton,
pp. 315). Direct excitation of fluorophores in an immobilized layer
extending from the biosensor results in improved signal to noise
ratio as interferences from background fluorescence in the bulk
environment are avoided.
[0127] One instrument used for fluorescence intensity measurements
is based on a fluorescence microscope as described elsewhere
(Brennan et al, 1990, Anal, Chim. Acta., 237: 253) and shown in
FIG. 4(a). An instrument as shown in FIG. 4(b) may also be used in
which the output from a suitable light source, for example an argon
ion laser, is directed into an optical fiber via a lens with a
numerical aperture which is equal to or greater than the numerical
aperture of the nucleic acid functionalized waveguide when in the
hybridization buffer solution used for analyte detection. The
excitation radiation may be coupled into a delivery fiber via a
twisted optical fiber waveguide assembly such that all modes
carried by the first fiber into which the excitation radiation was
first coupled would be delivered to the second fiber to provide
optimal excitation of fluorophores associated with the biosensor.
The excitation radiation may be totally internally reflected along
the length of the delivery fiber to a sensing fiber functionalized
with immobilized oligonucleotide and fluorophore. Coupling of the
radiation between fibers may be achieved by abutting the distal
terminus of the delivery fiber to the proximal terminus of the
sensing fiber in a suitable non-fluorescent fiber coupler. The
terminus of the coupler is preferentially designed as a
compression-fit end which provides a solution-tight seal to prevent
contaminants from diffusing into the fiber coupler and causing
drift in the analytical signal. The sensing fiber is situated
within in a small volume, stop-flow, hybridization chamber made of
a suitable inert material with good thermal conductivity (e.g.
stainless steel or titanium). The temperature of the hybridization
may be controlled by use of a suitable thermoelectric housing to
provide rapid thermostating to the desired temperature and computer
control. The temperature of the solutions in the hybridization cell
may be accurately determined (.+-.0.2.degree. C.) by use of a glass
encapsulated thermistor incorporated into the hybridization cell.
Solutions delivery to the hybridization cell and sensing fiber may
be done by use of a computer controlled pump (e.g. peristaltic
pump) where all solutions originate from a computer controlled
autosampler. Fluorescence emission from fluorophores associated
with immobilized nucleic acid complexes was totally internally
reflected within the sensing fiber. The portion of the light
coupled back into the delivery fiber was directed towards an
interference filter with the appropriate bandpass window for the
emission of the fluorophore used with the optical sensor.
Fluorescence radiation traversing the interference filter then
enters a photomultiplier tube to provide a quantitative measure of
the fluorescence intensity. In alternative embodiments, the
radiation source can be a frequency doubled laser, a semiconductor
laser, bright lamp or LED. Coupling into the waveguide can be
accomplished with fiber couplers, and the detector can be an
avalanche diode rather than a PMT.
[0128] In one embodiment of the invention the biosensor operates as
follows. The optical fiber with attached fluorescently labeled
single-stranded nucleic acid is placed in a flow through cell and
immersed in hybridization buffer solution. When single-stranded
nucleic acids or nucleic acid analogs which are complementary to
the immobilized strands are introduced to the flow cell,
hybridization occurs followed by intercalation (or other suitable
ligand binding motif) and enhanced fluorescence emission of the
attached fluorescent probe, as illustrated in FIG. 5. Fluorescence
intensity is monitored in a total internal reflection configuration
wherein the optical fiber and organic coating form a waveguide to
provide excitation to the surface immobilized nucleic acid and
fluorescent probe, as well as to collect fluorescence emission. By
monitoring the fluorescence intensity from the fiber, a measure of
the amount of target nucleic acid in solution can be
determined.
[0129] Regeneration of the biosensor can be achieved by thermal
methods such as by elevating the temperature within the
flow-through hybridization cell or by chaotropic methods in which
solutions of highly polarized salts alter the hydrogen bonding
structure of the solution to affect denaturation of the hybridized
complex. In either case, the complex stability in the system is
reduced to the point where hybridization is not energetically
favorable and the complement strands are dissociated from the
covalently immobilized oligomers and may be flushed out of the flow
cell. Regeneration methods as described herein can be employed to
recycle biosensors.
[0130] Formation of multi-stranded nucleic acids (i.e. nucleic acid
complexes composed of 3 or more strands), such as triplex nucleic
acids, may be determined from the temperature dependence of the
fluorescent signal. Normally, the fluorescence efficiency of a
fluorophore increases with decreasing temperature owing to the
reduced collisional deactivation as a consequence of the reduced
kinetic energy of the molecules surrounding the fluorophore.
Fluorescence efficiencies with negative temperature coefficients
are readily observed for fluorophores in solution and well as for
fluorophores intercalated into nucleic acids, as illustrated in
FIG. 6. When multi-strand formation occurs, (e.g. binding of a
third strand in the major groove of a double-helical nucleic acid)
exclusion of the bound ligand often follows as the partition
coefficient for the fluorophore in the multi-stranded nucleic acid
is often much reduced with respect to that of the same fluorophore
in double-stranded nucleic acid. The ligand exclusion process will
also show a temperature dependence where reduced ligand binding is
observed as the temperature of the system is decreased. As such, a
positive temperature coefficient of fluorescence intensity would be
observed for fluorophores associated with multi-stranded nucleic
acids as increasing amounts of fluorophore become excluded from the
highly-structured environment within the nucleic acid complex into
bulk solution where the probability for collisional quenching of
fluorescence is far greater. A net positive temperature coefficient
of fluorescence intensity would then be observed for a fluorescent
nucleic acid binding ligand in a multi-stranded nucleic acid. The
temperature at which multi-strand formation occurs could also be
assayed from the maxima in a fluorescence intensity versus
temperature plot where the temperature coefficient changes from
negative (for the dye bound in double-stranded nucleic acid) to
positive (for the dye being excluded from the multi-stranded
nucleic acid complex). This process is illustrated in FIGS. 7(a
& b) for triplex formation on the sensor surface with linear
and branched nucleic acids.
[0131] The biosensor of the present invention provides for rapid
clinical testing for viruses (e.g., HIV, T cell lymphotropic virus
1 and 2, hepatitis B and C), and pathogenic bacteria (e.g. E.
coli., Salmonella, Listeria, Chlamydia ssp., Trichomonas vaginalis,
Gradenerella vaginitis) as well as other microorganisms. Detection
of genetic disorders (e.g., cystic fibrosis and sickle-cell anemia)
and diseases such as cancer is also done with the method and
apparatus of the present invention as well as potential
therapeutics to treat such diseases (e.g. branched antisense
nucleic acids which inhibit expression of targeted nucleic acid
sequences via triplex formation with that particular sequence,
effectively shielding the genetic information from being read by
transcription enzymes).
[0132] The biosensor is useful for the monitoring of the in-vivo
response of bacteria to an antibiotic treatment to ensure the
efficacy of the treatment regime. The physician, based on past
experience, chooses both the antibiotic and the dosage to treat a
bacterial infection. Samples of the infecting organism would be
sent to the laboratory for MIC testing, Minimal Inhibitory
Concentration for the lowest concentration of that particular
antibiotic necessary to inhibit the growth of the infecting
organism. If the MIC of the in-vitro test is less than the dosage
given to the patient, the treatment is allowed to continue,
otherwise an increased dose and/or a change of antibiotic would be
ordered. The most significant problem of this approach is that of
the time required, wherein 1 to 3 days are needed to acquire the
MIC result. Secondarily, it is a test of an in-vitro response to
reflect an in-vivo situation. Using larger doses than necessary is
not a reasonable treatment, as most antibiotics are toxic to the
patient as well. Further the use of inappropriate antibiotics or
doses can encourage the development of drug resistance in the
infectious organisms. The biosensor described above overcomes these
problems by determining the concentration of one or more species of
bacteria present in the patient. Samples would be taken at
intervals and tested for the bacteria's concentration. The change
in the bacterial concentration over time would reflect the efficacy
of the antibiotic treatment against the infectious organism or
organisms. This ensures that an adequate amount of an appropriate
antibiotic would be provided to the patient without providing
excessive amounts of the antibiotic. The method described above may
be used in a variety of situations to monitor response of organisms
such as bacteria or fungi to drugs.
EXAMPLES
Example 1
[0133] Preparation of Fused silica Optical Fibers Derivatized with
Long Chain Aliphatic Spacer Molecules Terminated with a
5'-O-dimethoxytrityl-2'-deoxythymidine Nucleoside.
[0134] Plastic-clad silica optical fibers with a diameter of 400
.mu.m were purchased from 3M Specialty Optical Fiber (North York,
ON, Canada). The cladding on the fibers was mechanically removed,
and the fibers were cut to lengths of about 1 cm. One face on each
fiber was polished by suspending the fiber over (and placing the
end face of the fiber in contact with) the rotating plate of a
Thermolyne type 37600 speed controlled mixer (Sybron Corporation,
Dubuque, Iowa, USA) onto which 1200 grade emery paper was
immobilized. All fused silica optical fibers were cleaned using a
Harrick PDG-32G plasma cleaner (Harrick Scientific Corporation,
Ossining, N.Y., USA) before activation with aminopropyltriethoxy
silane (APTES).
[0135] The fibers were then washed with a 1:1 acetone/methanol
mixture and stored in a vacuum desiccator. The optical fibers were
plasma cleaned for 5 minutes at low power (40 W) and were placed in
a solution of 1:200 (v/v) aminopropyltriethoxy silane (APTES) in
dry toluene. This was done under a nitrogen atmosphere using
glassware which was oven dried and previously treated with
octadecyltrichlorosilane. The structure of the APTES coatings on
fused silica substrates has previously been investigated by
Vandenberg et al. (1991, J. Colloid and Interface Sci., 147: 103).
The method of Arnold and co-workers (1989, Collect. Czech. Chem.
Commun., 54: 523) was used to synthesize an aliphatic spacer arm
terminated with 5'-O-dimethoxytrityl-2'-deoxythymidine. In this
method 1,10-decanediol was condensed with succinic anhydride to
form 1,10 decanediol bis-succinate, as illustrated in FIG. 1(a).
The bis-succinate was reacted with N-hydroxysuccinimide and
5'-O-dimethoxytrityl-2'deoxythi- midine in the presence of
N,N'-dicyclohexyl-carbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) to yield a nucleoside functionalized spacer molecule. The
spacer was then attached to the surface of the APTES treated
optical fiber via amide formation.
Example 2
[0136] Preparation of Fused silica Optical Fibers Derivatized with
Glycidoxypropyltrimethoxysilane Extended with
Mono-Dimethoxytritylated Hexaethylene Glycol Substrate Linker
Molecules.
[0137] In order to grow oligonucleotides onto the surface of silica
substrates (such as fused silica) by automated solid phase
synthesis, the surface is functionalized with spacer molecules of
at least 25 .ANG. in length which had either an amine or a hydroxyl
functionality at the terminus of the spacer molecule. A chemically
resistant, non-hydrolyzable spacer molecule is employed. The method
used was a modification of that reported by U. Maskos and E. M.
Southern supra wherein the silica surface was treated with
glycidoxypropyltrimethoxysilane (GOPS), followed by extension via
treatment with hexaethylene glycol (HEG) under acidic conditions.
For the purpose of creating biosensors with higher sensitivity and
lower detection limits, this method is advantageous over the use of
hydrocarbon tethers. The water soluble HEG linker will provide a
more fluid environment (which should not self-assemble) so as to
improve the ability of the immobilized DNA strands to hybridize
with complementary material in solution (in terms of energetics and
kinetics). The hydrophilicity of the linker will also facilitate
the removal of adsorbed contaminants (e.g. proteins, organics)
which may occlude the surface and contribute to drift in the
fluorescence intensity. However, as HEG is bifunctional, there
exists the possibility of creating non-reactive closed-loop
structures which may significantly decrease the loading of
oligonucleotides on the surface of the fibers. In order to
eliminate this problem, one terminus of the HEG is protected with a
dimethoxytrityl functionality prior to extension with GOPS. This
strategy permits facile determination of the amount of support
linkers bound to the silica surface. Removal of the trityl
protecting groups by treatment with acid yields the highly colored
trityl cation, which can be quantitatively measured by monitoring
A.sub.(504 nm) of the deprotection solution. Knowing there is one
trityl group released per linker molecule attached to the surface,
the loading of HEG can easily be determined. Immobilization of a
protected linker molecule provides the additional advantage that
the hydroxyl groups produced after the attachment of the HEG to the
epoxide moiety and all other surface silanols can be capped to
prevent unwanted oligonucleotide growth at these sites. The
presence of side product oligonucleotides, which are prematurely
terminated due to the lack of a suitable support molecule, may
decrease the sensitivity and selectivity of the sensor. The
additional charge imparted from the anionic backbone of a side
product strand may inhibit hybridization between the analyte
strands and neighboring probe sequences. See: R. T. Pon Methods in
Molecular Bioloqy, Vol. 20: Protocols for Oligonucleotides and
Analogs, S. Agrawa, Ed, 1993, Humana Press, Inc. Totowa N.J. In
conjunction with the use of non-hydrolyzable spacer molecules,
t-butylphenoxyacetyl protected phosphoramidite synthons were
employed. This labile protecting group can be quickly removed (15
min @ 55.degree. C. or 120 min. at room temperature as compared to
12-16 hours @ 55.degree. C. using 27% aqueous ammonia) thereby
reducing the possibility of cleavage of the immobilized sequences
by hydrolysis of the silyl ether bonds which ultimately anchor the
strands to the fiber surface.
