Patent Application
20160115535
This invention relates to the field of biochemical analysis, more specifically, the use of patterned surfaces and microarrays for detection and quantification of biochemical targets, such as those associated with oligonucleotide detection and analysis.
The effective treatment of an infection requires microbial identification in order to prescribe an appropriate antimicrobial treatment. The traditional diagnostic approach cultures blood, sputum, urine, washes, swabs, or other bodily fluids to determine if microbes are present, and then examines phenotypes within the culture, such as morphology or antimicrobial susceptibility, for identification. This approach is slow. It often requires periods as long as 3-5 days to complete, during which time the prescription of antibiotics is under-informed and fraught with problems including serious threats to patient well-being.
Molecular diagnostics (MDx) have radically changed the process of clinical microbial identification (see, Deshpande et al., Expert Review of Molecular Diagnostics, 2012. 12(6): pp. 645-659; Millar et al., Current Issues in Molecular Biology, 2007. 9(1): pp. 21-40; and Muldrew, Current Opinion in Pediatrics, 2009. 21(1): pp. 102-111, the disclosures of all three of which are incorporated by reference herein). Importantly, MDx approaches are both specific and fast. They can identify microbes to the species and strain level based on biospecific markers over a time scale that can be as short as one hour. Consequently, clinicians can administer the most effective and appropriate antimicrobial treatment at an early time point with substantial implications both for patient well-being and for easing the burden on the health-care system.
Surface-patterned microgels provide a fundamentally different platform for the design and development of clinically relevant molecular diagnostics. Microgels can be localized on a surface in an array format with feature (spot) sizes as small as a few hundred nanometers, and they can be synthesized with reactive groups, such as biotin, which enable their functionalization with oligonucleotides or other biomolecules. Importantly, and unlike the vast majority of other surface-tethering methods, surface-patterned microgels not only physically separate the oligonucleotides from the underlying solid surface, but, because of the highly hydrated structure at the microgel surface, they tether the oligonucleotides in an environment much like the surrounding aqueous medium. This preserves their biospecific properties. Furthermore, in contrast to approaches that entrap probes (see, Livshits and Mirzabekov, Biophysical Journal, 1996. 71(5): pp. 2795-2801; Proudnikov et al., Analytical Biochemistry, 1998. 259(1): pp. 34-41; Rehman et al., Nucleic Acids Research, 1999. 27(2): pp. 649-655; Rendl et al., Langmuir, 2011. 27(10): pp. 6116-6123; and Rubina et al., Analytical Biochemistry, 2004. 325(1): pp. 92-106, the disclosures of all five of which are incorporated by reference herein) or primers within a hydrogel, microgel tethering positions the oligonucleotides at or near the gel surface rather than within the gel itself, thus providing maximum accessibility for hybridization and complexation.
While there are many possible ways to detect a hybridization event, fluorescence is among the most common. This is a simple mechanism that can be detected and processed using simple optics and electronics. The fluorescence-detection process can be further simplified by using self-reporting hybridization probes known as molecular beacons (see, Tyagi, S. and F. R. Kramer, Nature Biotechnology, 1996. 14(3): pp. 303-308, the disclosure of which is incorporated by reference herein) or other similar self-reporting probes. Molecular beacon probes fluoresce when hybridized to a complementary target, and they thus eliminate the additional need to label the target prior to, during, or after, hybridization to the probe. They furthermore enable real-time detection of the signal during, rather than after, an experiment or test, because the signal is emitted once a hybridization event occurs. Invented in the mid-1990's, they have been extensively used in basic scientific studies of cellular and sub-cellular processes, and they have played a key role in a number of highly successful commercial ventures associated with sequencing or detection (see, Tyagi, S. and F. R. Kramer, F1000 Medicine Reports, 2012. 4(1), the disclosure of which is incorporated by reference herein).
