Gradient resolved information platform Number:7,183,050 from the United States Patent and Trademark Office (PTO) owispatent

Title: Gradient resolved information platform

Abstract: Methods and devices for the detection and identification in a sample of one or more target molecules which bind to nucleic acid probe molecules are provided. The method includes contacting the sample with a surface that is coated with one or more gradients of nucleic acid or nucleic acid analog probe molecules that bind target molecules in the sample. Gradients are formed by varying a physical, structural or functional property of the probes on the surface; for example, the density of probe molecules bound to the surface. The coating layer or immobilization layer in which the gradient is formed is preferably continuous Determination of the location, speed and/or extent of hybridisation of a nucleic acid on a gradient surface is useful to identify target molecules bound to probes and/or to quantitatively measure the amount of the target in a sample.

Patent Number: 7,183,050 Issued on 02/27/2007 to Krull

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Inventors:	Krull; Ulrich J. (Mississauga, Ontario L5M 2Z8, CA)
Appl. No.:	10/126,504
Filed:	April 18, 2002

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Current U.S. Class:	435/6 ; 435/174; 435/283.1; 435/287.2; 536/23.1; 536/24.3
Current International Class:	C12Q 1/68 (20060101); C07H 21/04 (20060101); C12M 1/36 (20060101)
Field of Search:	435/6,174,283.1,287.2,287.9 536/23.1,24.3

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References Cited [Referenced By]

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U.S. Patent Documents

	5175209		December 1992		Beattie et al.
	5436327		July 1995		Southern et al.
	5599668		February 1997		Stimpson et al.
	5866330		February 1999		Kinzler et al.
	6045671		April 2000		Wu et al.
	6218116		April 2001		Ginot
	6271044		August 2001		Ballerstadt et al.
	6379897		April 2002		Weidenhammer et al.
	6471916		October 2002		Noblett
	6503711		January 2003		Krull et al.
	2002/0137074		September 2002		Piunno et al.
	2003/0157538		August 2003		Krull et al.

Foreign Patent Documents

	WO 95/26416		Oct., 1995		WO
	WO 97/33169		Sep., 1997		WO
	WO 98/00402		Jan., 1998		WO
	WO 98/47613		Oct., 1998		WO
	WO 98/58079		Dec., 1998		WO
	WO 99/35289		Jul., 1999		WO
	WO 00/04390		Jan., 2000		WO

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Primary Examiner: Forman; B J
Attorney, Agent or Firm: Christensen O'Connor Johnson Kindness PLLC

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Parent Case Text

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application takes priority under 35 USC 119(e) from U.S. provisional application Ser. No. 60/284,715, filed Apr. 18, 2001 which is incorporated by reference in its entirety to the extent not inconsistent with the disclosure herein.

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Claims

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The invention claimed is:

1. A hybridization platform comprising: a planar support; and an immobilized layer of single stranded nucleic acids or nucleic acid analogs formed on the surface of the planar support; wherein the immobilized layer is formed as either a) a continuous layer on the surface or b) as discrete regions on the surface; the nucleic acids or nucleic acid analogs being arranged as the continuous layer, or as each region, such that the one or more parameters selected from the density, sequence, orientation or structure of the nucleic acid or nucleic acid analogs are spatially varied as a continuum in the continuous layer or in each region in one or both of a first and second dimension across the surface of the planar support so as to form at least one gradient of the one or more parameters within the continuous layer or each region; and the one or more selected parameters includes the density of the nucleic acid or nucleic acid analogs.

2. The hybridization platform of claim 1 which comprises a first gradient and a second gradient, wherein the first gradient is formed by spatially varying the density of the nucleic acids or nucleic acid analogs.

3. The hybridization platform of claim 2 wherein the immobilized layer further comprises polyelectrolyte immobilized to the surface of the planar support such that a further gradient is formed by spatially varying the concentration of polyelectrolyte.

4. The hybridization platform of claim 2, wherein the at least on gradient spans an average nearest neighbour separation of from about 2 mm to over 40 nm.

5. The hybridization platform of claim 2 wherein the planar support is fused silica, quartz, silicon, glass, a plastic, a metal, a transparent electrode, a ceramic, a semiconductor, a conductive form of carbon, paper, a conductive polymer, or a waveguide operating in either the evanescent mode or direct mode of excitation.

6. The hybridization platform of claim 5 wherein the at least one gradient is formed by dip-casting, through gradients of light activation, by spraying, rolling, capping, sequence annealing, sequence degradation, sequence extension or combinations thereof.

7. The hybridization platform of claim 6 wherein the nucleic acids or nucleic acid analogs are immobilized to the surface by adsorption, absorption, ionic bonding, covalent bonding, avidin-biotin, or thiol-gold interactions.

8. The hybridization platform of claim 7 wherein the nucleic acids or nucleic acid analogs are immobilized to the surface by covalent bonding.

9. The hybridization platform of claim 5 wherein the planar support is a waveguide, operating in either the evanescent mode or direct mode of excitation.