[0138] i) Cleaning of Silica Substrates Prior to Functionalization
with GOPS:
[0139] The buffer coating was mechanically stripped from the
pre-cut optical fiber pieces (400 .mu.m diameter.times.44 mm) and
the cladding was dissolved by treatment with acetone. The fused
silica substrates, i.e., optical fibers or wafers, were added to a
1:1:5 (v/v) solution of 30% ammonium hydroxide/30% hydrogen
peroxide/water and the mixture was stirred at 80.degree. C. for
five minutes. The substrates were then removed and treated with a
solution of 1:1:5 (v/v) conc. HCl/30% hydrogen peroxide/water and
the mixture stirred at 80.degree. C. for five minutes. The
substrates were then sequentially washed with methanol, chloroform
and diethyl ether, respectively, and dried in-vacuo.
[0140] ii) Synthesis and Purification of Mono-dimethoxytritylated
Hexaethylene Glycol (DMT-HEG):
[0141] Dimethoxytrityl chloride (7.1 g) was dissolved in 10 ml of
dry pyridine and added dropwise to a stirred solution of
hexaethylene glycol (5.65 ml) in 5 ml of dry pyridine under an
argon atmosphere. Stirring was continued for 16 hours after which
time the reaction mixture was combined with 50 ml of
dichloromethane. The dichloromethane solution mixture was twice
shaken with 900 ml portions of 5% aqueous sodium bicarbonate and
then with three 900 ml portions of water in order to remove
unreacted HEG, pyridine, and pyridinium salts. The product was
purified by liquid chromatography using silica gel and a solvent
system of 0.1% triethylamine in 1:1 dichloromethane/diethyl ether.
The identity of the product was confirmed by proton NMR
spectroscopy (200 MHz).
[0142] iii) Functionalization of Fused Silica Substrates with
3-Glycidoxypropyltrimethoxysilane (GOPS):
[0143] The cleaned fused silica substrates were suspended in a
stirred solution composed of 40 ml xylene, 12 ml GOPS, and a trace
of Hunig's base at 80.degree. C. overnight. The fibers were then
sequentially washed with methanol, chloroform, ether, and then
dried in-vacuo.
[0144] iv) Linkage of DMT-HEG to GOPS Functionalized Silica
Substrates:
[0145] The GOPS functionalized fibers were suspended in a stirred
solution of 1:4:8 (v/v) DMT-HEG/diethyl ether/toluene containing a
catalytic amount of sodium hydride under an argon atmosphere. The
reaction mixture was stirred for 14 days after which time the
fibers were removed and washed sequentially with methanol,
chloroform, ether, and then dried in vacuo.
[0146] v) Capping of Unreacted Silanol and Hydroxyl Functionalities
with Chlorotrimethyl-silane:
[0147] The fused silica fibers functionalized with DMT-HEG were
suspended in a solution of 1:10 (v/v)
chlorotrimethylsilane/pyridine for 16 hours under an argon
atmosphere at room temperature.
Example 3
[0148] Preparation of Fused silica Optical Fibers Derivatized with
Mono-Dimethoxytritylated Hexaethylene Glycol Substrate Linker
Molecules via Mesylate Activation.
[0149] The details of the preparation of the fused silica
substrates and DMT-HEG synthesis are provided in example 2(i and
ii). DMT-HEG (0.5 g) was suspended in 50 ml anhydrous pyridine. The
solution was maintained under an anhydrous argon atmosphere and
stirred. while 1.2 equivalents of methanesulfonyl chloride was
added dropwise. The reaction allowed to proceed for 60 minutes at
room temperature with stirring. The cleaned fused silica and
silicon substrates were introduced into the solution containing the
mesylated DMT-HEG and the substrate functionalization reaction
allowed to proceed for 4 days with stirring at 40.degree. C. under
an argon atmosphere. Following the 4 day incubation period, the
solution was decanted away from the functionalized substrates which
were then washed with copious amounts of dichloromethane. Washings
were continued until no discernible absorbance at 504 nm was
observed from the wash solution made acidic by treatment with
trichloroacetic acid. The functionalized substrates were then
capped as per the methods of example 2(v) and stored in-vacuo and
over P.sub.2O.sub.5 until needed.
Example 4
[0150] Preparation of Fused silica Optical Fibers Derivatized with
Mono-Dimethoxytritylated Hexaethylene Glycol Phosphoramidite
Substrate Linker Molecules via Standard
.beta.-Cyanoethylphosphoramidite Coupling on an Automated
Synthesizer.
[0151] The details for the preparation of the fused silica
substrates and DMT-HEG are provided in example 2(i) and 2(ii),
respectively. DMT-HEG (0.5 g) was suspended in a solution
consisting of 12 ml of anhydrous THF and 4 ml of anhydrous
N,N-diisopropyl ethylamine. The solution was maintained under an
anhydrous argon atmosphere and stirred at all times. 1.1
equivalents of 2-cyanoethyl-N,N-diisoproylamino-phosphochloridite
was added dropwise to the DMT-HEG solution and the reaction allowed
to proceed for 90 minutes at room temperature. TLC analysis (1:1
CH.sub.2Cl.sub.2/diethyl ether) indicated quantitative formation of
the DMT-HEG phosphoramidite synthon (R.sub.f=0.7). The reaction
product was thrice extracted into ethyl acetate from a 5% sodium
bicarbonate solution. The organic phase was separated from the
aqueous layer, dried over NaSO.sub.4, filtered and the solvent
removed under reduced pressure. The product was then stored dry and
at -20.degree. C. under an anhydrous argon atmosphere until
required. Functionalization of fused silica substrates was then
done as part of a standard coupling cycle using the automated solid
phase DNA synthesizer and a 0.1 M solution of the DMT-HEG
phosphoramidite in anhydrous THF. The methods for automated
oligonucleotide synthesis are detailed in example 5. The capping
procedure detailed in example 2(v) was then done in order to block
any undesired reactive sites on the substrates.
Example 5
[0152] Synthesis of Oligonucleotides onto Substrate Linker
Functionalized Fused Silica Waveguides.
[0153] All DNA synthesis was done by the well established
.beta.-cyanoethylphosphoramidite method with an either an Applied
Biosystems 381A or 391 EP DNA Synthesizer using susbtrate linker
functionalized controlled-pore glass beads, fused silica optical
fibers, planar fused silica wafers, or planar silicon wafers.
Automated solid-phase DNA synthesis is well known and is described
in detail elsewhere (Beaucage et al., 1992, Tetrahedron Letters,
48: 2223-2311; Oligonucleotides and Analogues: A Practical
Approach, F. Eckstein, Ed. Oxford University Press, New York,
1991). The substrate linker functionalized optical fibers were
placed into an emptied Applied Biosystems (ABI) Oligonucleotide
Purification Cartridge column (OPC-column) or 10 .mu.mol scale
synthesis column with the dead volume being taken up by inert
polyethylene pieces. The end filter papers were replaced (ABI) and
the column ends were crimped closed using aluminum seals. Synthesis
of oligomers onto the optical fibers was carried out at the 0.2
.mu.mol. scale with a pulsed-delivery cycle in the "trityl off"
mode. The .beta.-cyanoethylphosphoramidite cycle was used as
supplied by ABI with the exception of extended nucleoside coupling
times (2-10 min.) and increased solution delivery times to
accommodate the larger synthesis columns. With the exception of
thymidine synthons, t-Butylphenoxyacetyl protected phosphoramidite
synthons were used in conjunction with a t-butylphenoxyacetic
anhydride capping solution as supplied by Millipore Inc.
[0154] In the case where polythymidilic acid oligonucleotides were
grown on the optical fibers, deprotection of the phosphate blocking
groups from the immobilized oligomer was achieved by standing the
fibers in a solution of 2:3 triethylamine/acetonitrile at room
temperature for 1.5 hours. This procedure caused the loss of the
phosphate blocking group via .beta.-elimination while not cleaving
the single-stranded DNA (ssDNA) from the optical fibers. In the
case where oligonucleotides containing bases other than thymine
were grown, the following protocol was used for phosphate and
nucleobase deprotection. A 30% ammonium hydroxide solution was
drawn up into the synthesis column containing the optical fibers
functionalized with immobilized oligonucleotides using a syringe
and a male-to-male luer adapter. The fibers submerged in ammonia
were allowed to stand for two hours at room temperature after which
time the ammonia solution was expelled from the column and the
contents of the column were washed five times with 5 ml portions of
sterile water. The deprotection solutions and washings were
collected and concentrated to a total volume of 1 ml. A.sub.260nm
of the concentrated deprotection solution was measured in order to
determine the quantity of DNA liberated from the fused silica
substrates. Based on the results of the trityl cation assay and
A.sub.260nm of the deprotection solution, it was found that
.about.20% of the oligomers remained attached to the surface
following the ammonia deprotection procedure. In the case where
oligonucleotides of mixed base sequence were grown onto optical
fibers, the oligonucleotide sequence was (5'-TAG GTG AGA CAT ATC
ACA GA-3'), which is a nucleic acid probe for the E03 forward
sequence of the Candida albicans genome.
[0155] Fibers coated in ssDNA were either stored dry or kept in a
solution of 1:1 ethanol/water. All fibers were cleaned prior to use
by sonication in a solution of 1:1 ethanol/water for 5 minutes in
order to remove any fluorescent contaminants adsorbed to the
surface of the fibers.
Example 6
[0156] Biosensor Characterization by Trityl Cation Assay.
[0157] All oligonucleotide syntheses were evaluated by
spectroscopic quantitation of trityl cation released during the
trichloroacetic acid treatment steps of the automated synthesis.
The collected fractions of trityl cation were diluted with 2.0 ml
of 5% TCA in 1,2-dichloroethane immediately prior to making
absorbance measurements. Absorption at 504 nm was measured in order
to determine the concentration and the total number of trityl
cation moieties released during each TCA deprotection step of the
synthesis. In this way, the total number of oligomers successfully
grown onto the solid supports was determined.
[0158] As there exists no discernible decrease in the amount of
trityl cation released during successive deprotection steps, it may
be safely assumed that the coupling efficiency of 99.5% or better
suggested by the manufacturer of the automated synthesizer (ABI)
was achieved. The results of a trityl cation assay for a synthesis
of dT.sub.20 onto optical fibers by the methods given in Examples 1
and 5 are shown in FIG. 8.
Example 7
[0159] Generation of Complementary and Non-complementary Nucleic
Acids.
[0160] Synthesis of dA.sub.20 and rA.sub.20 was done using a
conventional LCAA-CPG support with the
.beta.-cyanoethylphosphoramidite cycle supplied by ABI. A
nonadecamer of random base composition (dR.sub.19) was also
prepared by simultaneously introducing all four phosphoramidite
reagents to the column at each coupling step. Standard deprotection
with aqueous ammonia (29%, 1.5 ml, 24 h) was used to liberate the
oligomers from the solid support and remove the base protecting
groups. For the case of the rA.sub.20, deprotection of the
phosphate blocking groups, base protecting groups and cleavage from
the CPG support was done by treating the oligomers with 1.5 ml of a
solution consisting of 4 parts aqueous ammonia and 1 part ethanol
for 48 hours at room temperature. The aqueous solution containing
the. oligonucleotides was then collected, evaporated to dryness,
and the residue treated with 300 .mu.l of an anhydrous solution of
1 M tetra-N-butyl ammonium fluoride in THF overnight at room
temperature. After the incubation time, the reaction was quenched
by adding 1 ml of water to the reaction mixture. Crude oligomer was
purified by polyacrylamide gel electrophoresis and reverse phase
liquid chromatography or size exclusion chromatography.
