U.S. patent application number 09/971867 was filed with the patent office on 2003-06-05 for solid-phase chemical analysis using array hybridization facilitated by agitation during certrifuging.
Invention is credited to Gordon, Gary B..
Application Number | 20030104391 09/971867 |
Document ID | / |
Family ID | 24049470 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104391 |
Kind Code |
A1 |
Gordon, Gary B. |
June 5, 2003 |
Solid-phase chemical analysis using array hybridization facilitated
by agitation during certrifuging
Abstract
Array hybridization can be facilitated by agitating a reaction
cell subject to centrifugal force greater than 1G. A
two-dimensional hybridization array is preferably oriented
generally orthogonal to the centrifugal force. Agitation involves
titling the array back and forth about an axis, preferably parallel
to a centrifuge axis. The centrifugal force serves, in a sense, as
supergravity helping to overcome non-specific binding forces
(viscous forces and other forces at the liquid-solid boundary) that
limit the rate of liquid flow. Thus, the agitation rate and the
related replenishment rate can be increased. The agitation causes
the sample liquid to wash back and forth across the array, which
remains protected by a thin liquid film. The resulting "tidal"
motion, results in thorough mixing of the sample liquid. In
addition, since only a thin film is required over much of the
array, typically costly sample volume can be reduced. Thus, faster
hybridization with lower sample volumes can be achieved.
Inventors: |
Gordon, Gary B.; (Saratoga,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES
Legal Department, DL429
Intellectual Property Administration
P. O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
24049470 |
Appl. No.: |
09/971867 |
Filed: |
October 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09971867 |
Oct 4, 2001 |
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09514975 |
Feb 29, 2000 |
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6309875 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00707
20130101; B01J 2219/00529 20130101; B01J 2219/00722 20130101; B04B
5/02 20130101; C40B 40/06 20130101; B01J 2219/00659 20130101; C40B
60/14 20130101; B01J 2219/0049 20130101; B04B 9/10 20130101; B01J
2219/00596 20130101; B01J 2219/00608 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. An apparatus for array hybridization comprising: a reaction cell
for confining sample liquid and a gas, said reaction cell having an
array of probes for hybridizing with respective complementary
molecular components of said liquid sample; a centrifuge for
rotating said reaction cell so as to impose a centrifugal force
greater than 1 G on said sample liquid, said centrifuge having a
rotor that rotates about a centrifuge axis; an agitator for
rotating said reaction cell relative to said rotor so that said
sample liquid moves relative to said array, said agitator being
mechanically coupled to said centrifuge and said reaction cell.
2. An apparatus as recited in claim 1 wherein said agitator rotates
said reaction cell about an axis more orthogonal to than along said
centrifugal force.
3. An apparatus as recited in claim 2 wherein said agitator changes
direction of rotation of said reaction cell relative to said rotor
periodically so as to define an agitation cycle rate.
4. An apparatus as recited in claim 3 wherein said rotor has a
rotation rate greater than said agitation cycle rate.
5. An apparatus as recited in claim 2 wherein said agitation means
rotates said reaction cell about an axis that extends more parallel
to said centrifuge axis than orthogonal to it.
6. An apparatus as recited in claim 5 wherein said array extends
more orthogonal to than parallel to said centrifugal force so that
said centrifugal force forces said sample liquid against said
array.
7. An apparatus as recited in claim 6 wherein said agitation means
rotates said reaction cell about said agitation axis so that said
centrifugal force forces liquid in said reaction cell away from
said array.
8. An array hybridization method comprising the steps of:
introducing sample liquid into a reaction cell so that some
interior volume is partially occupied by sample liquid and
partially occupied by gas; centrifuging said sample liquid by
rotating said reaction cell having a probe array so that
centrifugal forces urges said sample liquid against said array; and
agitating said sample liquid in said reaction cell during said
centrifuging so that said sample liquid moves relative to said
array.
9. An array hybridization method as recited in claim 8 wherein said
agitation involves rotating said sample cell about an agitation
axis that is more orthogonal to than along said centrifugal
force.
10. An array hybridization method as recited in claim 9 wherein
said agitating involves periodically changing the direction of
rotation about said agitation axis so as to define an agitation
cycle rate.