10. The hybridization platform of claim 2 wherein the first and second gradients are formed in orthogonal dimensions of the surface.

11. The hybridization platform of claim 2 wherein the second gradient is formed by varying the sequence of the nucleic acids or nucleic acid analogs.

12. The hybridization platform of claim 2 wherein the immobilized layer further comprises detectable labels that indicate binding of target molecules to the nucleic acids or nucleic acid analogs.

13. The hybridization platform of claim 2 further comprising detectable labels bound to the nucleic acids or nucleic acid analogs.

14. The hybridization platform of claim 13 wherein the detectable labels are fluorescent labels tethered to the nucleic acid or nucleic acid analogs.

15. The hybridization platform of claim 2 further comprising fluorescent labels immobilized to the surface.

16. The hybridization platform of claim 2 further comprising a further gradient formed by varying the density of detectable labels tethered to the nucleic acids or nucleic acid analogs.

17. The hybridization platform of claim 16 wherein the detectable labels are fluorescent molecules.

18. The hybridization platform of claim 2 further comprising a reference region in the at least one gradient.

19. The hybridization platform of claim 2 wherein the second gradient is formed by varying the length of the nucleic acids or nucleic acid analogs in the immobilized layer.

20. The hybridization platform of claim 19 wherein the second gradient is formed by varying the length of the nucleic acids or nucleic acid analogs in single base increments.

21. The hybridization platform of claim 20 wherein the second gradient spans 1,000 or more bases.

22. The hybridization platform of claim 20 wherein the second gradient spans 100 or more bases.

23. The hybridization platform of claim 20 wherein the second gradient spans 10 to about 50 bases.

24. The hybridization platform of claim 1 wherein the immobilized layer is formed as a continuous layer on the surface.

25. The hybridization platform of claim 1 wherein the immobilized layer is formed as discrete regions on the surface.

26. A biosensor for detection of one or more target molecules comprising: a planar support; and an immobilized layer of single stranded nucleic acids or nucleic acid analogs formed on the surface of the planar support and capable of hybridizing to the one or more target molecules; wherein the immobilized layer is formed as either a) a continuous layer on the surface or b) as discrete regions on the surface; the nucleic acids or nucleic acid analogs being arranged as the continuous layer, or as each region, such that the one or more parameters selected from the density, sequence, orientation or structure of the nucleic acid or nucleic acid analogs are spatially varied as a continuum in the continuous layer or in each region in one or both of a first and second dimension across the surface of the planar support so as to form at least one gradient of the one or more parameters within the continuous layer or each region; and the one or more selected parameters includes the density of the nucleic acid or nucleic acid analogs.

27. The biosensor of claim 26 further comprising detectable labels for indicating binding of the one or more target molecules to the nucleic acids or nucleic acid analogs.

28. A kit for conducting an assay for detecting and quantifying one or more target molecules in a sample which comprises: a planar support; and an immobilized layer of single stranded nucleic acids or nucleic acid analogs formed on the surface of the planar support and capable of hybridizing to the one or more target molecules; wherein the immobilized layer is formed as either a) a continuous layer on the surface or b) as discrete regions on the surface; the nucleic acids or nucleic acid analogs being arranged as the continuous layer, or as each region, such that the one or more parameters selected from the density, sequence, orientation or structure of the nucleic acid or nucleic acid analogs are spatially varied as a continuum in the continuous layer or in each region in one or both of a first and second dimension across the surface of the planar support so as to form at least one gradient of the one or more parameters within the continuous layer or each region; and the one or more selected parameters includes the density of the nucleic acid or nucleic acid analogs.

29. The kit of claim 28 further comprising one or more reagents to detect and quantify the one or more target molecules hybridized to the nucleic acids or nucleic acid analogs.

30. A method of forming a hybridization platform comprising: providing a planar support, and forming on the surface of the planar support an immobilized layer of single stranded nucleic acids or nucleic acid analogs, the immobilized layer being formed either as a) a continuous layer on the surface or b) as discrete regions on the surface, the continuous layer or each region being arranged such that one or more parameters selected from the density, sequence, orientation or structure of the nucleic acid or nucleic acid analogs are spatially varied as a continuum in the continuous layer or in each region in one or both of a first and second dimension across the surface of the planar support so as to form at least one gradient of the one or more parameters within the continuous layer or each region, wherein the one or more selected parameters includes the density of the nucleic acids or nucleic acid analogs.

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Description

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BACKGROUND OF INVENTION

High-density arrays of oligonucleotide probes have been fabricated using spotting technology, spraying technology, electrostatic attraction, and high-resolution photolithography in combination with solid-phase oligonucleotide synthesis. Such forms of DNA detection technology, which are often associated with chip-based structures and microarrays, may be used for parallel DNA hybridisation analysis, directly yielding sequence information from genomic DNA fragments. Prior to sequence identification, the nucleic acid targets are commonly fluorescently labelled. This can occur prior to or after hybridisation to the oligonucleotide array, via direct chemical modification of the target strand or by use of an intercalant or groove-binding dye subsequent to hybridisation on the DNA microarray. The hybridisation 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 small quantities of a particular nucleic acid sequence as is done in the field of dedicated biosensors, this technology has focused on sequence analysis of nucleic acids in suitably high copy number so as to sufficiently occupy the oligonucleotide array.