Example 8
[0161] Detection and Quantification of Complement DNA (cDNA),
Complement RNA (cRNA) and Non-complement Nucleic Acid by the
Optical Sensor Fabricated by the Procedures of Examples 1 and
5.
[0162] An optical fiber functionalized with polythymidilic acid
icosanucleotide was selected at random from the batch of fibers
(ca. 25) created in example 1 and was positioned under the
objective of the microscope, as illustrated in FIG. 4(a). In this
orientation the incident laser radiation entered the proximal
terminus and was totally internally reflected throughout the fiber.
The majority of the fiber was submerged in a hybridization buffer
solution consisting of 1.0 M NaCl and 50 mM sodium phosphate (pH
7.4) in sterile water contained within a 4 ml plastic cuvette.
Hybridization buffer was passed through an acrodisc.RTM. filter
immediately prior to introduction to the cuvette. Emitted
fluorescent radiation, from the stimulated fluorescent molecules
associated with the double-stranded nucleic acids, was directed
back towards the microscope by total internal reflection. The
emission from the fluorescent molecules was separated from the
excitation radiation by a dichroic mirror and directed to a
photomultiplier tube. The photomultiplier tube provided
measurements of the intensity of fluorescence emission.
Fluorescence intensity values are reported with the system at
25.degree. C. to avoid inconsistencies cause by the temperature
dependence on fluorescence quantum efficiency and as relative
quantities, thereby obviating the need to control experimental
parameters such as laser intensity, optical alignment and
photomultiplier tube (PMT) gain which are beyond accurate control
from day to day.
[0163] In order to affect hybridization with the immobilized
nucleic acid strands, dA.sub.20 ssDNA was added to the plastic
cuvette containing the suspended fiber in fresh hybridization
buffer at a temperature of 85.degree. C. This temperature was
chosen as it is sufficiently greater than the 60.degree. C. duplex
melting temperature (T.sub.m, the temperature at which half of all
the duplexes present are dissociated) and is well below the boiling
point of the buffer. Incubation at temperatures below T.sub.m has
been shown to cause incomplete hybridization wherein only a
fraction of the bases on each strand interact to form partially
hybridized complexes (Rubin et al, 1989, Nucleic Acid and
Monoclonal Antibody Probes, B. Swaminathan and G. Prakash, Eds.,
Marcel Dekker, Inc., New York, pp. 185-219). Though covalent
immobilization of ssDNA removes one degree of freedom from the
oligomer, hybridization at temperatures initially above the duplex
T.sub.m ensures the formation of duplexes with the greatest
possible extent of overlap. In all cases, no appreciable intensity
change from that of the baseline was observed after the 90 minute
incubation period. The solution was allowed to stand and cool to
room temperature (25.degree. C.) between 30 and 90 minutes after
which the fiber was flushed with 60 ml of hybridization buffer
(25.degree. C.) to remove the excess strands.
[0164] Intercalation of the fluorophore into the dsDNA was achieved
by injecting 10 .mu.l of a 1 mg.multidot.ml.sup.-1 aqueous solution
of ethidium bromide (EB) into the cuvette and allowing the solution
to stand for 15 minutes followed by washing the fiber by flushing
the cuvette with 60 ml of fresh hybridization buffer (25.degree.
C.).
[0165] The response of the fiber optic DNA biosensor to EB and cDNA
is shown in FIG. 9. As a control experiment, 10 .mu.L of a 1
mg.multidot.ml.sup.-1 aqueous solution of EB was added to the
cuvette in which the fiber functionalized with ssDNA was suspended.
After 15 minutes, 60 ml of fresh hybridization buffer (25.degree.
C.) was flushed through the cuvette in order to remove any
non-specifically bound ethidium cation and no discernible increase
in fluorescence intensity from the fiber was observed. A 104.+-.15%
increase in the fluorescence intensity was observed from the fiber
which was exposed to 189 ng.multidot.ml.sup.-1 of cDNA and stained
with EB relative to the baseline value for the cleaned sensor with
only ssDNA on the waveguide surface. The fiber was regenerated by
flushing the cuvette and optical sensor 30 ml of hot (85.degree.
C.) buffer solution over a period of c.a. 30 seconds and the system
allowed to stand for five minutes. After the five minute wait, an
additional 30 ml of hot buffer was flushed through the cuvette to
wash away the dissociated cDNA strands. This procedure is known to
melt DNA duplexes as the buffer temperature was well above the
T.sub.m of the dsDNA. The fluorescence intensity returned, within
experimental uncertainty, to the initial intensity observed at the
beginning of the experiment. The same hybridization experiment was
repeated where the optical sensor was exposed to 757
ng.multidot.ml.sup.-1 of cDNA during the hybridization procedure.
This yielded an increase of 720.+-.20% in the observed fluorescence
intensity from the sensor with respect to the baseline value. In
contrast, a similar concentration of non-complementary sequences (a
nonadecamer of random base sequence) gave essentially no response,
as shown in FIG. 9(a).
[0166] A 3.8 ng.multidot..mu.l.sup.-1 solution of rA.sub.20 (450
.mu.l) was introduced into a cuvette containing hot hybridization
buffer and the sensor to provide a 570 ng ml.sup.-1 solution of
cRNA. The same hybridization and staining procedure as used for
cDNA was followed. The response profile for this hybridization
procedure is shown in FIG. 9(b). A comparison of response of the
biosensor with immobilized dT.sub.20 to cDNA and cRNA agree to
within experimental error.
Example 9
[0167] Effective of Ethidium Bromide (EB) Staining Time and
Concentration
[0168] The staining time of the sensor with EB, was changed after
each hybridization with cDNA. For each determination, injections of
30 .mu.l of 56.8 .mu.g.multidot.ml.sup.-1 solution of aqueous
dA.sub.20 were made and the hot hybridization buffer in the
cuvette, which contained the cDNA strands, was allowed to cool to
room temperature over a time of 30 minutes. A 1
mg.multidot.ml.sup.-1 solution of EB in water (10 .mu.l) was added
to the cuvette after each hybridization to provide an EB
concentration of 8.4.times.10.sup.-3 M. A staining time of 20 min.
with 8.4.times.10.sup.-3 M EB was required to generate .gtoreq.99%
of the full signal, as shown in FIG. 10(a).
[0169] To study the effect of EB concentration during dsDNA
staining, all hybridization parameters were the same as those used
to study staining time and a staining time of 20 min. was used.
Staining with EB solutions of concentrations of
8.5.times.10.sup.-3M or greater were required to generate
.gtoreq.99% of the full staining in 20 min., as shown in FIG.
10(b).
Example 10
[0170] Long Term and Thermal Stability of the Nucleic Acid
Sensor.
[0171] The robustness of the optical sensors, and DNA as a
biorecognition element, was made evident by the maintenance of
activity after long term storage and vigorous cleaning conditions.
Fibers that were stored for over 1 year in vacuo, in 1:1
ethanol/water solutions, absolute ethanol, or dry at -20.degree. C.
provided identical response characteristics to freshly prepared
fibers. Adsorbed fluorescent contaminants which were accumulated
through long term storage were completely removed (as confirmed
through fluorescence microscopy) by sonicating the fibers in a
solution of 1:1 ethanol/water with full maintenance of activity and
sensitivity. FIG. 11(a) shows the response of a 1 month old fiber
(stored in vacuo) used with no cleaning of the surface and (b) an
11 month old fiber (stored dry at -20.degree. C.) which had been
cleaned by sonication in ethanol solution. It should be noted that
the sensitivity of the cleaned 11 month old fiber is identical to
that of 1 month old fibers cleaned by the same procedure (data not
shown). Cleaning of the sensor by sonication prior to use has
consistently been observed to increase the sensitivity of the
device by a factor of c.a. 2.5. The sensors have provided
femtomolar detection limits and a response which is linear with the
concentration of cDNA (M.W.=6199 g.multidot.mol.sup.-1). The
regression lines shown in FIG. 11 show good fits to the data points
with r.sup.2 values of 0.965 and 0.968 for the 1 month and 11
month-old fibers, respectively. From this data, the sensitivity of
the optical sensor (11 month-old, fabricated by the protocols of
examples 1 and 5) was determined to be an increase in fluorescence
intensity of 203% per 100 ng.multidot.ml.sup.-1 of cDNA with a
measured limit of detection of 86 ng.multidot.ml.sup.-1.
Maintenance of calibration has been observed for all experiments
done thus far in which as many as 5 regenerations have been done
over durations of up to 12 hours.
[0172] The ability to clean and sterilize a bioprobe or biosensor
device so that it may be usable in an on-line configuration is a
significant advantage. As the specific binding properties of
nucleic acids are based on secondary structure, the use of nucleic
acids in biosensor fabrication leads to devices which are not only
stable to prolonged storage, but also to harsh washing conditions
and sterilization. A summary of the effects of cleaning by
sonication in absolute ethanol (15 minutes) and autoclaving
(120.degree. C. for 20 minutes at 4 atmospheres pressure in sterile
water) on the response of the sensors to (.about.400
ng.multidot.mL.sup.-1) is shown in the Table which follows. Both
sonication in ethanol and autoclaving are observed to improve the
response of the sensor, most likely through the removal of
contaminants on the surface of the sensor (stored dry for 11
months, or stored in ethanol).
1TABLE 1 Effect of storage conditions, cleaning, and sterilization
on sensor response to 400 ng.multidot.ml.sup.-1 cDNA. Cleaning/
Sterilization Relative Fluorescence Storage Conditions Conditions
Intensity Increase (%) 1:1 Ethanol/Water (25.degree. C.) -- 333
.+-. 20 95% Ethanol (25.degree. C.) -- 395 .+-. 20 Dry (-20.degree.
C.) -- 341 .+-. 20 1:1 Ethanol/Water (25.degree. C.) Autoclave 430
.+-. 20 1:1 Ethanol/Water (25.degree. C.) Sonication 453 .+-.
20
Example 11
[0173] Thermal Denaturation Studies of the cDNA: Immobilized DNA
Complex on the Sensors Created from the Protocols of Examples 1 and
5 and Comparison to that of the Same Oligonucleotide Complex in
Solution.
[0174] i) Thermal Denaturation Investigations of Aqueous dT.sub.20
with Aqueous dA.sub.20.
[0175] Equimolar amounts of each oligomer in hybridization buffer
(1 M NaCl, 10 mM PO.sub.4, pH 7.0) were mixed so that the final
concentration was approximately 1 .mu.M in each strand. Prior to
thermal melt studies the oligonucleotide mixture was heated briefly
to 80.degree. C. and slowly cooled to 20.degree. C. in order to
hybridize all of the strands. The samples were held at the low
temperature limit for 15 minutes prior to initiating melt studies,
to allow for thermal equilibration. The temperature was then ramped
at 0.5.degree. C. intervals at a rate of 0.5.degree. C./min. While
the absorbance was recorded at 260 nm.
[0176] ii) Melt-Curve Investigations of Immobilized dT.sub.20 with
cDNA.
[0177] Sequences of dT.sub.20 were immobilized onto planar fused
silica wafers (5 mm.times.10 mm.times.1 mm) according to the
protocols in examples 1 and 5. The immobilized dT.sub.20 was
hybridized with complementary dA.sub.20 sequences by immersing the
wafer in a 56.8 ng.multidot.ml.sup.-1 solution of dA.sub.20 at
85.degree. C. and allowing the immersed wafer to cool to room
temperature (25.degree. C.). The wafer was then removed from the
cDNA solution and washed with room temperature hybridization buffer
solution. The wafer was then suspended in a quartz cuvette that was
placed in the temperature controlled cuvette housing of the UV-vis
spectrometer. The placement of the wafer was adjusted so that it
rested in the path of the light beam. The dead volume beneath the
wafer was taken up by inert packing material. Absorption spectra
were collected at approximately 2.degree. C. increments of
temperature in the range from 29.degree. C. to 76.degree. C. The
temperature in the cuvette was set by programming an external
circulating bath to a specific temperature and the temperature of
the buffer solution surrounding the fused silica wafer was
quantitatively measured using a silanized glass encapsulated
thermistor. Measurements of absorption at each temperature were
done by integrating 100 spectra in the wavelength range between 220
nm and 320 nm.