11. An array hybridization method as recited in claim 11 wherein
said centrifuging involves rotating said reaction cell at a
centrifuge rate greater than said agitation rate.
12. An array hybridization method as recited in claim 10 wherein
said agitation involves rotating said sample cell about an
agitation axis that extends more parallel to said centrifuge
axis.
13. An array hybridization method as recited in claim 12 wherein
said array extends more orthogonal to centrifugal than along it so
that said centrifugal forces urges said sample liquid against said
array.
14. An array hybridization method as recited in claim 13 further
comprising a step of removing sample liquid from said reaction
cell, said removing step involving rotating said reaction cell by
rotating it about said agitation axis so that said centrifugal
force urges said fluid in said reaction cell away from said
array.
15. An array hybridization method as recited in claim 8 wherein
said reaction cell is filled at most half way with sample liquid.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to solid-phase
chemistry and, more particularly, to chemical and biochemical
reactions between a molecules bound to a substrate surface and
molecules in a liquid, as in array hybridization. A major objective
of the present invention is to provide for more rapid array
hybridization.
[0002] Solid-phase chemistry involves chemical or biochemical
reaction between components in a fluid and molecular moieties
present on a substrate surface. Solid-phase chemical reactions can
involve: the synthesis of a surface-bound oligonucleotide or
peptide, the generation of combinatorial "libraries" of
surface-bound molecular moieties, and hybridization assays in which
a component present in a fluid sample hybridizes to a complementary
molecular moiety bound to a substrate surface.
[0003] Hybridization reactions between surface-bound molecular
probes and target molecules in a sample liquid may be used to
detect the presence of particular biomaterials including
biopolymers and the like. The surface-bound probes may be
oligonucleotides, peptides, polypeptides, proteins, antibodies or
other molecules coverable of reacting with target molecules in
solution. Such reactions form the basis for many of the methods and
devices used in the new field of genomics to probe nucleic acid
sequences for novel genes, gene fragments, gene variants and
mutations.
[0004] The ability to clone and synthesize nucleotide sequences has
led to the development of a number of techniques for disease
diagnosis and genetic analysis. Genetic analysis, including
correlation of genotypes and phenotypes, contributes to the
information necessary for elucidating metabolic pathways, for
understanding biological functions, and for revealing changes in
genes that confer disease. New methods of diagnosis of diseases,
such as AIDS, cancer, sickle cell anemia, cystic fibrosis,
diabetes, muscular dystrophy, and the like, rely on the detection
of mutations present in certain nucleotide sequences. Many of these
techniques generally involve hybridization between a target
nucleotide sequence and a complementary probe, offering a
convenient and reliable means for the isolation, identification,
and analysis of nucleotides.
[0005] In biological chip or "biochip" arrays, a plurality of
probes, at least two of which are different, are arranged in a
spatially defined and physically addressable manner on a substrate
surface. Such "biochip" arrays have become an increasingly
important tool in the biotechnology industry and related fields, as
they find use in a variety of applications, including gene
expression analysis, drug screening, nucleic acid sequencing,
mutation analysis, and the like. Substrate-bound biopolymer arrays,
particularly oligonucleotide, DNA and RNA arrays, may be used in
screening studies for determination of binding affinity and in
diagnostic applications, e.g., to detect the presence of a nucleic
acid containing a specific, known oligonucleotide sequence.
[0006] Regardless of the context, all chemical or biochemical
reactions between components in a fluid and molecular moieties
present on a substrate surface require that there be adequate
contact between the fluid's components and the surface-bound
molecular moieties. To this end, a number of approaches have been
proposed to facilitate mixing of fluid components during solid
phase chemical or biochemical reactions so that a substantially
homogeneous fluid contacts the reactive surface. Most recently, a
great deal of attention has focused on improving hybridization
assays using various mixing techniques.
[0007] Inadequate mixing is a particular problem in chemical and
biological assays where very small samples of chemical,
biochemical, or biological fluids are typically involved.