Despite their many successes, molecular beacon probes have thus far been used almost exclusively untethered in solution. While they can be immobilized on solid surfaces by several mechanisms (see, Du et al., J Am Chem Soc, 2005. 127(21): pp. 7932-40; Du et al., J Am Chem Soc, 2003. 125(14): pp. 4012-13; Liu et al., Analytical Biochemistry, 2000. 283(1): pp. 56-63; Martinez et al., Anal Chem, 2009. 81(9): pp. 3448-54; Situma et al., Analytical Biochemistry, 2007. 363(1): pp. 35-45; Song et al., Angew Chem Int Ed Engl, 2009. 48(46): pp. 8670-4; Stoermer et al., J Am Chem Soc, 2006. 128(51): pp. 16892-903, the disclosures of all seven of which are incorporated by reference herein), their performance when tethered has typically been much poorer than that when free in solution. The background fluorescence associated with surface-tethered beacons has traditionally been high, so the signal above background is low. This phenomenon has been attributed to the fact the beacon's probe sequence, fluorophore, or quencher can nonspecifically interact with the substrate to produce background fluorescence without a biospecific hybridization event. Consequently, molecular beacon probes have not been extensively used in microarray formats.
The problem of preserving high signal-to-background when tethering molecular beacon probes to a surface has been addressed (see, Dai et al., Soft Matter, 2012. 8(11): pp. 3067-3076; Dai et al., WO/2013/089888, the disclosures of both of which are incorporated by reference herein) by tethering the beacons to highly hydrated microgels patterned by a focused electron beam on a glass or Si substrate (see, Wang et al., J Polymer Science, Part B: Polymer Physics, 2013, the disclosure of which is incorporated by reference herein). This important gel-tethering approach brings at least two significant advantages. First, the beacons are grafted to the outermost surface of the microgel. In contrast, trapping oligonucleotide probes within a gel not only restricts the possible conformations of the various biomolecules but also slows the reaction kinetics because of the need for diffusion through tortuous pathways within the gel prior to binding. Both of these constraints are eliminated when the probes are tethered to the surface of the gel. Second, the nature of the e-beam patterned microgels is such that the crosslink density at the outer surface gradually approaches zero, so the probes that are tethered at the outer surface find themselves extremely unconstrained and in the most water-like environment possible. Hence, their performance can approach that of untethered beacons in aqueous solution where the probes are unrestricted.
Microgels can be particles of any shape whose equivalent diameter is approximately 0.1-100 μm. Surface-pattern microgels can be created by techniques of photolithography (see, Revzin et al., Langmuir, 2001. 17(18) 5440-5447, the disclosure of which is incorporated by reference herein). However, e-beam patterning of microgels brings particular flexibility to the creation of the surface-patterned microgels for use in self-reporting, multiplexed, microarrays needed to interrogate a physiological sample such as blood with a large set of questions. Unlike spotting methods that create individual array spots with diameters of approximately 100-500 μm (see, Rubina et al., Analytical Biochemistry, 2004, 325(1), 92-106, the disclosure of which is incorporated by reference herein), e-beam patterning can create discrete microarray spots with diameters ranging from sub-micron (one microgel) to tens/hundreds of microns (multiple overlapping microgels). Individual array spots can be positioned in user-defined shapes, at specific locations on a surface, at controllable distances from each other, and in any number of patterns, including 1-D or 2-D arrays within a microfluidic channel or a chamber. Furthermore, discrete spots within the array can be differentially functionalized. For example, functionalization at the scale of approximately 1-10 μm can be achieved using a method such as dip-pen nanolithography or at the scale of approximately 10-100 μm using a position-sensitive microspotting robot.
Molecular beacons can be tethered to a microgel by a number of chemistries. Among the successful chemistries is the biotin-streptavidin interaction. Microgels can be synthesized using biotinylated PEG (B-PEG) homopolymer and then activated by exposure to streptavidin. Biotinylated oligonucleotide(s) can subsequently be deposited onto the activated microgel spot(s) by one of the micro or nano-spotting methods. For microgels e-beam synthesized by e-beam under typical conditions (e.g., 100 nm films of 5 kDa B-PEG homopolymer and 50 fC of 2 keV focused electrons), each microgel can be created so that it presents about 11,000 active binding sites (see, Dai et al., Soft Matter, 2012. 8(11): pp. 3067-3076; Dai et al., WO/2013/089888, the disclosures of which are incorporated by reference herein).