[0178] iii) Hypochromicity and Melt-Curve Thermodynamics.
[0179] The transition between an ordered duplex state and the
disordered denatured state for systems of complementary nucleotides
can be monitored and analyzed by UV-Visible absorbance spectroscopy
to determine the duplex melting temperature (T.sub.m). The extent
of hybridization (i.e. the number of base pairs formed per duplex)
was determined by a comparison of melt profiles for the immobilized
oligonucleotides to similar reported values and dA.sub.20+dT.sub.20
in solution.
[0180] The fraction of single strands present in the system at any
temperature (f.sub.SS(T)) may be determined through the use of the
following equation: 4 f ss ( T ) = A ( T ) - A ds ( T ) A ss ( T )
- A ds ( T )
[0181] where A(T), A.sub.SS(T), and A.sub.ds(T) are the absorbances
of the experimentally obtained melting curve, the upper baseline
(single stranded oligomers), and the lower baseline (double
stranded oligomers) respectively at temperature T (Nelson, J. W.;
Martin, F. H.; Tinoco Jr., I. Biopolymers 1981, 20, 2509-2531.). By
plotting f.sub.SS against temperature, the duplex melting
temperature can be obtained by determining the temperature at which
f.sub.SS=0.5.
[0182] iv) Melt-Curve Studies of Support Bound Duplex DNA and
Aqueous Phase DNA.
[0183] The purpose of the thermal denaturation studies was to
examine whether linkage of an oligonucleotide to a solid support
through a terminal nucleotide phosphate would cause the loss of
degrees of freedom with respect to the availability of each
nucleotide to partake in formation of the double stranded
structure. Melt profiles for the thermal denaturation of dsDNA
immobilized on the surface of a fused silica wafer and dsDNA in
solution were obtained, and the results of these investigations are
summarized in FIG. 12. The duplex melting temperature of the
immobilized strands with aqueous phase complement strands was
62.4.+-.0.3.degree. C. The T.sub.m value for the aqueous phase
dA.sub.20+dT.sub.20 duplex was determined to be 60.5.degree. C.
using the software supplied by Varian. Kibler-Herzog et. al.
(Kibler-Herzog, L.; Zon, G.; Whittier, G.; Mizan, S.; Wilson, W. D.
Anti-Cancer Drug Design 1993, 8, 65-79.) have reported the melting
temperature of a dA.sub.19+dT.sub.19 duplex in 1.02 M NaCl to be
61.1.degree. C. This suggests that for the immobilized oligomers
investigated in this work, the extent of hybridization was complete
with base pairing of 20 bases per strand. The small differences in
the three T.sub.m values may be accounted for by the fact that each
of these experiments was done on a different instrument at
different times, and the salt concentration used in this work was
slightly lower than that used by Kibler-Herzog et al. As the duplex
stability in low ionic strength buffers is less than that in high
ionic strength buffers, it would be expected that the melting
temperature of the immobilized dT.sub.20+dA.sub.20 duplex would be
higher in the buffer of lower stringency (Puglisi, J. D.; Tinoco
Jr., I.; Methods in Enzymology, 1989, 180, 304-325.). In addition
to this, a greater value of T.sub.m for the immobilized duplex over
the aqueous phase duplexes should not be considered unusual as only
one of the strands will experience a significant gain in entropy
upon melting of the immobilized duplex. These factors lead to the
conclusion that, within experimental uncertainty, the immobilized
dT.sub.20+dA.sub.20 duplexes were more stable, if not as stable, as
the aqueous phase dA.sub.20+dT.sub.20 and dA.sub.19+dT.sub.19
duplexes. This also suggests that no hindrance of duplex formation
is observed with respect to the availability of the bases for
hybridization. This result is in accord with the investigations of
Wolf et al. (Wolf, S. F.; Haines, L.; Fisch, J.; Kremsky, J. N.;
Dougherty, J. P.; Jacobs, K. Nucleic Acids Research 1987, 15,
2911-2926.), in which oligonucleotides bound to solid supports via
a long chain aliphatic tether at the strand termini (3'-end) were
not observed to be hindered with respect to hybridization
efficiency
Example 12
[0184] Detection of cDNA with and Optical Sensor Functionalized
with an Oligonucleotide Probe Sequence for Candida albicans.
[0185] Optical Sensors were created by the protocols in examples 2
and 5 where the oligonucleotide sequence (5'-TAG GTG AGA CAT ATC
ACA GA-3'), which is a nucleic. acid probe for the E03 forward
sequence of the Candida albicans genome, was assembled onto the
substrate linker functionalized fibers. Hybridization and staining
protocols as reported in example 9 were followed. The response of
the sensor to cDNA (20 nucleotides in length) is shown in FIG. 13.
Linear calibration (r.sup.2=0.988), good sensitivity (100%
fluorescence intensity increase per 89 pM increase in concentration
in the 4 ml of solution surrounding the optical sensor) and low
detection limits (6.times.10.sup.10 molecules) were observed for
the device.
Example 13
[0186] Fabrication of Optical Sensors with Immobilized
Polythymidilic Acid Icosanucleotides Functionalized at the
5'-terminus with N5-Tethered 3,8-Diamino-6-phenylphenanthridium
Cation.
[0187] i) Synthesis of methyl-(12-hydroxy)dodecanoate.
[0188] 12-hydroxydodecanoic acid (5 g) was dissolved in 100 ml of
dry methanol to which was added a solution of p-toluenesulfonic
acid (88 mg) in 5 ml of methanol drop-wise over a 15 minute
time-span. The solution was refluxed for 16 hours after which time
the solvent was removed under reduced pressure. The product was
then twice extracted into chloroform from a 5% aqueous solution of
sodium bicarbonate. The organic phase was recovered, dried over
NaSO.sub.4, and the solvent removed under reduced pressure.
[0189] ii) Tosylation of methyl-(12-hydroxy)dodecanoate
[0190] Methyl-(12-hydroxy)dodecanoate (1.6 g, 7 mmol) was placed in
and oven dried flask cooled under anhydrous argon and treated with
3 ml of a solution of p-toluenesulfonyl chloride (1 eq., 7 mmol,
1.31 g) in dry pyridine. The solution was stirred at 25.degree. C.
under an inert atmosphere for 16 hours. The solvent was then
removed under reduced pressure and the tosylated product was stored
dry at -20.degree. C. until needed.
[0191] iii) N-Alkylation of 3,8-dinitro-6-phenyl-phenanthridine
with the tosylate of methyl-(12-hydroxy)dodecanoate.
[0192] 3,8-Dinitro-6-phenyl-phenanthridine (3.5 mmol, 1.2 g) was
combined with the tosylated methyl-(12-hydroxy)dodecanoate (7 mmol,
2.7 g) in dry nitrobenzene and the solution stirred for 6 hours at
160.degree. C. under an argon atmosphere. The alkylated quaternary
ammonium salt was precipitated from the mother liquor by addition
of diethyl ether and collected by filtration. The product was
further purified by silica gel column chromatography (25% methanol
in chloroform) and recovered as a dark purple solid.
[0193] iv) Reduction of
3,8-dinitro-5-methyldodecanoate-6-phenyl-phenanthr- idium
Chloride
[0194] 3,8-Dinitro-5-methyldodecanoate-6-phenyl-phenanthridium
chloride (1.3 mmol, 0.72 g) was dissolved in 10 ml of THF and
stirred over NiCl.sub.2.6H.sub.2O (10.68 g) and powdered Al (0.81
g). Water (0.3 ml) was then added to initiate the formation of the
black Ni/Al catalyst and the reaction allowed to proceed for 15
minutes. The solution containing the reduced product was recovered
by filtration, followed by removal of the solvent under reduced
pressure. The product was purified by silica gel column
chromatography (25% methanol in chloroform) and recovered as a dark
purple solid (4%, 0.03 g).
[0195] v) Tritylation of
3,8-diamino-5-methyldodecanoate-6-phenyl-phenanth- ridium
Chloride
[0196] 3,8-diamino-5-methyldodecanoate-6-phenyl-phenanthridium
Chloride (0.03 g, 62 .parallel.mol) was dissolved in dry pyridine
and treated with dimethoxytrityl chloride (3 eq, 68 mg) suspended
in dry pyridine (4 ml). The reaction was allowed to proceed for 16
hours at 25.degree. C. with stirring under an inert atmosphere. The
solvent was then removed under resuced pressure and the product
purified (58%, 36 .mu.mol) by reverse phase HPLC (isocratic elution
with 1:1 methanol/water).
[0197] vi) Deprotection of the methyl-ester Protecting Group on
3,8-Bis(dimethoxytritylamino)-5-methyldodecanoate-6-phenyl-phenanthridium
Chloride
[0198]
3,8-Bis(dimethoxytritylamino)-5-methyidodecanoate-6-phenyl-phenanth-
ridium chloride (36 .mu.mol) was suspended in 80 ml of a solution
of 1:3 water/methanol. The solution was degassed and treated with
KOH (4 eq., 160 .mu.mol) for 16 hours with stirring at 25.degree.
C. The reaction was quenched and the pH neutralized by treatment
with HCl (1 eq, 15 .mu.l of conc.).
[0199] vii) Synthesis of 5'-aminohexyl-dT.sub.20 Functionalized
Optical Sensors.
[0200] DMT-HEG-GOPS functionalized optical fibers (prepared
according to the method of Examples 2) were functionalized with
polythymidilic acid icosanucleotide (according to the method of
example 5) terminated an N-trifluoroacetamide protected aminohexyl
moiety at the 5'-end by use of the commercially available Aminolink
2.RTM. phosphoramidite synthon from ABI. Deprotection of the
phosphate blocking groups from the immobilized oligomers was
achieved by standing the fibers in a solution of 2:3 (v/v)
triethylamine/acetonitrile at room. temperature for 1.5 hours.
Removal of the trifluoroacetamide protecting group on the
aminohexyl functionality located at the 5'-end of the immobilized
strands was done by exposing the fibers to a 10.sup.-3 M solution
of sodium borohydride in absolute ethanol for 1 hour at room
temperature. The fibers were then washed once in a solution of
10.sup.-3 M HCl followed by washing with copious amounts of sterile
water.
[0201] viii) Attachment of the Trityl-Protected Tethered Ethidium
Analogue to the Aminohexyl Functionalized Optical Fibers:
[0202] The fully deprotected fused silica optical fibers
functionalized with 5'-aminohexyl polythymidilic acid
icosanucleotides were immersed in a solution containing 5 mg of the
DMT-protected tethered ethidium analogue, 40 .mu.l of
1-methylimidazole, and 1.91 g of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 50 ml of water.
After a 7 day incubation period at room temperature, the fibers
were washed five times each with 50 ml portions of water, ethanol,
and dichloromethane, respectively. The proportion
dye-functionalized oligonucleotides was determined by measurement
of the amount of dimethoxytrityl released from each detritylation
step during automated synthesis and that from the deprotection
procedure used to restore the primary amine moieties on the dye.
From these assays it was determined that 63% of the immobilized
oligonucleotides were functionalized with tethered dye.
[0203] ix) Characterization of the Fluorescence Response of the
Reagentless Sensors with Tethered Fluorophore:
[0204] The response of the reagentless sensor to 720 ng of
complement DNA is shown in FIG. 14. Hybridization was done at
40.degree. C. in a buffer consisting of 1 M NaCl and 50 mM
phosphate (pH 7.0). It should be noted that this sensor has a
significantly improved response time over the sensors without
tethered dye. 99% of the full analytical signal was reached in c.a.
6 minutes after injection of the complementary strands for the
reagentless system while 45 minutes was required for full signal
generation by the sensors without tethered dye (see FIG.
10{a}).
Example 14
[0205] Detection of TAT Triple-Helical DNA Using a Fiber Optic
Biosensor
[0206] i) Background
[0207] One important avenue not yet explored by the fiber-optic
nucleic acid biosensor community is the investigation of
triple-stranded oligonucleotide formation. Typically, a number of
spectroscopic techniques (CD, NMR, UV and fluorescence
spectroscopy) in addition to gel mobility shift assays need to be
implemented in order to study the formation of triple-helical
nucleic acids. However, each of these methods have problems in
terms of either the amount of material that is required for
analysis (NMR, CD, and gel mobility assays), or that they are
limited to investigations of only certain triplex systems (e.g.
only TAT triplexes can be monitored by UV absorption spectroscopy
at 284 nm).