Inhomogeneous solutions resulting from inadequate mixing can lead
to poor hybridization kinetics, low efficiency, low sensitivity,
and low yield. With inadequate mixing, diffusion becomes the only
means of transporting the reactants in the mobile phase to the
interface or surface containing the immobilized reactants. In such
a case, the mobile phase can become depleted of reactants near the
substrate as mobile molecules become bound to the immobile phase.
Also, if the cover is not exactly parallel to the plane of the
substrate, the height of the fluid film above the probe array will
vary across the array. Since the concentration of target molecules
will initially be constant throughout the solution, there will be
more target molecules in regions where the film is thicker than in
regions where it is thinner, leading to artifactual gradients in
the hybridization signal.
[0008] As array density is ever increasing, and the need for faster
and more accurate hybridization assays is ongoing, there is
currently a great deal of emphasis on improving "mixing" of sample
liquid during hybridization and, correspondingly, in maximizing
contact between the components of the sample liquid and the
entirety of the array surface.
[0009] The Affymetrix GeneChip.RTM. Fluidics Station hybridization
and wash instrument includes a means for pumping a sample liquid
back and forth across an array on a substrate surface while the
substrate is mounted in a holder. While this method provides for
mixing of components within the sample liquid, there are
disadvantages that can adversely affect the accuracy of the
hybridization reaction. That is, the method is prone to
contamination because of the number and variety of materials that
come into contact with the sample liquid, i.e., adhesives, various
plastic components, and the like. In addition, large sample volumes
(greater than 200 microliters (.mu.l) are required, and temperature
control is poor.
[0010] In U.S. Pat. No. 4,849,340 to Oberhardt, an alternative
means is disclosed for mixing components in a fluid during an assay
performed in an enclosed chamber. Oberhardt discloses an apparatus
comprising a base, an overlay and a cover which when combined
define a sample well, a channel, and a reaction space. Fluids
introduced into the sample well flow by coverillary action to the
reaction space. Mixing of fluids within the reaction space is
effected using mechanical or electromechanical means to create
forced convection currents. Again, large sample volumes are
required (100 to 200 .mu.l) because of the need to maintain a gap
between the base and the cover during mixing. Additionally, the
method relies on coverillary action to promote fluid flow, and
mixing may thus be slow and incomplete, particularly when viscous
reagents are used.
[0011] U.S. Pat. No. 5,192,503 to McGrath et al. discloses an
apparatus for conducting an in situ assay of a tissue section
mounted on a slide. A seal member, mounted on a plate, forms a
closed periphery and encloses and defines an interior region on the
slide that forms a reaction chamber. A plate covers the slide and
seal member. The joined plate and slide together form a probe clip.
The reaction chamber may comprise a single chamber or two chambers.
In the one-chamber embodiment a time-release material, such as
gelatin, is applied over the probe, allowing time for reaction of
the tissue sample with reagents before the probe is released and
thus able to react with the tissue sample.
[0012] In the two-chamber embodiment, the probe reaction chamber
defined by the closed periphery of a first seal member is divided
into two regions by a raised portion of the plate, a mixing chamber
and a reaction chamber. At least one end of this raised portion
does not contact the first seal member, thereby leaving a channel
available for fluid flow. Probe compounds placed in the mixing
chamber do not mix with the fluid reagents in the reaction chamber
until fluid is induced to flow between the two chambers via a
channel in a gap left between the raised portion and the seal
member. Fluid flow may be induced by rotating the probe clip to a
substantially vertical orientation, allowing fluid reagents from
the reaction chamber to flow into the mixing chamber and mix with
the probe compounds. Re-orienting the probe clip to the horizontal
causes the mixed probe and fluid reagent to flow to the reaction
chamber for reaction with a tissue section therein. Thus, the
position and flow of fluid reagents and probes in the reaction
chamber and the mixing chamber is controlled by gravity.
Optionally, both gravity-controlled flow and use of a time-release
agent such as gelatin may be used at the same time to regulate the
mixing of reagent fluids and probes. Like the Oberhardt device, the
McGrath et al. apparatus is disadvantageous when viscous solutions
are used or rapid mixing is required, insofar as mixing depends
upon gravity to induce flow.