Other spatially resolved approaches for development of microarray technologies have also been introduced where electrochemical manipulation of hybridisation at spots or pads of DNA can be done, and where the tips of fibres that form a fibre optic bundle are altered to house addressable discrete DNA microbeads. Further examples of spatially resolved devices include the use of spots of nucleic acids that are deposited onto a glass or fused silica surface by pin spotting or piezo-based ink jets, spatially resolved electrochemical analysis as found in Light-Addressable Potentiometric Analysis (LAPS) technology, and spatially resolved Surface Plasmon Resonance for pads that are located over conductive metals.

In all these cases, the concept is that individual independent spots, beads or pads of nucleic acid are deposited across a surface, and that the immobilized chemistry in each spot, bead or pad is consistent and discrete. In array technologies, each spot, bead or pad typically has a plurality of bound nucleic acid molecules and each spot, bead or pad can contain one or more, although typically a relatively small number of, different bound nucleic acids. The purpose of these arrays is to achieve detection of multiple targets, whether they be pathogenic organisms, mutations or combinations of genes that are concurrently up and down regulated. This is achieved in any one analysis by looking at alterations of a pattern of discrete signals on a surface. The approach is based on study of the results of many partially-selective reactions, where ideally the chemistry of each reaction can be defined and controlled. The problem with such approaches is that it is virtually impossible to select a stringency that is concurrently suitable for optimization of hybridisation at each and every spot, bead or pad, and the approach therefore incorporates a lack of selectivity by design. Furthermore, such detection devices are generally not amenable to providing absolute quantitative results and are not usually reusable.

Approaches to sensor development have basically taken two distinctive paths:

1) The use of one type of ssDNA sequence on a relatively large surface area for biosensor preparation.

2) The use of microarrays of many different ssDNA sequences, each different ssDNA sequence being immobilized in a small, discrete surface area, with many different ssDNA sites being distributed over a large surface area. (More recently, microarrays composed of discrete areas in which a relatively small number of different ss DNA are immobilized have been employed.)

Two common platforms used for development of DNA biosensors are Surface Plasmon Resonance Spectroscopy (SPR) and Total Internal Reflection Fluorescence Spectroscopy (TIRF). SPR can detect surface binding interactions in real time without the use of labels. SPR instrumentation is commercially available and Pharmacia's BIAcore.TM. instrument is in common use in many laboratories to investigate the kinetics of interfacial nucleic acid hybridisation, formation of triple-stranded complexes, to develop assays for selective detection of polymerase chain reaction (PCR) amplified nucleotides (N. Bianchi, C. Rutigliano, M. Tomassetti, G. Feriotto, F. Zorzato, and R. Gambari, Clinical and Diagnostic Virology 8, pp. 199 208, 1997) and to investigate the use of peptide nucleic acid (PNA) capture probes to enhance selectivity. The BlAcore system has been used by several groups for the monitoring of DNA-DNA interactions in real time (P. Nilsson, B. Persson, M. Uhlin, and P. Nygren, Analytical Biochemistry 224, pp. 400 408, 1995; M. Tosu, M. Gotoh, K. Saito, M. Shimizu, Nucleic Acids Symposium Series 31, pp. 121 122, 1994). The association and dissociation kinetics of target oligonucleotides composed of either complementary sequences or mismatched bases have been monitored. The authors claimed that differences in kinetic parameters could be detected for non-complementary strands as well as for various 20-mers containing two, four or six mismatched base pairs. The time required for each analysis was reported to be 15 20 minutes and the results showed promise for real-time interaction analysis for such processes as gene assembly, DNA polymerase activity, and sequencing experiments. Bier and Scheller (F. F. Bier and F. W. Scheller, Biosensors and Bioelectronics 11, pp. 669 674, 1996) used SPR to study the interaction of the restriction endonuclease EcoRE, a DNA modifying enzyme. The action of the enzyme was observed by measuring the loss of bound DNA after a short incubation with the enzyme.