[0208] Various groups have developed methods for triplex detection
({i} Geselowitx, D. A.; Neumann, R. D. Bioconjugate Chem., 1995, 6,
502. {ii} Bates, P. J., Dosanjh, H. S.; Kumar, S.; Jenkins, T. C.;
Laughton, C. A.; Neidle, S. Nucleic Acids Res., 1995, 23, 3627.).
The use of nucleic acid binding ligands to identify DNA structures
and morphology is one such method. Many ligands are known to
interact in a noncovalent manner with the target oligonucleotide.
Binding modes can be characterized as: (i) intercalation of the
ligand, in which typically a planar aromatic moiety slides between
the DNA bases--stabilized by .pi.-.pi. stacking and dipole
interactions, or (ii) minor or major groove interaction which is
stabilized by hydrogen bonding, hydrophobic and/or electrostatic
interactions (Long, E. C.; Barton, J. K. Acc. Chem. Res. 1990, 23,
273.). Ethidium bromide binds to both duplexes and triplexes via an
intercalative mode (Waring, M. J. Biochim. Biophys. Acta, 1966,
114, 234.), and this has been studied extensively by fluorescence
methods. The fluorescence quantum efficiency of the ethidium cation
increases when intercalated into duplexes (LePecq, J. B.; Paoletti,
C. J. Mol. Biol., 1967, 27, 87.) and triplexes (Mergny, J. L.;
Collier, D.; Rouge, M.; Montenay-Garestier, T.; Hln, C. Nucleic
Acids Research, 1991, 19, 1521., Scaria, P. V.; Shafer, R. H. J.
Biol. Chem., 1991, 266, 5417), however, it has been shown that
there is a marked difference in the binding efficiency and hence
fluorescence intensity between the two types of complexes. LePecq
and Paoletti were the first to observe that the fluorescence
enhancement of ethidium during interaction with the duplex (poly
rA).multidot.(poly rU) was significantly greater than for binding
to the triplex (poly rA).multidot.(poly rU).sub.2 (LePecq, J. B.;
Paoletti, C. C. R. Acad. Sc. Paris 1965, 260, 7033.). More recent
studies have confirmed that the fluorescence intensity of
intercalated ethidium bromide is greater for duplexes than
triplexes of ribonucleic acid, and that a temperature dependence
exists for deoxyribonucleic acids (Mergny, J. L.; Collier, D.;
Rouge, M.; Montenay-Garestier, T.; Hlne, C. Nucleic Acids Res.,
1991, 19, 1521., Scaria, P. V.; Shafer, R. H. J. Biol. Chem., 1991,
266, 5417., Fang, Y.; Bai, C. L.; Zhang, P. C.; Cao, E. H.; Tang,
Y. Q. Science In China (Ser. B), 1994, 37, 1306.). The results of
molecular modeling studies suggest that a reduced binding affinity
of ethidium for triplexes (relative to duplexes) exists due to the
energetic cost of destacking base triplets as compared to
successive base pairs (Sun, J. S.; Lavery, R.; Chomilier, J.;
Zakrzewska, K.; Montenay-Garestier, T.; Hlne, C. J. Biomol. Struct.
Dynam., 1991, 9, 425.). This is partially offset by the quantum
efficiency of ethidium bromide in triplex DNA which is greater than
that for duplex DNA. Short homopolymeric T*AT triplexes have been
the subject of seminal fluorescence studies. Letsinger et al.
(Salunkhe, M., Wu, T. & Letsinger, R. L. (1992) J. Am. Chem.
Soc. 114, 8768-8772.) have shown that for parallel T*AT triplexes,
the fluorescence intensity of ethidium cation decreases
dramatically in comparison to fluorescence intensity of the ligand
bound to AT duplexes. Independent confirmation of decreased
fluorescence intensity for ethidium bound to parallel T*AT
triplexes (2.times.dT.sub.10:dA.sub.10) relative to duplexes
(dT.sub.10:dA.sub.10) has appeared (Fang, Y.; Bai, C. L.; Zhang, P.
C.; Cao, E. H.; Tang, Y. Q. Science In China (Ser. B), 1994, 37,
1306.).
[0209] We chose to investigate both parallel and antiparallel TAT
triplexes as these sequences have been well documented in the
literature (Plum, G. E.; Pilch, D. S.; Singleton, S. F.; Breslauer,
K. J. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 319, and
references therein). Branched nucleic acids as described by Damha
et al. (Hudson, R. H. E.; Damha, M. J. Nucleic Acids Res. Symp.
Ser. 1993, 29, 97., R. Hudson, A. Uddin, and M. Damha J. Am. Chem.
Soc., 1995, 117, 12470.) were used in this study as the unique
architecture of bNAs has been utilized to stabilize
reversed-Hoogsteen and Hoogsteen TAT triplexes. Investigations were
also done to determine the best oligonucleotide orientation on the
support for the detection of TAT triplexes. The motivation behind
this research endeavor was then to create a rapid, reliable,
reproducible assay for the detection of triple-helical nucleic acid
formation. The development of a triple-helical assay, is an
extension of work initiated for the detection of nucleic
hybridization (Watson-Crick motif) using fiber optic total internal
reflection fluorecence (TIRF) sensors functionalized with
single-stranded deoxyribonucleic acid (ssDNA) probes.
[0210] ii) Synthesis of Oligonucleotides on Optical Fibers.
[0211] Optical fibers were activated by protocols given in example
2 and polyadenilic decanucleotides were assembled onto the
substrate linker molecules on the fiber surface as per the methods
given in example 5. Two batches of fiber were created, the first
using commercially available
N.sup.6-phenoxyacetyl-5'-O-DMT-2'deoxyadenosine-3'-O-[(.beta.-cyanoethyl)-
N,N-diisopropyl]phosphoramidite from Millipore Inc. to assemble
decanucleotides with the 5'-terminus oriented away from the fiber
surface.
N.sup.6-phenoxyacetyl-3'-O-DMT-2'deoxyadenosine-5'-O-[(.beta.-cy-
anoethyl)N,N-diisopropyl] phosphoramidite was prepared via standard
protocols and used to grow oligonucleotide on the functionalized
fibers in a reversed (fiber.fwdarw.substrate
linker.fwdarw.5'-dA10-3') orientation.
[0212] iii) Synthesis of Branched Oligonucleotides.
[0213] The "V" branched sequence 1 (FIG. 15) was synthesized on an
Applied Biosystems 381A instrument using a 1 .mu.mol scale
synthesis cycle and .beta.-cyanoethylphosphoramidite chemistry.
Purification, desalting, and analysis of the branched
oligonucleotide 1 by polyacrylamide gel electrophoresis was
accomplished by our detailed protocols (Damha, M. J., Ganeshan, K.,
Hudson, R. H. E., Zabarylo, S. V., (1992) Nucleic Acids Res 20,
6565-6573; Damha, M. J.; Ogilvie, K. K. In Methods in Molecular
Biology, Vol. 20: Protocols for Oligonucleotides and Analogs;
Agrawal, S., Ed.; Humana Press, Inc.: Totowa, N.J., 1993, pp
81-114). Typical yields of this branched oligomer were 5-15
A.sub.260 units (15-25%). The complement dA.sub.10 for thermal
denaturation studies was obtained from Dalton Laboratories
(Toronto, Canada).
[0214] iv) Thermal Denaturation Profiles.
[0215] Absorbance versus temperature profiles of the nucleic acid
complexes were measured at 260 nm using a Varian Cary I UV-VIS
spectrophotometer equipped with a variable temperature cell holder
controlled by an external variable temperature circulating bath.
Data were collected with the spectrophotometer set on dual beam
optical mode to reduce optical drift. The data were collected at
260 nm at 0.5.degree. C. intervals with an equilibration time of 60
s for each measurement point. Absorption coefficients of the
branched molecules were assumed to be similar to their
corresponding linear sequences and were calculated from those of
mononucleotides and dinucleotides according to the nearest-neighbor
approximation (Puglisi, J. D.; Tinoco, I., Jr. In Methods in
Enzymology; Dahlberg, J. E., Abelson, J. N., Eds.; Academic Press,
Inc.: San Diego, 1989; Vol. 180, 304.). Samples for thermal
denaturation analysis were prepared by mixing the pyrimidine
containing strand with the target (2 mM), lyophilizing the solution
to dryness, and dissolving the oligomers in 10 mM Tris, 50 mM
MgCl.sub.2, pH 7.3 adjusted with HCl. The mixtures were then
transferred to Hellma QS-1.000-104 cells. Oligonucleotide solutions
were heated to 80.degree. C. for 15 min and then slowly cooled to
room temperature prior to melting experiments. Normalized plots
were constructed according to the method of Kibler-Herzog et al.
(Kibler-Herzog, L.; Zon, G.; Whiftierm, G.; Shaikh, M.; Wilson, W.
D. Anti-Cancer Drug Des. 1993, 8, 65.) based on
{(A.sub.t-A.sub.o)/(A.sub.f-A.sub.o)}: where A.sub.o is the initial
absorbance, A.sub.f is the final absorbance and A.sub.t is the
absorbance at any temperature. All complexes showed sharp melting
transitions. The melting temperature (T.sub.m) was determined from
the first derivative of each thermal curve. A precision in T.sub.m
values, determined from variance in repeated experiments, of
0.5.degree. C. or better was obtained for all of the denaturation
profiles investigated.
[0216] v) Instrument Setup and Fluorescent Measurements
[0217] The laser radiation exiting the immersion lens of the
fluorescence microscope (as described in Example 9) was coupled
into a delivery fiber of similar numerical aperture (0.48) aligned
beneath the objective, as illustrated in FIG. 4(c). The light was
totally internally reflected along the delivery fiber to a sensing
fiber functionalized with immobilized oligonucleotide. Coupling of
the radiation between fibers was achieved by abutting the distal
terminus of the delivery fiber to the proximal terminus of the
sensing fiber. A loss in optical transmission of no greater than 2%
was observed for the coupled system. The termini of the teflon
fiber coupler were designed as compression-fit ends which provided
a solution-tight seal that prevented contaminants from diffusing
into the fiber coupler and causing drift in the analytical signal.
The sensing fiber was placed in a small volume, stop-flow,
stainless steel hybridization chamber (1.5 mm i.d..times.48 mm)
which provided a solution volume of 79 .mu.l exposed to the sensing
fiber. The temperature of the hybridization cell was controlled by
placing the cell in a thermostated housing in which glycol
solutions from external variable temperature circulating baths were
made to flow. The temperature of the solutions in the hybridization
cell were accurately determined (.+-.0.2.degree. C.) by use of a
glass encapsulated thermistor incorporated into the hybridization
cell and in contact with the solution at the exit of the
hybridization chamber. Solutions containing hybridization buffer,
ethidium bromide, and complementary nucleic acid sequences were
delivered to the hybridization cell and sensing fiber by use of a
peristaltic pump. Fluorescence emission from ethidium bromide that
was intercalated into immobilized nucleic acid complexes was
totally internally reflected within the sensing fiber. The portion
of the light coupled back into the delivery fiber was directed
towards the microscope objective where it was collimated and
directed to the dichroic mirror. The fluorescence radiation was of
longer wavelength (.lambda..sub.max=595 nm) than the dichroic
cut-off, and was transmitted through the mirror and directed
towards a photomultiplier tube, where the fluorescence intensity
could be quantitatively measured. Drift caused by variations in the
efficiency of optical coupling, laser intensity and photomultiplier
gain were obviated by normalization of all signals to that of a
standard solution of ethidium bromide at 25.degree. C. prior to and
at the completion of each analysis.
[0218] vi) PAGE Mobility Retardation Assay.
[0219] The solutions of oligonucleotides were lyophilized to
dryness, incubated in 10 .mu.L of 30% sucrose with 50 mM MgCl.sub.2
at 75.degree. C. for 15 min., and then cooled to room temperature
slowly. After a 4 day incubation at 4.degree. C., the samples were
loaded onto the gel. The running buffer contained 90 mM tris-borate
buffer (pH 8.0). The non-denaturing 15% polyacrylamide gels
contained 90 mM tris-borate (pH 8.0) and 50 mM MgCl.sub.2. The
native gels were run at 12.5 mA for 12 h. Following
electrophoresis, the gels were covered with Saran Wrap.RTM. and
photographed with a Polaroid MP4 Land Camera over a fluorescent TLC
plate (Merck, distributed by EM Science, Gibbstown, N.J.)
illuminated by a UV lamp (Mineralight lamp, Model UVG-54, San
Gabriel, Calif.). Instant Sheet Film (#52, medium contrast, ISO
400/21.degree. C.) was used and the exposure (f4.5, 1.5 s) made
through a Kodak Wratten gelatin filter (#58). The gels were
subsequently stained for 5 min in a 5 .mu.g/ml solution of ethidium
bromide and destained in distilled water for 30 s. The gels were
then covered with Saran Wrap.RTM., illuminated by a UV lamp and
photographed (f4.5, 2 s) through a Hoya orange filter over a white
background.