[0013] A method for more thorough mixing of components in a sample
liquid during a solid phase chemical or biochemical reaction is
disclosed in commonly assigned, co-pending U.S. patent application
Ser. No. 09/343,372 to Schembri et al., filed Jun. 30, 1999
("Apparatus and Method for Conducting Chemical or Biochemical
Reactions on a Solid Surface Within an Enclosed Chamber"). That
method involves mixing a very thin film of fluid in a chamber,
wherein an air bubble is incorporated therein and, when used in
hybridization, a surfactant is preferably present as well. However,
non-specific binding between the sample liquid and reaction cell
surfaces, as well as liquid viscosity limit the rate the air bubble
can be moved in the reaction cell. This in turn limits the rate at
which mixing can occur, and thus the hybridization rate.
[0014] In FIG. 3, reaction cell 30 is being rotated
counterclockwise as indicated by arrows 50. Centrifugal force
arrows 45 are shown broken into a component 51 along array 40 and a
component 53 orthogonal to array 40. The component 51 along array
40 urges sample liquid 39 toward (left) end 47. This movement is
indicated by arrows 55, which show a tapered liquid profile flowing
toward (left) end 47. A curved arrow 47 shows a return motion for
liquid sample 39. This return motion provides for highly desirably
vertical mixing.
[0015] The vertical mixing assures that every target molecule
spends some time close enough to array 40 for binding to occur. The
centrifugal force 45 helps overcome the inertia of the liquid and
its non-specific binding forces with the substrate so that a high
agitation rate can be maintained. The advantages of the invention
can be understood with the following, admittedly approximate,
understanding of the hybridization process.
[0016] When the agitation rate is doubled, each target molecule is
likely to be found half as far from a respective probe for half the
time. When it is half as far, it is four times as likely to
hybridize. However, the interval over which it can hybridize is
half as long. Thus, in principle, doubling the agitation rate
doubles the hybridization rate. This linear relationship applies
until non-specific binding fluid forces prevent sample liquid from
completing its motion across the array. The stronger the
centrifugal force, the higher the agitation rate can be raised
before this limiting consideration applies. Thus, the centrifuge
rate can be increased until the forces involved adversely affect
specific binding or threaten the integrity of the hybridized or
non-hybridized species.
[0017] In FIG. 1, the agitation axes are parallel to the centrifuge
axis and the hybridization arrays are generally orthogonal to the
centrifugal force. In other embodiments, the hybridization arrays
are also generally orthogonal to the centrifugal force, but the
agitation axes are not parallel to the centrifuge axis. For
example, the agitation axes can be circumferentially (in other
words, "tangentially") oriented relative to the centrifuge
axis.
[0018] Particularly with a circumferentially oriented agitation
axis, but also other cases in which the array is orthogonal to the
centrifugal force, the substrate can be curved cylindrically, for
example, along a radius slightly less than (e.g., 90% of) the
distance between the agitation axis and the centrifuge axis. In
this case, the centrifugal force is more orthogonal to the array
away from the array center and even at the extremes of the
agitation motion. This provides a more uniform sample liquid
distribution across the array, which in turn allows less sample
liquid to be used without risking drying of the array. In addition,
the agitation is gentler on the sample.
[0019] Reaction cells 30 of FIG. 1 are oriented so that arrays 40
generally orthogonal to the centrifugal force. Oblique orientations
are also provided for. For example, reactions cells can be oriented
so that they are more orthogonal to the centrifugal force than
along it. However, reaction cells 530 of FIG. 4 represent another
case in which reaction cells are oriented both along and orthogonal
to the centrifugal force.
[0020] FIG. 4 shows system AP1 with reaction cells 530 oriented
parallel to turntable 17. Reaction cells 530 are similar to
reaction cells 30 and likewise include a probe array, in this case,
probe array 540. Centrifugal force 545 urges sample liquid 549
radially outward, so that gas with cell 530 is radially inward of
liquid 539. In this case, the agitation axis is perpendicular and
through the center of array 540.
[0021] In greater detail with reference to FIG. 3, reaction cell 30
includes substrate 31 that preferably has a substantially planar
surface, with at least a portion of the surface representing a
reaction area (hybridization array 40) on which the chemical or
biochemical reactions are conducted, and cover 33, optimally of
plastic, having a peripheral lip which sealingly contacts the
substrate surface about the reaction area, and wherein the cover
and the reaction area of the substrate surface form an enclosure
having an interior space that serves as the reaction chamber. The
chamber is adapted to retain a quantity of fluid so that the fluid
is in contact with the reaction area of the substrate surface and
the inner surface of the cover.