Numerous evanescent wave fibre optic DNA sensors have been reported in the literature. The evanescent field typically penetrates about 200 nm to 400 nm (typically less than 1 .mu.m) into the surrounding medium when using visible radiation, conferring surface selectivity (W. F. Love, L. J. Button, and R. E. Slovacek, in Biosensors with Fibre Optics. Eds. Wise and Wingard, pp. 139 180, The Humana Press Inc., 1991). The first such fibre optic DNA sensor was reported by Squirrell in 1992 (C. R. Graham, D. Leslie, and D. J. Squirrell, Biosensors and Bioelectronics 7, pp. 487 493, 1992). Preliminary experiments using covalently immobilised probe oligonucleotides and fluorescein-labelled complementary strands gave fast (60 second) detection in the nanomolar range with a linear response curve, but were not as sensitive as radio labelling techniques. Analysis of 204-base oligonucleotides showed that the detection of PCR products was feasible. Abel (A. P. Abel, M. G. Weller, G. L. Duveneck, M. Ehrat, and H. M. Widmer, Analytical Chemistry 68, pp. 2905 2912, 1996) operated a similar system in a competitive binding mode.

Sensitivity of evanescent biosensors may be significantly improved by use of mono-modal optical fibres (T. R. Glass, S. Lackie, and T. Hirschfeld, Applied Optics, 26, pp. 2181 2187, 1987). With use of mono-modal fibres, up to 10% of the optical power may be present in the evanescent field. Bier (F. Kleinjung, F. F. Bier, A. Warsinke, and F. W. Scheller, Anal. Chimica Acta 350, pp. 51 58, 1997), used two strategies for immobilisation of oligonucleotides to monomodal optical fibres: direct coupling to amino-activated surfaces or coupling via the avidin-biotin bridge. Using the fluorescent double-stranded ligands YOYO and picogreen, detection limits of 30 fM (3.2 amol) were achieved. These are the lowest detection limits reported to date for fibre optic DNA biosensors. The sensor was also able to detect single base pair mismatches in the target sequence.

A second major route to production of devices for DNA analysis involves placement of arrays of different sequences across surfaces, or at the tips of fibre-optic bundles (Michael, K. L., Taylor, L. C., Schultz, S. L., Walt, D. R., Anal. Chem. 1998, 70, 1242 1248). Automated oligonucleotide synthesis has seen commercial application by Fodor and Affymetrix (E. L. Sheldon, J. Briggs, R. Bryan, M. Cronin, M. Oval, G. McGall, E. Gentalen, C. G. Miyada, R. Masino, D. Modlin, A. Pease, D. Solas and S. P. A. Fodor, Clinical Chemistry 39, pp. 718 719, 1993; G. H. McGall, A. D. Barone, M. Diggelmann, S. P. A. Fodor, E. Gentalen and N. Ngo, JACS 119, pp. 5081 5090, 1997), where photolithography techniques have been used to grow arrays of oligonucleotides on DNA "chips". This involves the activation of glass surfaces and then extension of the surface with a hexaethyleneglycol-type linker. The terminal groups of the linker are blocked with photolabile protecting groups. These groups are then removed from predefined regions by selectively exposing the surface with light through photolithographic masks, followed by oligonucleotide addition. This has been done using phosphoramidites with photolabile protecting groups in the 5'-hydroxyl position, or more recently with conventional DMT protected phosphoramidites in combination with polymeric semiconductor photoresist films (G. McGall, J. Labadie, P. Brock, G. Wallraff, T. Nguyen, and W. Hinsberg, PNAS, 93, pp. 13555 13560, 1996). The phosphoramidites react only with the sites that were previously exposed to light. The process is repeated with different lithographic masks until the desired oligonucleotides are obtained. The number of oligonucleotide probes that can be immobilised is limited by the size of the chip and the lithographic resolution (M. Chee, R. Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stem, J. Winkler, D. J. Lockhart, M. S. Morris and S. P. A. Fodor, Science 274, pp. 610 614, 1996). It has been reported that chips with 136,528 unique oligonucleotides have been synthesized on a 13 cm.sup.2 chip.

Another approach involves placing aminated polypropylene sheets in a Southern Array Maker (SAM) and then standard phosphoramidite chemistry is applied to 64 distinct and independent channels producing 64 independent oligonucleotides (M. J. O'Donnell-Maloney and D. P. Little, Genetic Analysis: Biomolecular Engineering 13, pp. 151 157, 1996). Other methods involve a piezoelectric ink-jet dispenser that delivers discreet droplets of reagent to chip surfaces, or delivery by "printing" using bundles of capillaries or pins.

SUMMARY OF THE INVENTION

The present invention provides a very different approach to detection and quantitative measurement of nucleic acids, nucleic acid analogs, and agents that bind to or associate with nucleic acids or nucleic acid analogs, which uses spatially-resolved analysis of binding of such molecules to a surface carrying one or more spatially-distributed gradients of selectivity. In this approach, a surface carries one or more gradients of probe molecules wherein the gradient is formed by spatiality varying one or more physical, structural or functional properties of the probe molecules. For example, gradients of probe density (e.g., low to high density) and/or probe structure (e.g., sequence variation, different fictionalisation of probes) and/or the orientation of bound probes with respect to each other. The surface carrying the spatially-distributed gradient(s) is contacted with a sample to allow binding of targets in the sample to probes in the gradient. The surface is treated to remove non-selectively bound targets or optionally to adjust selectively of binding. Any spatially-resolved method is then employed to detect the selective binding of targets to the surface. The detection of patterns of binding to the one or more gradients allows the detection and identification of targets present in a sample. Additionally, detection of patterns of binding to the gradient as a function of contact time with the sample, or assay conditions including, among others, temperature, and washing conditions, e.g., salt concentration. Further, differential binding of different target molecules in a sample to the gradient can provide for separation of target molecules in a mixture.