[0220] vii) Parallel and Antiparallel TAT Triplex
Considerations.
[0221] In the formation of the intermolecular triplex
2.times.dT.sub.10/dA.sub.10 triplex, the third dT.sub.10 strand
interacts by means of Hoogsteen hydrogen bonds with the dA.sub.10
strand in target Watson-Crick duplex, and is oriented parallel to
it. In melting experiments (Mg.sup.+2 buffer), the triplex
2.times.dT.sub.10/dA.sub.10 has two clearly resolved transitions,
one for dissociation of the third strand from the duplex, i.e.,
dT.sub.10*dA.sub.10/dT.sub.10.fwdarw.dT.sub-
.10+dA.sub.10/dT.sub.10 (T.sub.m 18.degree. C.), and one for
dissociation of the duplex into its component strands, i.e.,
dA.sub.10/dT.sub.10.fwdar- w.dA.sub.10+dT.sub.10 (T.sub.m
32.degree. C.). Thus for this complex, association of the third
(dT.sub.10) strand with the duplex (dA.sub.10/dT.sub.10) is
thermodynamically weaker than duplex formation itself.
[0222] Branched oligonucleotides are useful probes for stabilizing
triplex DNA (R. Hudson, A. Uddin, and M. Damha J. Am. Chem. Soc.,
1995, 117, 12470.). The branched oligomer 1 (FIG. 15) for instance,
binds to dA.sub.10 to give a novel TAT triple-stranded complex in
which both dT.sub.10 strands are antiparallel to the purine
(dA.sub.10) strand. Although this motif had been observed for TAT
bases in complexes dominated by pur.circle-solid.pur/py bonding
(e.g. G.circle-solid.GC, A.circle-solid.AT) ({i} Moser, H. E.;
Dervan, P. B. Science, 1987, 238, 645. {ii} Strobel, S. A.;
Doucettestamm, L. A.; Riba, L.; Housman, D. E.; Dervan, P. B.
Science, 1991, 254, 1639 {iii} Hoogsteen, K. Acta Crystallogr.
1959, 12, 822-823; (b) Felsenfeld, G.; Davies, D. R.; Rich, A. J.
Am. Chem. Soc. 1957, 79, 2023-2024; {iv} R. Hudson, A. Uddin, and
M. Damha J. Am. Chem. Soc., 1995, 117, 12470.), it had not been
observed previously for dT.sub.n/dA.sub.n complexes. The formation
of this triplex was induced by linkage of two dT.sub.10 strands
through their 5' ends via coupling to riboadenosine at the
neighboring 2' and 3' oxygen atoms (FIG. 15). This arrangement
causes the initial direction of the two dT.sub.10 strands to be
parallel, and forces the formation of a triplex in which the third
dT.sub.10 strand runs antiparallel to the dA.sub.10 strand, and is
bound to it via reversed-Hoogsteen interactions. Thermal
denaturation profiles of a mixture of 1 and dA.sub.10 (1:1) in
Mg.sup.+2 buffer, show a single transition from bound to unbound
complex, consistent with its formation involving one rather than
two bimolecular reaction steps, ie., 1+dA.sub.10.fwdarw.triplex
1/dA.sub.10 (T.sub.m 35.degree. C.).
[0223] viii) Triplex Studies Using Derivatized Optical Fibers with
Normal (Fiber-3'-dA.sub.10-5') Oligonucleotide Orientation.
[0224] Characterization of the triple-helical complexes via thermal
denaturation studies were done. The dA.sub.10 was grown in the
conventional 3'-to-5' direction from the fiber surface. Solutions
of ethidium bromide, ethidium bromide with dT.sub.10, or ethidium
bromide with 1 were heated in the hybridization chamber containing
the decaadenylic acid functionalized optical fibers. Upon slow
cooling, fluorescent measurements were taken at various
temperatures. FIG. 16(a), illustrates that as the
dT.sub.10:dA.sub.10 duplex was formed by lowering the temperature,
there was an increase in the fluorescence intensity corresponding
to ethidium bromide intercalation and quantum yield enhancement of
the ligand in this complex. After further lowering of the
temperature, we observed a decrease in the fluorescence intensity
with decreasing temperature, indicative of exclusion of the ligand
as a result of triplex formation (2.times.dT.sub.10:dA.sub.10).
This process is illustrated in FIG. 7(a). In order to verify that
triplex formation was alone responsible for the exclusion of the
ethidium cation and hence the decrease in fluorescence intensity
from the fiber, a control experiment was done using optical fibers
functionalized with a twenty-nucleotide probe sequence of mixed
base composition incapable of forming triplex structures. The
hybridization experiment was done using the same methodology as for
the decaadenylic acid functionalized fibers with the exception of
the hybridization buffer (1 M NaCl, 50 mM PO.sub.4, pH 7.0). Having
this nucleic acid sequence and buffer composition, only
double-stranded complexes could form between the immobilized probe
sequence and the complementary sequence. As can be seen in FIG. 6,
a fluorescence intensity with a negative temperature coefficient
was observed for the duplex system over the temperature range
studied. The denaturation temperature for this system of nucleic
acids and hybridization buffer was determined to be 73.degree. C.
by UV-visible thermal denaturation studies. Only double-stranded
complexes existed over the temperature range investigated, as
indicated by the enhanced fluorescence intensity for the experiment
using the ethidium bromide and complementary oligonucleotide. The
control experiment with ethidium bromide and no complementary
oligonucleotide showed no such dramatic increase in intensity.
[0225] Upon exposure of the optical sensor to the
reversed-Hoogsteen forming 1, no significant increase in
fluorescence intensity over that of the ethidium bromide alone in
solution was observed. The geometrical constraints of compound 1
are such that, if a complex is formed with the immobilized
dA.sub.10 strand in this particular (fiber-3'-5') orientation, the
branch-point riboadenosine should be oriented toward the fiber
surface thus presenting a steric barrier to triplex formation. In
order to test whether steric interference surrounding the
branch-point prevented triple-helical formation, an optical fiber
having a dA.sub.10 strand in the opposite orientation from the
surface (i.e. fiber-5'.fwdarw.3') was synthesized.
[0226] ix) Triplex Studies Using Derivatized Optical Fibers with
Reversed (Fiber-5'-dA.sub.10-3') Oliqonucleotide Orientation.
[0227] From FIG. 16(b), the fluorescent intensity versus
temperature profile indicated that with dT.sub.10 there was an
initial increase in fluorescence indicative of duplex formation.
This was followed by a decrease in the intensity which was
indicative that triplex formation had occurred. Upon treatment of
the optical sensor with 1, a decrease in fluorescence intensity for
the reverse Hoogsteen complex at temperatures below the T.sub.m
(35.degree. C.) was observed FIG. 16(c), which is consistent with
triplex formation. From the data of Scaria and Shafer (Scaria, P.
V.; Shafer, R. H. J. Biol. Chem., 1991, 266, 5417), it can be
inferred that a temperature below 25.degree. C. is required for the
ethidium cation exclusion process to dominate the fluorescence
intensity. Given that the quantity of ethidium cation that can be
accommodated by triplexes is lower than that for duplexes (where
intercalation occurs once every 2.8 base triplets and once per 2.4
base pairs at 25.degree. C.) a 14% reduction in the amount of
intercalated ethidium upon triple-strand formation results (Scaria,
P. V.; Shafer, R. H. J. Biol. Chem., 1991, 266, 5417). However,
within the triplex structure the fluorescence quantum yield of the
intercalated ethidium cation has been observed to increase by 19%
for the S.sub.1.fwdarw.S.sub.0 transition, thereby resulting in an
overall change in fluorescence intensity of +2.3%. Therefore,
direct correlation between the T.sub.m for triplex formation and
the onset of decreased fluorescence intensity from the optical
sensor will be observed for systems of nucleic acids which have
T.sub.m values at or below 25.degree. C. This is consistent with
our findings, FIGS. 16(a and b) where the decrease in fluorescence
intensity from the sensor correlates well with the 17.degree. C.
T.sub.m for triplex formation. As can be seen in FIG. 16(c),
although the transition for triple-strand formation between 1 and
the immobilized dA.sub.10 occurs at 35.degree. C., a decrease in
fluorescence intensity was not observed until the system was cooled
to below 25.degree. C. In this regard, our fluorescence studies
involving ethidium bromide binding to triple-helices is in full
agreement with several earlier findings. However, our system is
then limited in terms of being able to identify only the duplex to
triplex transition temperature for nucleic acid systems with
T.sub.m values at or below 25.degree. C.
[0228] x) PAGE Mobility Retardation Assay.
[0229] Gel-shift experiments provided us with the opportunity to
confirm the interaction of ethidium bromide with the complexes
observed in these studies. The electrophoretic mobility of the
Watson-Crick base paired dT.sub.10/dA.sub.10 duplex, (both the
Hoogsteen and reverse-Hoogsteen paired TAT triplexes, and that of
their component strands), was studied at 4.degree. C. in a buffer
containing magnesium. Following electrophoresis, the gels were
visualized by UV shadowing, and by staining with ethidium bromide,
as shown in FIGS. 17(a and b), respectively. The Hoogsteen triplex
migrated more slowly than the duplex due to the presence of the
third dT.sub.10 strand. The reversed-Hoogsteen triplex has the
slowest mobility of all, and is characteristic of branched nucleic
acid structures [ref Hudson and Damha, JACS 1993; Wallace, J. C.;
Edmons, M. PNAS vol. 80, 950-954, 1983). Association of 1 and
dA.sub.10 was quantitative as evidenced by the complete
disappearance of compound 1 and dA.sub.10, when mixed in equimolar
amounts. The stoichiometry of interaction between dT.sub.10 and
dA.sub.10 for the duplex and Hoogsteen triplex was also confirmed
by studies at different concentrations of the two oligonucleotides.
When the gel shown in FIG. 17(a) was stained with ethidium bromide
and illuminated by a UV lamp, fluorescence was observed only in the
bands corresponding to the complexes (not single strands). This is
consistent with the well-known intercalation mechanism of ethidium
bromide (Lim, C. S. BioTechniques, 1994, 17, 626.). As previously
suggested by the biosensor studies, the 1/dA.sub.10 reverse
Hoogsteen triplex gave the lowest fluorescence intensity, which
could be related to the limited ability of ethidium to bind to this
complex.
Example 15
[0230] Optical Sensors Which Function by the Intrinsic Mode of
Operation.
[0231] Background
[0232] Angularly dependent light scattering experiments were done
to determine the refractive index of oligonucleotide monolayers
covalently immobilized onto fused silica substrates. With knowledge
of the refractive index of the immobilized oligonucleotide film,
the mode of operation of the devices, namely, intrinsic or
evanescent total internal reflection fluorescence, can be
elucidated. The concept of the experiments done is based on
classical optical theory with respect to how alterations in the
direction of a ray or collimated beam of light traversing an
interface between two dielectric materials may be predicted based
on the difference in the refractive index of the two materials, and
vice versa. In particular, Snell's law of refraction states that
for a ray of light traveling in a plane normal to that defined by
the interface between two materials of refractive index n.sub.1 and
n.sub.2, the angular trajectory of the transmitted ray,
.theta..sub.t, from the normal to the interfacial plane will differ
from that of the incident ray, .theta..sub.i, by a quantity
dependent on the difference in refractive index of the two
materials. This can be solved for mathematically using the
following equation (Ohanian, H. C.; Physics, W. W. Norton and
Company, New York (1985) p. 837):
n.sub.1.multidot.sin(.theta..sub.i)=n.sub.2.multidot.sin(.theta..sub.t)
(4)
[0233] FIG. 18(a) illustrates this concept for the case where the
upper medium is fused silica and the lower medium is the ambient,
as characterized by n.sub.Fused Silica and n.sub.Ambient,
respectively, where n.sub.Fused Silica>n.sub.Ambient. As shown
in FIGS. 18(a) and 18(b), as the angle of incidence is increased,
the angle of the refracted beam will be deflected by increasing
amounts toward the interface, where at all times
.theta..sub.i<.theta..sub.t. This trend is continued to the
point where the refracted beam is directed into the interfacial
plane (i.e. .theta..sub.t=90.degree.). The angle of incidence for
which this occurs is known as the critical angle, .theta..sub.c,
and can be calculated from the following relation: 5 c = sin - 1 (
n Ambient n Fused Silica ) ( 5 )
[0234] For the case where .theta..sub.j.gtoreq..theta..sub.c, the
incident ray undergoes total internal reflection (TIR) at the
interface. The angle of the reflected beam with respect to the
normal to the interface is then equal to that of the angle of
incidence for all .theta..sub.i.gtoreq..the- ta..sub.c, as
illustrated in FIG. 18(c).