[0022] The reaction cell also includes a fastening means (not
shown) effective to press the cover and the substrate together,
i.e., to immobilize the cover on the substrate, thereby forming a
watertight, temporary seal therebetween. The fastening means
ensures stable, effective and secure positioning of the cover over
the substrate. Optional gasket means adjacent the surface of the
cover may be included to aid in equalizing the pressure provided by
the fastening means. The optional gasket may be, for example,
placed between the cover and the rigid frame to provide compliance
in the system and to even the pressure applied to the cover and the
substrate. The apparatus further comprises fluid transfer means
which enables introduction of fluid from the exterior of the
apparatus to the reaction chamber, and removal therefrom. In a
preferred embodiment, the fluid introduction means comprises one or
more ports in the cover.
[0023] It is preferred that the cover be made of plastic and the
substrate of glass, plastic, fused silica or silicon, the seal
between plastic and either glass, plastic, fused silica or silicon
being advantageous for producing the apparatus of the invention.
The cover material should be thermally stable, chemically inert,
and preferably non-stick. Furthermore, when the apparatus is used
in hybridization, the cover should be comprised of a material that
is chemically and physically stable under conditions employed in
hybridization. In a preferred embodiment, the plastic cover is
polypropylene, polyethylene or acrylonitrile-butadiene-styrene
("ABS"). In the most preferred embodiment, the plastic cover is
comprised of polypropylene. The cover may be constructed by
machining or molding technologies.
[0024] As noted above, the cover preferably has a lip along the
perimeter of the cover bordering a recessed portion that comprises
the major portion of the area of the inner face of the cover.
Applying pressure to the outer face of the cover directly above the
perimeter lip is required to form the tight seal between the cover
and the substrate. Any means that presses the lip of the cover
securely to the substrate is suitable. Such pressure may be applied
evenly by, for example, clamps, a press, or by coverturing the
substrate and cover within a two-part rigid frame and compressing
the two together to supply an even pressure to the cover and
substrate. If desired, the peripheral lip of the cover may be
modified to provide for an improved seal; for example, one or more
continuous ridges can be incorporated into the lip so that the
pressure supplied to the cover is higher at those locations and
preferentially causes them to compress. In any of these
embodiments, the reaction cell may be re-used, as the peripheral
seal is temporary and the fastening means may be removed when
desired. Thus, the reaction cell may be readily disassembled after
use, cleaned, and re-assembled (with alternate components, such as
a different substrate, if desired) so that some or all of the
components of the reaction cell may be re-used.
[0025] This reaction cell interior height may range from about
0.002" to 0.02" (50 .mu.m to 500 .mu.m). The dimension of the
cover, the peripheral lip, and the reaction area are such that the
reaction area is generally in the range of about 4 mm.sup.2 to 500
mm.sup.2, preferably about 20 mm.sup.2 to 350 mm.sup.2, and the
reaction chamber has a volume in the range of about 0.2 .mu.l to
about 312 .mu.l, preferably about 1 .mu.l to 200 .mu.l.
[0026] Hybridization array 40 has a plurality of molecular probes
bound thereto. Preferably, the molecular probes are arranged in a
spatially defined and physically addressable manner, i.e., are
present in one or more "arrays." In a most preferred embodiment,
the probes are oligonucleotide probes (including cDNA molecules or
PCR products), although other biomolecules, e.g., oligopeptides and
the like, may serves as probes as well.
[0027] The term "hybridization" as used herein means binding
between complementary or partially complementary molecules, as
between the sense and anti-sense strands of double-stranded DNA.
Such binding is commonly non-covalent binding, and is specific
enough that such binding may be used to differentiate between
highly complementary molecules and others less complementary.
Examples of highly complementary molecules include complementary
oligonucleotides, DNA, RNA, and the like, which comprise a region
of nucleotides arranged in the nucleotide sequence that is exactly
complementary to a probe; examples of less complementary
oligonucleotides include ones with nucleotide sequences comprising
one or more nucleotides not in the sequence exactly complementary
to a probe oligonucleotide.