Because the concentration of targets (e.g., microbial RNA) in a specimen such as blood is typically low, amplification is used to generate detectable signals. So-called target amplification creates copies of the target RNA (or DNA). PCR is one-such amplification method. It has been extensively used in a variety of formats. Nucleic Acid Sequence Based Amplification (NASBA) is another established but less-common method (see, Deiman et al., Applied Biochemistry and Biotechnology—Part B Molecular Biotechnology, 2002. 20(2): pp. 163-179, the disclosure of which is incorporated by reference herein). It directly amplifies RNA, and, unlike RT-PCR, is not susceptible to contamination by genomic DNA. Furthermore, NASBA is isothermal, and a technology based on NASBA thus eliminates the added complexity of thermal cycling required for PCR. Many different amplification methods are available including, for example, Transcription-Mediated Amplification (TMA), Strand Displacement Amplification (SDA) and many variations of PCR.
In the art, the NASBA process has been implemented with the reagents dissolved in one or more solutions. The NASBA process uses three enzymes: Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT); RNase H; and T7 RNA Polymerase (T7 Poly). It also uses two primers: the P2 forward primer and the P1 reverse primer. In a solution-based NASBA process, these reagents are mixed with nucleic acids and strain-specific RNA (target RNA+) isolated from target microbes in the physiological sample. Using strain-specific target RNA+ isolated during sample preparation, the initiation phase produces ds-DNA templates with a T7 promoter region from which the target antisense RNA (RNA-) is produced. The cyclic phase uses this target RNA− to make additional ds-DNA from which more copies of RNA− amplicons are created at an increasing rate.
Multiplexing imposes important challenges to an amplification process. When probing for multiple target microbes in a single reaction chamber, primer sets needed to amplify each particular target must be in that chamber. As the number of possible targets increases, the number of primers and their overall concentration increases. When the primers are untethered and free in solution, they can complex to form, for example, primer-dimers rendering them ineffective for the amplification process. When performing a multiplexed amplification process in a single solution, the amplification efficiency thus decreases as the degree of multiplexing increases. One method of circumventing this problem is simply to create individual reaction chambers, each of which contains only one primer set and the corresponding probe, and all of which contain a sample of the parent target-containing solution. Such an approach has been used successfully in commercial PCR-based systems.
Embodiments of the present invention provide gel-tethered molecular beacon probes and corresponding amplification primers, microarrays that include discrete regions each with biospecific combinations of gel-tethered probes and primers, and related methods of analysis and detection of multiple biological targets using such probes, primers and arrays.
In one aspect, the present invention provides a device for detecting multiple biological targets (e.g., nucleic acids or peptides). Embodiments of the device include a plurality of quenched molecular beacon probes capable of binding in a complementary manner to molecules that are complementary to the probes, a plurality of amplification primers, and an array of spatially-separated structures on a solid surface. The conformational changes in the probes that take place during binding cause the probes to fluoresce. In embodiments of the present invention, the complementary molecules are uniquely identified with the target molecules in such a way that the fluorescence of the probe signals the presence of the biological target.
In a second aspect, the present invention provides a conjugate for amplifying and detecting a target. Embodiments of the conjugate have: (i) a gel having a diffuse surface; (ii) a molecular beacon probe that is linked to the gel; and (iii) one or more amplification primers that are linked to the same gel.
In a third aspect of the present invention, the aforementioned device, one or more conjugates, or an array described above is used in a method for detecting the presence of one or more targets in a biological sample. Embodiments of methods according to the present invention include the steps of (a) providing a device, one or more conjugates, or an array described above; (b) contacting the device, the one or more conjugates, or the array with the biological sample for a period of time under conditions permitting binding between the one or more targets and the corresponding molecular beacon/hairpin probes or to the corresponding amplification primers; and (c) determining the presence of the binding between the one or more targets and the corresponding probes thereby detecting the presence of the one or more targets.
A fourth aspect of the present invention provides methods for making a conjugate described above. Methods according to the present invention include the steps of: (i) providing a gel that contains a first member of an affinity pair; (ii) contacting the gel with molecular beacon probes specific for a target, each molecular beacon probe comprising a second member of the affinity pair, for a period of time under conditions permitting binding between the first member and second member; (iii) removing molecular beacon probes that do not bind to the first member; (iv) contacting the gel with amplification primers that produce targets specific to the molecular beacon probes, each primer comprising another member of the affinity pair, for a period of time under conditions permitting binding between the first member and second member; and (v) removing primers that do not bind to the first member.