[0235] If a detector for optical radiation was placed directly
beneath the intersection point of the light ray with the interface
and intensity was recorded as a function of incidence angle, a
continuously decreasing intensity with increasing incidence angle
would be observed. This observation is the result of the refracted
beam being increasingly deflected out of the optical axis of the
detector. An illustration depicting the trends in detector response
is included on the right hand side of each ray diagram in FIGS.
18-20. As the refracted beam closely approaches the interface
between the two dielectric materials, a local maxima in the
detector response would be observed as a result of the beam being
scattered by surface roughness and other imperfections at the
interface. This would continue until .theta..sub.i=.theta..sub.c,
after which point the incident beam would undergo TIR and hence
provide negligible amounts of signal to the detector, with the
exception of that which leaks out via scatter from imperfections at
the interface and within the waveguiding media. As such, the
critical angle for TIR can be determined directly from this point
on the plot of scatter intensity versus incidence angle (FIG.
18(c)). For the case where the refractive index of one of the media
is known, the refractive index of the other can be solved using
equation (5).
[0236] A three-layer model must be considered for the case where a
thin film of organic material is placed at the interface, as shown
in FIGS. 19 and 20. Each medium type is herein characterized by the
refractive index of the material, as given by n.sub.Fused Silica,
n.sub.Film, and n.sub.Ambient, respectively, for the fused silica,
organic film, and ambient. The interaction of a light ray at each
interface must be considered independently. An incident ray in the
fused silica medium at an angle .theta..sub.j relative to the
interfacial normal will be refracted to a differing angle after
traversing each interface. The propagation angle of the ray will
then be .theta..sub.t.sub..sub.Film and
.theta..sub.t.sub..sub.Ambient in the organic film and ambient
media, respectively, relative to the interfacial normal.
[0237] For the case where n.sub.Fused
Silica>n.sub.Film>n.sub.Ambien- t, the reciprocal trend will
be observed with respect to the propagation direction of the
refracted rays, where .theta..sub.i<.theta..sub.t.sub-
..sub.Film<.theta..sub.t.sub..sub.Ambient, as shown in FIG. 19.
As .theta..sub.i is increased, a local maxima in the detector
response will be observed as .theta..sub.t.sub..sub.Film passes
through the critical angle for TIR at the film-ambient interface.
This is illustrated in FIGS. 19 (b and c). A second local maxima
will be revealed as .theta..sub.i passes through the critical angle
for TIR at the fused silica-film interface, as shown in FIG. 19(d).
Given n.sub.Fused Silica and n.sub.Ambient, the refractive index of
the organic film can be directly determined from analysis of traces
of scatter intensity versus .theta..sub.i. The critical angle for
the TIR at the fused silica-film interface can be directly obtained
from the point where the second local maxima intersects the
baseline scatter intensity, as described previously for the
two-layer model and shown in FIG. 19(d). By substituting
.theta..sub.c.sub..sub.Fused Silica/Film and n.sub.Fused Silica
into equation 5, n.sub.Film can be solved for directly.
Verification of this result can be acquired by substituting the
calculated value of n.sub.Film and n.sub.Ambient, and into equation
5 to determine the value of .theta..sub.c.sub..sub.Film/Ambient.
Using the value of .theta..sub.i from the point where the first
local maxima intersects the baseline scatter intensity, n.sub.Fused
Silica, n.sub.Film and equation 4, a second method for calculating
.theta..sub.c.sub..sub.Film/Ambient is provided. The goodness of
agreement between the two values of
.theta..sub.c.sub..sub.Film/Ambient would indicate the validity of
the calculated value of n.sub.Film.
[0238] For the case where n.sub.Fused
Silica<n.sub.Film>n.sub.Ambien- t, an estimate of the value
for n.sub.Film may be attained provided the values of n.sub.Fused
Silica, n.sub.Ambient are known. A direct determination of
n.sub.Film cannot be achieved in this case as TIR will not occur at
the interface between the fused silica and organic film for light
incident in the fused silica, yielding no mechanism for the
determination of .theta..sub.t.sub..sub.Film. The ray diagrams and
detector response trends for this scenario are shown in FIG. 20. A
slight underestimate for the value of n.sub.Film may be had by
assuming that .theta..sub.c.sub..sub.Film/Ambient is equal to the
value of .theta..sub.i at the termination point of the local maxima
from the plot of scatter intensity versus incidence angle. This
overestimate of .theta..sub.c.sub..sub.Film/Ambient and
n.sub.Ambient can be used in equation 5 to provide an underestimate
of the value of n.sub.Film. This value of n.sub.Film along with
those for n.sub.Fused Silica and .theta..sub.i can then be
substituted into equation 4 to provide an underestimate of
.theta..sub.c.sub..sub.Film/Ambient. This underestimate of
.theta..sub.c.sub..sub.Film/Ambient and n.sub.Ambient can again be
used in equation 5 to provide an overestimate of n.sub.Film. An
average of these two values should provide a good estimate of the
true value of n.sub.Film to within the uncertainty limits set by
the low and high value extremes.
[0239] Materials and Methods
[0240] Planar Suprasil.RTM. fused silica wafers (Heraeus Amersil,
Duluth, Ga., USA) with dimensions of 10.times.5.times.1 mm, a
refractive index of 1.46008 and a surface flatness of 10
waves/inch, were functionalized with substrate linker molecules by
the methods of examples 2 and 3. Similarly, silicon wafers (Heraeus
Amersil, Duluth, Ga., USA) with dimensions of 10.times.5.times.1 mm
were functionalized with substrate linker molecules by the methods
of example 3. Polythymidilic acid icosanucleotides were then
assembled onto the functionalized wafers by automated solid-phase
oligonucleotide synthesis, as per the methods provided in example
5. All water used in the light scattering experiments was obtained
from a Milli-Q 5 stage cartridge purification system (Millipore
Corp., Mississauga, ON, Canada) and had a specific resistance not
less than 18 M.OMEGA..multidot.cm. The hybridization buffer was the
same as that used for hybridization experiments on optical fibers
and described in example 8. The refractive index of the
hybridization buffer was determined by use of an Bausch & Lomb
Abbe-3L Refractometer (Fisher Scientific, Nepean, ON, CA) to within
the reported accuracy of 0.0001. Octadecyltrichlorosilane (OTS),
ethylene glycol, hexadecane, carbon tetrachloride, chloroform and
cyclohexane were of analytical grade or better from Aldrich
Chemical Co. (St. Louis, Mo., USA) and used as received unless
stated otherwise.
[0241] OTS Functionalization of Fused Silica Wafers
[0242] Fused silica wafers were cleaned by treatment with solutions
of NH.sub.4OH/H.sub.2O/H.sub.2O.sub.2 and HCl/H.sub.2O/
H.sub.2O.sub.2 respectively, as per the method detailed in example
2 (i). Prior to use, carbontetrachloride and chloroform were dried
by reflux over P.sub.2O.sub.5 under an argon atmosphere followed by
distillation under the same conditions. Functionalization of the
substrates with OTS monolayers was then done as per the methods of
von Tscharner and McConnell (von Tscharner, V. and McConnell, H.
M., Biophys. J., 36 (1981) 421) and as described in the following.
The cleaned substrates were treated with a solution of 80%
hexadecane, 12% carbon tetrachloride, 8% chloroform and 0.1% OTS
(v/v) for 15 minutes at 25.degree. C. with stirring under an
anhydrous argon atmosphere. The reaction mixture was then decanted
and the functionalized fused silica wafers were then washed thrice
with distilled chloroform and stored in-vacuo and over
P.sub.2O.sub.5 until required.
[0243] Instrumentation Used for Light Scattering Experiments
[0244] Wafers were placed in a custom-built stop-flow cell, beneath
a Harrick EA 7X89 fused silica hemispherical prism with a radius of
8 mm (Harrick Scientific Corp., Ossington, N.Y., USA), as
illustrated in FIG. 21. Optical contact between the fused silica
hemispherical prism and fused silica wafer functionalized with an
oligonucleotide monolayer was made by applying a thin film of
fluorescence free Zeiss Immersionsoel 518C refractive index
matching oil (n=1.515, Carl Zeiss Canada Ltd., Don Mills, ON, CA)
at the interface between the two. The other face of the wafer was
exposed to a solution compartment with dimensions of
9.times.2.times.1 mm (l.times.w.times.h). The flow cell was mounted
at the vertex of a modified goniometer element obtained from a type
43702-200E Thin Film Ellipsometer (Rudolph Research Corporation,
Flanders, N.J., USA) with an angular accuracy and precision of
0.005.degree.. 543 nm optical radiation from a Gre-Ne.TM. Laser
(Melles Griot, Carlsbad, Calif., USA, 1 mW output power, 1.5 mm
beam diameter, 0.01 mrad beam divergence) mounted on one arm of the
goniometer was passed through the hemispherical prism and impinged
on the planar fused silica wafer. The hemispherical prism
guaranteed that alterations in the incidence angle owing to
refraction at the air-prism interface were eliminated as the beam
invariably entered the prism normal to the prism/air interface. A
M062-FC03 Slo-Syn stepping motor (Superior Electric Co., Bristol,
Conn., USA, 200 steps per revolution) was coupled via a set of
gears to the screw shaft of the goniometer used to drive the
pivoting mechanism of the goniometer arms. The gear ratio used
provided the motor with a 7.times. mechanical advantage so as to
reduce the load on the motor and prevent it from slipping. TTL
signals from a standard PC parallel interface were used to operate
the advance mechanism of the stepper motor so as to offer accurate
control of the angle of the goniometer arms and incidence of the
laser beam. One end of a fiber-optic bundle (Oriel Corp, Stratford,
Conn., USA, model no. 77533) was mounted in the base of the flow
cell ca. 1 mm from the exposed face of the fused silica wafer. The
other terminus of the fiber bundle was directed to a 630 nm
long-pass colloidially-colored glass filter (Schott Glass
Technologies, Duryea, Pa., USA) placed before the window of an
R-928 photomultiplier tube (Hamamatsu Corp., Bridgewater, N.J.,
USA) operated using a DZ-112 Photoelectric Indicator (Rudolph
Research, Flanders, N.J., USA). The long-pass filter provided for
attenuation of the light intensity transmitted by the fiber bundle
by a factor of 10.sup.5. This was done in order to prevent an
overload condition in the PMT form occurring, guarantee the
linearity of response and preserve the useful lifetime of the
detector. The current from the PMT was converted to an analog
voltage output (0-5 VDC) from the signal processing electronics
contained within the Photoelectric Indicator and passed to a 12 bit
analog to digital converter (Metra-Byte, Taunton, Mass., USA) for
data acquisition on a PC computer using software created in-house
to acquire plots of intensity versus incidence angle.
[0245] Results and Discussion
[0246] In order to test the validity of the light scattering
approach for refractive index determination, samples of known
refractive index were introduced into the flow cell beneath the
hemispherical prism and analyzed by ramping the incidence angle of
the laser mounted on the goniometer arm from low to high incidence
angles while recording the observed scatter intensity. The results
of experiments done using samples of air (n=1.0003), water
(n=1.33), and cyclohexane (n=1.4266) are shown in FIGS. 22(a-c),
respectively and summarized in Table 2. Knowing the refractive
index value of the prism material (fused silica, n=1.46) and the
analyte, equation 5 was used to determine the critical angle values
for TIR in each system. Good correlation with the predicted values
was observed in all three cases with no more than 1% error between
the experimental and theoretical values for .theta..sub.c. An
unmodified fused silica wafer was then coupled to the base of the
prism and the same three control samples were again analyzed.