In a specific exemplary embodiment this new method as applied to nucleic acid probe molecules is herein designated Gradient Resolved Information Platform (GRIP) which is based on a surface that is coated with an immobilized layer of nucleic acid molecules, which comprises at least one gradient of a varying physical, structural or functional property of the probe molecules. A surface can include one or more gradients of such properties, including, among others, gradients of probe density and/or probe sequence and/or probe orientation and/or probe structure. The methods are particularly useful with surfaces having one or more spatially-distributed gradients of single-stranded nucleic acid or single-stranded nucleic acid analog probe molecules.

The location, extent of binding or hybridisation, and speed of binding or hybridisation on such a surface by a target molecule is useful to isolate one or more targets and/or identify one or more bound targets and optionally to quantitatively measure the amount of one or more targets in a sample. The location and speed of signal development (e.g., of a label sensitive to binding or hybridisation) will be dependent on the stability of the hybrid or complex formed, which is in turn dependent on the density, sequence (or structure) and availability of the immobilized probes, e.g., single-stranded nucleic acid. Such an approach to detection adapts to alterations of the conditions of stringency (e.g., hybridisation or binding conditions), and nucleic acids or other target molecules in a mixture can each optimally bind at any one stringency by localizing to the area of highest energetic stabilization. Identification and quantification is based on the spatially resolved signal location and signal magnitude within any gradient. Many different device technologies that can spatially resolve a signal magnitude or rate of signal appearance can be used for detection of target binding.

The surfaces and substrates of this invention that carry one or more gradients of immobilized probe properties are useful in methods for the detection of one or more target molecules in a sample. Targets are detected by detecting their binding to the probe gradient. Spatially-resolved detection of target binding to determine the location in the gradient where the target binds facilitates identification of the bound target and quantitation of the amount of target (or relative amounts of targets) in a sample. The invention also provides kits for the detection of target molecules which comprise one or more substrates at least one of which substrates carries one or more gradients of immobilized probe which binds to the target.

More specifically, surfaces and substrates of this invention that carry one or more gradients of immobilised probe nucleic acids or nucleic acid analogues are useful in methods for the detection of one or more target nucleic acids that are at least in part complementary in sequence to a probe on the surface or substrate. Such surfaces and substrates are useful in hybridisation assays for detection of target nucleic acids in which the target binds to the gradient on surface or substrate. Spatially-resolved detection of target binding facilitates identification of the bound target and quantitation of the amount of target (or relative amounts of targets) in a sample. The invention also provides kits for the detection of target nucleic acids which comprise one or more substrates at least one of which substrates carries one or more gradients of immobilized nucleic acid probes which binds to a target.

In a specific example, fluorescent dyes that associate with the formation of double-stranded DNA (dsDNA) can be used to detect hybridisation of target nucleic acids to immobilized probe nucleic acids. Such dyes can be free in solution, can be associated with the target nucleic acid, or can be associated with single-stranded DNA (ssDNA) probe molecules on a surface. The intensity distribution of a pattern and the location of the pattern of the fluorescence upon hybridisation of immobilized probe molecules with target DNA can be used to identify and quantify one or more targets.

A further feature that can be included in the methods of this invention is immobilization of one or more references or markers, such as a known sequence of ssDNA in defined spatial zones on the gradient surfaces.

The new technology described herein provides for at least one and preferably a multi-dimensional distribution of selective chemistry at a surface, in such a way that the chemical coating layer on the surface operates to provide one or more gradients of selectivity in one or more directions on the surface. The coating layer or immobilization layer in which the gradient is formed is preferably continuous, but may be composed of discrete bands, spots or regions. A gradient is formed in a selected spatial distinguishable pattern on the surface, and preferably is formed along a dimension of the surface, e.g., along the length or width of a rectangular surface. Where two or more gradients are present the pattern of each gradient is distinct and identifiable. For example, two gradients on a rectangular surface are preferably formed in orthogonal directions or dimensions on the surface, e.g., along the length and width, respectively of a surface. In another example, a gradient can be formed with respect to a point on a surface or other geometric shape, e.g., varying as a function of radial distance from the point or varying linearly from a line on the surface. Any given surface may contain more than one gradient formed from more than one point, line and/or other geometric shape on the surface. For example, a radially varying gradient originating at a point on the surface may be combined with a linearly varying gradient originating from a line on (or an edge of) the surface.

The surfaces of this invention with one or more gradients of bound or immobilized probe molecules can be employed for the separation, isolation and/or detection and identification of one or more target molecules which can bind to probe molecules in a gradient on the surface.