[0028] For use in hybridization, the interior of the reaction cell,
in other words, the "hybridization chamber," is filled with a
sample liquid comprising a target molecule which may hybridize to a
surface-bound molecular probe, and with a surfactant of a type and
present at a concentration effective to substantially reduce
nonspecific binding and promote mixing of components within the
sample liquid. The surfactant is selected from the group consisting
of anionic surfactants, cationic surfactants, amphoteric
surfactants, nonionic surfactants, and combinations thereof, with
anionic surfactants and polymeric nonionic surfactants particularly
preferred. Suitable anionic surfactants include, but are not
limited to, the sodium, potassium, ammonium and lithium salts of
lauryl sulfate, with lithium lauryl sulfate most preferred. A
preferred polymeric nonionic surfactant is polyethylene oxide, with
particularly preferred polyethylene oxides comprising an
alkylphenol ethylene oxide condensate. Such surfactants may be
obtained commercially under the trade name "Triton" from the Sigma
Chemical Company (St. Louis, Mo.), and including, for example,
Triton X-100 (octylphenol ethylene oxide condensate) and Triton
X-102 (also an octylphenol ethylene oxide condensate). More
specifically, Triton X surfactants have been described as having
the formula: 1
[0029] in which N for Triton X-100 has an average of about 9.5
units per molecule while for Triton X-102 N is an average of about
12.5 units per molecule. Further information on both Triton X-100
and Triton X-102 can be found at the following Internet addresses:
"www.sigma-aldrich.com/sigm- a/proddata/t6878.htm" and
"www.sigma-aldrich.com/sigma/proddata/t6878x.htm- ".
[0030] The surfactant generally represents between about 0.1 wt. %
and 10 wt. % of the sample liquid, preferably between about 0.5 wt.
% and 5 wt. % of the sample liquid, more preferably between about
0.75 wt. % and 5 wt. % of the sample liquid; however, it should be
emphasized that the exact concentration will vary with the
surfactant selected, and those skilled in the art may readily
optimize the concentration with respect to the desired results,
i.e., reduction of nonspecific binding and facilitation of mixing
within the sample liquid. An exemplary sample liquid will contain
between about 0.1 wt. % and about 1 wt. % of polyethylene oxide and
between about 0.05 wt. % and about 1 wt. % lithium lauryl
sulfate.
[0031] The invention is particularly useful in conjunction with
substrate surfaces functionalized with silane mixtures, as
described in co-pending, commonly assigned U.S. patent application
Ser. No. 09/145,015, filed Sep. 1, 1998, and entitled
"Functionalization of Substrate Surfaces with Silane Mixtures."
That method provides a functionalized surface on a substrate with
low surface energy. The method for preparing such a surface
comprises contacting a substrate having reactive hydrophilic
moieties on its surface with a derivatizing composition comprising
silane-containing groups R.sup.1-Si(R.sup.LR.sup.xR.sup.y) and
R.sup.2-(L).sub.n--Si(R.sup.LR.sup.xR.sup.y) under reaction
conditions effective to couple the silanes to the substrate. This
provides --Si-R.sup.1 and --Si--(L).sub.n-R.sup.2 groups on the
substrate. The R.sup.L, which may be the same or different, are
leaving groups, the R.sup.x and R.sup.y which may also be the same
or different, are either leaving groups, like R.sup.L, or are lower
alkyl, R.sup.1 is a chemically inert moiety that upon binding to
the substrate surface lowers the surface energy thereof, n is 0 or
1, L is a linking group, and R.sup.2 comprises either a functional
group enabling covalent binding of a molecular moiety or a group
that may be modified to provide such a functional group.
[0032] The ratio of the silanes in the derivatizing composition
determines the surface energy of the functionalized substrate and
the density of molecular moieties that can ultimately be bound to
the substrate surface. When used in conjunction with the present
invention, the surface-bound molecular probes are bound to the
R.sup.2 moieties provided by the second silane-containing group.
These and other variations upon and modifications to the disclosed
embodiments are provided for by the present invention, the scope of
which is defined by the following claims.
* * * * *