[0247] Identical results within the resolution of the experimental
technique were observed between the theoretical and observed values
of critical angle for analyses done with and without the fused
silica wafer. This indicated that no additional modifications to
the instrument or correction factors would need be applied as a
result of moving the intersection point of the laser beam 1 mm
below the base of the prism. Hybridization buffer (n=1.35) was also
analyzed by the light scattering method (FIG. 22(g)) and provided
good agreement with the calculated value for .theta..sub.c based on
the refractive index of the buffer as determined using a standard
Abbe refractometer.
2TABLE 2 Summary of the Results from the Angularly Dependent Light
Scattering Experiments and Correlation with Controls of Known
Refractive Index. Interface Type .eta..sub.Ambient Experimental
.theta..sub.c Calculated .theta..sub.c % Error Fused Silica 1.0003
42.8.degree. 43.2.degree. 0.9 Prism-Air Fused Silica 1.33
65.6.degree. 65.6.degree. 0.0 Prism-Water Fused Silica 1.427
77.8.degree. 77.8.degree. 0.0 Prism- Cyclohexane Fused Silica 1.35
67.7.degree. 67.6.degree. 0.1 Prism-Hybrid- ization Buffer Fused
Silica 1.0003 43.2.degree. 43.2.degree. 0.0 Wafer-Air Fused Silica
1.33 65.6.degree. 65.6.degree. 0.0 Wafer-Water Fused Silica 1.427
77.9.degree. 77.8.degree. 0.1 Wafer- Cyclohexane
[0248] Experiments were then done using films of organic media of
known refractive index in order to test the validity of the
technique as applied to the previously described the three-layer
model. Thin films (10-50 .mu.m) of refractive index matching oil
and ethylene glycol were applied to the exposed surface of the
hemispherical prism and analysis was then done where air was used
as the ambient in both cases. The results of the light scattering
experiments for these samples are shown in FIGS. 23a and 23b,
respectively. As can be seen in FIG. 23a, the experimentally
determined value of .theta..sub.C.sub..sub.Film/Ambient
=41.7.degree. for the oil-air interface, based on a value of
.theta..sub.i of 43.5.degree., and that predicted from theory, well
agree after taking into account refraction of the beam upon
traversing the fused silica-oil interface. However, for this
particular example, the two-step approximation method for
determination of n.sub.Film cannot be used in this example. The
first assumption that .theta..sub.i=.theta..sub- .t used in this
treatment leads to a first estimate of n.sub.Film which is lower
than that of n.sub.fused silica. This would lead to the result that
the transmitted beam in the oil would be refracted away from the
normal as opposed to towards the normal, as would be the case for
n.sub.Film>n.sub.Fused Silica. This causes the next
approximation of n.sub.Film to be a more exaggerated underestimate
of the true value. As such, films of refractive index slightly
greater than that of the fused silica substrate cannot be solved
for.
[0249] The results for the light scattering experiment using an
ethylene glycol film provided very good agreement between the
values of .theta..sub.c at each interface with respect to that
calculated from theory. Of significance in this plot of scatter
intensity versus incidence angle is the appearance of two distinct
maxima. The observation of the two maxima concurs with that
proposed for the three-layer model (FIG. 19) for the case where
n.sub.Fused Silica>n.sub.Film>n.sub.Am- bient. As such,
information with regard to whether the refractive index of the
organic film is greater than or less than that of the substrate
material can be obtained by quick inspection.
[0250] A monolayer film of OTS was covalently attached to the
surface of a fused silica wafer by a method previously shown to
provide dense surface packing and a theoretical refractive index in
the range of 1.4-1.6. ({a}Ducharme, D. et al., J. Phys. Chem, 94
(1990) 1925. {b} von Tscharner, V. and McConnell, H. M.; Biophys
J., 36 (1981) 421). The results of the light scattering experiments
are shown in FIGS. 23(c) and 23(d), respectively, for OTS
functionalized fused silica wafers exposed to air and water as the
ambient. Using the following rearrangement of equation 5: 6 n Film
= n Ambient sin c Film / Ambient ( 6 )
[0251] the value of the refractive index for the OTS monolayer
could be solved for. Values of 1.44 and 1.45 for the refractive
index of the monolayer were determined from the analyses using air
and water as the ambient, respectively. Given that the refractive
index values determined for the OTS monolayer differed by .about.1%
with that of the fused silica substrate, the same limitation as
observed for the refractive index matching oil layer applies
herein. As such, it can only be assumed that the refractive index
of the OTS overlayer is only slightly greater than that of the
fused silica. This is reinforced by the fact that of only one local
maxima in the plot of scatter intensity versus incidence angle was
observed. If the film refractive index was indeed less than that of
the fused silica substrate then two local maximas should have been
observed, in accord with the three-layer model concept and as
clearly demonstrated by the experiment using ethylene glycol for
the film (FIG. 23(b)).
[0252] Samples of fused silica wafer functionalized with substrate
linker molecules by the methods of example 2 and 3 onto which
polythymidilic acid icosanucleotides were assembled by the method
of example 5 were analyzed by the angularly dependent light
scattering technique. The results of the analysis are shown in
FIGS. 24(a) and 24(b) for samples prepared by the mesylate
activation scheme as detailed in example 3. The results for samples
prepared by the GOPS-HEG protocol given in example 2 are shown in
FIG. 24(c). For the samples prepared by mesylate activation for
which water and 3:1 ethylene glycol in water solution was used as
the ambient, a value of 1.57 was determined in both cases for the
underestimate of n.sub.film, based on the assumption that
.theta..sub.t.sub..sub.Film=.theta..sub.i. Subsequent to the
recalculation of .theta.t.sub..sub.Film based on the underestimated
value of n.sub.Film, overestimates for n.sub.Film of 1.67 and 1.68,
respectively, were determined from the cases where water and 3:1
ethylene glycol in water were used as the ambient. This provided an
average value for n.sub.Film of 1.62.+-.0.05. Similarly, analysis
of the fused silica wafer functionalized with polythymidilic acid
icosanucleotide on GOPS-HEG substrate linkers (example 2) yielded
an average value for n.sub.Film of 1.48.+-.0.01. The fact that
estimates of n.sub.Film for both types of nucleic acid--substrate
linker overcoating could be solved for strongly reinforces the fact
that these overlayers on the fused silica substrates indeed posses
a larger value of refractive index than that of the substrates onto
which they are immobilized.
[0253] In addition to the light scattering experiments,
ellipsometry was done in order to provide secondary confirmation of
the experimentally determined values of the refractive index for
the oligonucleotide monolayers. Ellipsometry was done on samples of
silicon wafer functionalized with substrate linker molecules by the
methods of example 3 onto which molecules of polythymidilic acid
icosanucleotide were assembled by automated solid-phase
oligonucleotide synthesis as detailed in example 5. Silicon wafers
were necessarily used as the substrate material for these
experiments as the fused silica substrates used for the light
scattering experiments provide little reflection of the laser beam
incident at an angle 70.degree. in the ambient. The surface of the
silicon wafers was made similar to that of fused silica via the
cleaning procedure used prior to functionalization of the
substrate. This cleaning procedure is known to provided a layer of
oxidized silicon at the surface of the silicon wafers (Kern, W. and
Puotinen, D. A.; RCA Review, 31 (1970) 187-206). As such, silanol
moieties then present at the oxidized silicon-ambient interface
provide attachment points for the substrate linker molecules.
[0254] McCracken (F. L. McCracken, NBS Technical Note 479,
Washington DC (1969)) has developed software capable of providing
values of thickness and refractive index from ellipsometric
measurements of thin films using the exact Drude equations for
ellipsometry. The Film 85 software provided with the AutoEL-II null
reflection ellipsometer (Rudolph Research Corp., Flanders, N.J.,
USA) was based on that originally developed by McCracken and used
for the analysis of ellipsometric data from the experiments
described herein. Ellipsometric analysis of the cleaned substrate
revealed the formation of a 20 .ANG. thick layer of oxidized
silicon on the surface of the wafers. Three silicon wafers
functionalized with substrate linker and oligonucleotide where then
analyzed. Ten different locations on the wafer surfaces were chosen
at random and the results of the ellipsometric analysis are
summarized below in Table 3.
3TABLE 3 Results of Ellipsometric Analysis of Oxidized Silicon
Substrates Functionalized with Substrate Linker Molecules by the
Methods of Example 3 and Polythymidilic Acid Icosanucleotide by the
Methods of Example 5. Film Refractive Film Thickness (.ANG.) Index
Estimated Estimated Using the Using the Iterative Calculation
Iterative Calculation Corrected Refractive Sample Method of Method
of Index for a Film of Number Program 12. Program 12. 100.ANG.
Thickness. 1 84 2.090 1.76 2 103 1.764 1.81 3 113 1.372 1.55 4 108
1.402 1.51 5 75 2.115 1.59 6 72 2.189 1.58 7 76 2.091 1.59 8 82
2.129 1.75 9 81 2.119 1.72 10 73 2.132 1.56 Average Value .+-.
.sigma. 1.6 .+-. 0.1
[0255] As can be seen by inspection of the data shown in Table 3,
determination of thickness and refractive index concurrently via
the iterative process provides a large egree of variation. This is
based largely on the fact that the covalently immobilized nucleic
acid membrane system is not ideal for ellipsometric analysis in
that it violates many of the assumptions of the Drude equations. Of
particular significance is the fact that a densely packed
oligonucleotide film with the nucleic acid strands oriented
perpendicular to the air-film boundary would be uniaxially
anisotropic. This would cause alterations in the speed of the p-
and s-polarized components of the light beams upon passage through
the oligonucleotide film. This effect has been known to produce
relative errors in thickness of up to 10% (R. M. A. Azzam and N. M.
Bashara, Ellipsometry and Polarized Light, North Holland Publishing
Company, New York (1977)).
[0256] A better estimate of the refractive index of the immobilized
nucleic acid films may be achieved by application of Maxwell-Garnet
theory (R. M. A. Azzam and N. M. Bashara, Ellipsometry and
Polarized Light, North Holland Publishing Company, New York (1977),
p. 359). The concept of Maxwell-Garnet theory, as applied herein,
is based on the notion that a partially formed monolayer film of
coverage .THETA. is optically equivalent to a fully formed
monolayer film of refractive index (n.sub.Film) and relative
thickness (T.sub.t) such that the observed film thickness (T) is
related to that of the fully formed film by:
T=.THETA.T.sub.f (7)
[0257] Likewise, the same scaling factor, .THETA., can be applied
to the refractive index value given from ellipsometric analysis so
that values more representative of that for the actual immobilized
layers can be obtained. The results after applying this correction
are given in Table 3 and provided an average value of 1.6.+-.0.1
for n.sub.Film.
[0258] The good correlation between the result of the light
scattering experiments and ellipsometry provides unequivocal
evidence that monolayers of oligonucleotides can be assembled onto
substrate linker functionalized fused silica substrates of higher
refractive index than that of the substrate material.
Oligonucleotides assembled onto fused silica wafers functionalized
with substrate linker molecules via the mesylate activation scheme,
as outlined in example 3 were observed to provide immobilized
nucleic acid monolayers with a refractive index of 1.62.+-.0.05 by
light scattering investigations. This correlated well to the
refractive index value of 1.6.+-.0.1 obtained by ellipsometric
investigations. Light scattering investigation of oligonucleotides
assembled onto fused silica wafers functionalized with substrate
linker molecules via the methods of example 2, revealed a nucleic
acid film refractive index of 1.48.+-.0.01, which also is higher
than that of the fused silica substrates onto which they are
covalently attached. As such, optical sensors created by the
methods reported herein will then function by and provide the
signal throughput advantages associated with the intrinsic TIRF
motif described previously.
[0259] The present invention has been described in terms of
particular embodiments found or proposed by the present inventor to
comprise preferred modes for the practice of the invention. It will
be appreciated by those of skill in the art that, in light of the
present disclosure, numerous modifications and changes can be made
in the particular embodiments exemplified without departing from
the intended scope of the invention. All such modifications are
intended to be included within the scope of the appended
claims.
[0260] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety. This application also claims priority
from U.S. application Ser. No. 60/050,970 and Canadian application
no. 2,208,165, both of which are incorporated by reference.
Sequence CWU 1
1
1 1 20 DNA Candida albicans 1 taggtgagac atatcacaga 20
* * * * *