The distributed chemistry on the surfaces of this invention provides the advantages that one or more target molecules in a sample can be separated by binding to different locations in the gradient on the surface; that a target molecule can be identified by determining the location of its binding within or on a gradient (by comparison to a known reference or marker, for example), and the quantity of the target molecule present in a sample can be determined by following signal magnitude in time(e.g., by detection of label as a function of time). Conventional spatially resolved imaging techniques (e.g. confocal microscopy, diode array, CCD, etc.), can be used in combination with the surface gradients of this invention to determine quantitative results with automatic correction for any changes of solution conditions (stringency). Various analytical techniques that offer spatially-resolved signal analysis can also be used (e.g. Surface plasmon resonance, electrochemistry, acoustic technologies, thermal analysis, surface enhanced Raman spectroscopy, surface potential measurement devices, mass spectrometry, fibre-optic bundles).

Other advantages of the invention include reversibility of chemistry, the ability to use tethered markers and/or mixed markers (for example, fluorescent dyes that preferentially associate with dsDNA), the use of calibration and referencing signals (e.g., the use of internal reference sequences and internal standards) that appear concurrently with the analytical signal.

In addition, the use of surface gradients is compatible with a variety of optical techniques, which allow the use of a waveguiding approach to improve signal generation and signal recovery (i.e. high sensitivity and low detection limit). The surfaces comprising gradients of probe molecules of this invention can function on many different device platforms, are suitable for concurrent assay of multiple nucleic acid targets, are suitable for determining the degree of selectivity to targets, and can operate in mixtures where there are multiple targets of differing lengths and where sample clean-up may not be complete.

The surfaces of this invention preferably have at least one substantially continuous gradient, wherein the average value of the parameter, upon which the gradient is based, is varied continuously in a defined pattern on a surface. Preferred surfaces of this invention contain at least one gradient of varying average density of bound probe molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a surface showing a two-dimensional gradient, where in one dimension the density of probe molecules is varied, and in the other dimension, the length of the immobilized sequence is varied.

FIG. 2 illustrating a surface based on a two-dimensional gradient exemplifying the partitioning of a target (signal at left) into one zone, and a reference sequence onto a reference zone (signal at right).

FIGS. 3A I illustrate binding of target nucleic acid to gradient surfaces as described in Example 2. FIG. 3A illustrates fluorescence from an ITO slide in which a continuous gradient formed by electrochemical hydroxylation, GOPS treatment (reflux in toluene), then washing with methanol, DCM, and ether (no immobilization of probe, no hybridisation to target, no label); FIG. 3B illustrates fluorescence from an ITO slide prepared as in FIG. 3A with immobilization of dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4) as in Example 2 followed by washing with sterile water; FIG. 3C illustrates fluorescence from an ITO slide in which a continuous gradient is formed by electrochemical hydroxylation, (with no treatment with GOPS), followed by immobilization of dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4) by spotting; and treatment with dA.sub.20-Cy.sub.5, followed by washing with PBS (No significant fluorescence is observed); FIG. 3D illustrates fluorescence from an ITO slide in which a continuous gradient is formed by electrochemical hydroxylation, followed by GOPS treatment (reflux in toluene), followed by immobilization of dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4) by spotting, followed by treatment with dA.sub.20-Cy.sub.5, and washing with PBS; FIG. 3E illustrates fluorescence from an ITO slide in which a continuous gradient is formed by GOPS treatment (neat with Hunig's base, 110.degree. C., 60 min) after hydroxylation by plasma cleaning (15 min), followed by immobilization of dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4) by spotting; treatment with dA.sub.20-Cy.sub.5, and washing with PBS; FIG. 3F illustrates fluorescence from an ITO slide in which a continuous gradient is formed by homogeneous hydroxylation by the electrochemical method followed by treatment as for FIG. 3D; FIG. 3G illustrates fluorescence from an ITO slide in which a continuous gradient is formed by electrochemical hydroxylation, followed by treatment with GOPS (reflux in toluene), immobilization of dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4), followed by treatment with dA.sub.20-Cy.sub.5, and washing with PBS where DNA to be immobilized is not spotted, but spread along the slide; FIG. 3H illustrates fluorescence from an ITO slide in which a continuous gradient is prepared as for 3F, but only the upper half part (in red) is electrochemically homogeneously hydroxylated before GOPS treatment, immobilizing dT.sub.20-C.sub.12--NH.sub.2 (0.05M-H.sub.2SO.sub.4) by spreading over entire surface; FIG. 3I illustrative results for hybridisation of partially complementary DNA dT.sub.8A.sub.3T.sub.9-Cy.sub.5, to immobilized dT.sub.20, high contrast.

DETAILED DESCRIPTION OF THE INVENTION

Many microarray and biosensor platforms have been described to detect DNA hybridisation at interfaces. All of these approaches are predicated on the use of immobilised single-stranded nucleic acid (e.g., ssDNA) or a nucleic acid analog, and each is constrained by the physical chemistry of hybridisation in the environment defined by a surface. Thermodynamic considerations are often used to evaluate selectivity, and it is clear from this perspective that selectivity is not just a function of the nucleic acid sequence that is used to define a probe molecule. The thermodynamic stability of dsDNA is also dependent on nearest-neighbour interactions, e.g., between immobilized probes, including the extent of surface occupancy by ss nucleic acid and ds nucleic acid. This has consequences in terms of both selectivity and quantitative binding (equilibrium partitioning based on thermodynamic stability), and each can change as a result of the extent of formation of hybrids during an analytical experiment. Similar arguments apply for the kinetics of hybridisation and denaturation.

A fundamental issue is whether there can be confidence in assignment of sequence identification, and in quantitative analysis, when using markers (eg. fluorescent dyes, radiolabels, etc.) to detect the presence of dsDNA. Biosensors that are based on the use of one or a few sequences of ssDNA, or a device that is covered with many pads each containing one different ssDNA, cannot deal with the problems of selectivity and the thermodynamics of binding. At best, it might be possible to lay down a layer of ssDNA of known average density, and this can be used for calibration of concentration and selectivity over a narrow range of solution conditions and target DNA concentrations.

A solution to this problem provided by this invention is based on a blend of concepts; the distributed chemistry approach as found in microarrays combined with the careful control of the spatial distribution of probe molecules on a surface. What can solve the aforementioned limitations is the device strategy of this invention that relies on the use of gradients of properties of the immobilized probe molecule which can affect target binding to probe, for example gradients of ssDNA density and sequence length as shown in FIG. 1. Such gradients can be grown on a substrate surface in a continuum across a device surface, with for example immobilized probe density varying in one dimension. Two or more gradients can be formed on a single surface, for example, with probe density varying in one dimension and sequence length varying in a second dimension. The result is a surface carrying one or more spatially-distributed gradients that will maximize thermodynamic stability of target binding in defined zones or bands. The binding pattern of targets can then be observed as a one- or multi-dimensional spatially resolved image with a signal intensity gradient profile, e. g., using fluorescence labelling, as shown in FIG. 2.

A reference zone can optionally be built along a surface. A reference standard can be added to the sample (internal standard), or can be built onto the surface (FIG. 2), so that the reference standard binds at the surface within a defined zone or band that can be easily identified and the signal measured quantitatively for comparison purposes. The location of binding and the signal magnitude of the reference standard serves to calibrate for environmental conditions of ionic strength, pH, temperature and even non-selective adsorption of interferents. A comparison of the reference signal to the analytical signal can be done by ratioing or other background correction techniques, and this provides for confidence in sequence assignment of a real sample as well as quantitative analysis.

The use of large surface areas and large area-to-volume ratios (sample volume) means that such surfaces can react very quickly for signal generation from target binding. Many signal transduction methods are well-known in the art and used in known assays which detect the selective binding of a probe molecule to a target molecule. These methods can be employed in the methods of this invention, and include, but are not limited to, detection of fluorescence from labelled target, fluorescent intercalators, fluorescent groove binders, molecular beacons, radioisotopes, surface potentials, coloured products, enzyme labelled targets, antibody labelled targets, and gold particle labelled targets.

In one embodiment of this invention, light emitted from fluorophores at a surface of a sensing device of this invention which carried one or more gradients can be monitored by a photomultiplier tube (PMT), a vidicon tube, a CCD or any other suitable one-dimensional scanning device or two- or multi-dimensional light detection equipment. In one specific application, the use of tethered fluorescent dyes permits fast, sensitive detection, and a regenerable device technology that can be used to measure multiple samples. Microscopy or waveguides can be used to collect fluorescence emission, followed by known chemometric methods for signal processing to discern patterns of binding from spatially resolved signals that are based on intensity, wavelength and time-resolved spectroscopy.

Gradients of probe molecule density (including mixtures of molecules such as ssDNA and other polyelectrolytes as spacers), sequence length, orientation and structure (eg. types of structures: aptamers, hairpins, lariats, and related structures ) can be generated on surfaces by many different methods. As one example, a density gradient of hexaethyleneglycol (HEG) linker that is used as a template for subsequent immobilization of ssDNA is described herein. A gradient of density of HEG can be immobilized by allowing the HEG to react along different areas of a surface under suitable chemical conditions for different periods of time or at different concentrations. Control of reaction time can for example be achieved by controlling the speed of removal of a surface from a reactive HEG solution by dip-casting technology. Other techniques include gradient spraying or rolling, differential electrochemical reaction across a surface as can be done using a resistance drop, or any other means of controlling reaction time as a function of a dimension of the surface. Once the density gradient is established with a linker, automated nucleic acid synthesis, or single step oligonucleotide immobilization can then proceed to form a gradient of immobilized nucleic acid probes. A gradient density of reactive probe molecules can be immobilized to a surface in a manner similar to that described for HEG.

Another example that provides for a gradient of HEG is to immobilize a constant density of HEG that has a protecting group at the terminus, and then to remove the protecting group as a gradient across the surface. This creation of a gradient of capped HEG insures that further coupling of probe molecules is controlled spatially, as the probe molecules can only be immobilized at uncapped HEG linkers.

Another example of a parameter that can be varied to create a gradient of selectivity is sequence length of probe molecules. For example, this can be achieved by use of enzyme assembly of segments of nucleic acid, or by enzymatic disassembly of oligonucleotides, using an approach that controls reaction time. This control can be achieved using dip-casting methodology, spraying, rolling, and other methods.

Another parameter that can provide a gradient of selectivity is the orientation of immobilized probe molecules. The orientation of probe molecules can be controlled by density manipulation, and by selection of nucleotide sequences so that folding and bending can be induced in the probe structure.

A further example of a parameter that can provide a gradient of selectivity is the distribution of formal surface charge or dielectric constant. For example, charged linker molecules (with carboxymethyl or amine moieties), or a mixture of charged and uncharged linker molecules, can be deposited on a surface as a gradient using controlled dip casting methods, gradient spraying or rolling as discussed above. The gradient of electrostatic fields influences the alignment and mobility of probe molecules that are subsequently immobilized, and can affect the thermodynamic stability and kinetics of formation and dissociation of probe-target hybrids.

Another alternative is to generate a gradient of reporter molecules or markers that can report the presence of hybridisation as a function of location in the gradient, for example, a gradient of tethered intercalating dyes can be generated on a surface using methods which control the rate of the tethering reaction.

The surfaces that can be used to support gradients of nucleic acids or nucleic acid analogs are many, and include, but are not limited to, fused silica, quartz, silicon, plastics, glass, gold, metals, transparent electrodes (e.g., indium tin oxide or related materials), ceramics (e.g., metal oxides), paper, conductive carbon, and conductive polymers. Immobilization of the nucleic acids and nucleic acid analogs or other probe molecules can be achieved by covalent bonding, adsorption, biotin-avidin linkage, thiol-gold interactions and any other method that can attach the materials at a controllable density, sequence length or orientation. The surface may be of any shape convenient for conducting spatially resolved imaging or detection. Gradients may be formed as a function of any dimension of the substrate, e.g., along a length or a width of a substrate, extending radially from a point on a surface or extending linearly from a straight line on a surface.

Gradients can be achieved by templating the surface with activation sites or linkers for attachment of nucleic acids and nucleic acid analogs, and by controlling the sequence length, sequence type, and orientation of probes, across a surface. Methods of preparation of gradients include but are not limited to use of controlled dip-casting and use of controlled reaction time, generation of gradients of light intensity in a photocatalyzed immobilization, generation of mechanical gradients achieved by spraying and/or rolling, control of capping reagents and cap densities, control of sequence length by annealing of segments of nucleic acids, and removal of portions of sequences by degradation methods.

While not necessary, it is possible to immobilize probe molecules onto a linker or spacer. In such a case, the length of the spacer between the substrate and the first nucleoside is chosen to be sufficiently long so that the environment of the terminal nucleoside is fluid enough to permit efficient coupling with oligonucleotides, or 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 (>99.5%) synthon coupling yields in automated nucleic acid synthesis. Because the linker is terminated by a protected nucleoside, any reactive sites on the support that would lead to the production of unwanted side products during automated synthesis can be eliminated by treating the derivatised supports with a surface-capping agent such as acetic anhydride prior to synthesis. Using such an approach, a gradient of capping on a surface can be created by controlling the time of reaction along the surface.

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 functional amphiphilic support derivatisation agent is created by condensing aminopropyltriethoxysilane (APTES) with 12-nitrododecanoic acid. Similarly, a surface can be activated with other reagents such as glycidoxypropyltrimethoxysilane (GOPS) according to the method of Watterson et al. (2000, Langmuir, 16: 4984). This yields a substrate derivatised with short spacer molecules with terminal epoxide moieties. 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. 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 NucI. Acids Res., 20(7). 1679). Polyether chains provide for hydration, flexibility for molecular motion, and improved biocompatibility in terms of minimization of non-selective binding to biological compounds. This support is then used directly for oligonucleotide attachment by automated synthesis wherein an ammonolysis resistant phosphoramidite linkage is made between the activated support and the first nucleotide, or for direct immobilization of a oligonucleotide. 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.

Since polyethylene glycols are bifunctional, there exists the possibility of creating non-reactive closed-loop structures that may significantly decrease the amount of loading of oligonucleotides on the surface of an optical fibre. To eliminate any such problem, one terminus of the polyether can be protected with a suitable blocking group, for example, with a dimethoxytrityl (DMT) functionality, prior to extension of the glycidoxypropyltrimethoxysilane. 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+).

Mono-dimethoxytrityl protected polyethylene glycols may be introduced onto the surface by a number of methods. Surfaces that are 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