A fifth aspect of the present invention provides methods for making an array, such as the arrays discussed above. Such methods include the steps of: (1) obtaining a plurality of conjugates described above, which are specific for a plurality of targets, respectively; (ii) obtaining a support that has a plurality of unique locations; and (iii) depositing said plurality of conjugates on the plurality of unique locations, respectively.
For a better understanding of the present invention, reference is made to the following detailed description of the exemplary embodiments considered in conjunction with the accompanying drawings, in which:
Embodiments of the present invention provide gel-tethered molecular beacon probes and corresponding amplification primers, microarrays that include discrete regions each with biospecific combinations of gel-tethered probes and primers, and related methods of analysis and detection of multiple biological targets using such probes, primers and arrays.
In one aspect, the present invention provides a device for detecting multiple biological targets (e.g., nucleic acids or peptides). Embodiments of the device include a plurality of quenched molecular beacon probes capable of binding in a complementary manner to molecules that are complementary to the probes, a plurality of amplification primers, and an array of spatially-separated structures on a solid surface. The conformational changes in the probes that take place during binding cause the probes to fluoresce. In embodiments of the present invention, the complementary molecules are uniquely identified with the target molecules in such a way that the fluorescence of the probe signals the presence of the biological target. In embodiments of the device, each structure contains a member selected from the group consisting of hydrogels, microgels, hydrogel coatings and hydrogel-like coatings, each having chemically or physically incorporated therein a linking moiety. The structure may be a surface-patterned microgel (e.g., an electron-beam surfaced patterned microgel). The microgel may comprise poly(ethylene glycol) or other polymers from which gels can be formed. In some embodiments, the structure is biotinylated, e-beam patterned poly(ethylene glycol) microgel.
In a second aspect, the present invention provides a conjugate for amplifying and detecting a target. Embodiments of the conjugate have: (i) a gel having a diffuse surface; (ii) a molecular beacon probe that is linked to the gel; and (iii) one or more amplification primers that are linked to the same gel. The molecular beacon probe is specific for the target. The amplification primers are specific to produce that same target. In embodiments of the present invention, the linkage may be a covalent or non-covalent chemical bond. In one embodiment, the molecular beacon probe and the primers are linked to the gel via an affinity pair. In embodiments of the present invention, the members of each affinity pair may be in a pair format (e.g., biotin-streptavidin). In embodiments of the present invention, the members of each affinity pair may be in a sandwich format (e.g., biotin-streptavidin-biotin). In embodiments of the present invention, the molecular beacon probe and primers are linked to a biotin, and the gel presents at or near its surface, biotin or streptavidin so that probe and gel are linked via the biotin-streptavidin pair. In other embodiments, the probe and corresponding primers are linked to the gel by a covalent pathway provided that the chemistry of the linking is orthogonal to other functional groups on the conjugate and on the probe.
In some embodiments of the present invention, the conjugate includes poly(ethylene glycol), poly(ethylene oxide), or other water-soluble polymers including: poly(acids), such as poly(acrylic acid), poly(methacrylic acid), poly(N-isoacrylimide), and poly(vinyl pyrrolidone); as well as co-polymers of these and other polymer moieties. In some embodiments, the conjugate is a microgel. In some embodiments, each microgel is linked to 5,000 or more (e.g., 6,000, 10,000, 15,000, or other numbers of similar magnitude) molecular beacon probes and corresponding amplification primers. In some embodiments, the microgel is 100-10,000 nm in diameter, (e.g., 100-1,000 nm in diameter). In some embodiments, (e.g., where the microgel is bound to a solid substrate), the hydrated microgel is 50-500 (e.g., 60-120) nm or more in height.
In some embodiments, when hydrated in an aqueous medium, the conjugate includes a transition region from the aqueous medium at the conjugate surface to a pure hydrated region within the conjugate in which the conjugate becomes gradually more crosslinked. In some embodiments, the transition region has a thickness at least equal to the combined length of the probe and the affinity pair or to the length of a ds-DNA bridge tethered at each end by the specific amplification primers, each with an affinity pair. In some such embodiments, the molecular beacon probes and the amplification primers are in the most water-like environment possible while remaining tethered to the gel. In such embodiments, the signal-to-background (SBR) ratio of the molecular beacon probe for the target can be 5 or greater, e.g., as high as 10, 20, 30 or greater. In some such embodiments, the SBR is 10% or greater (e.g. 20%, 30%, 40%) of a reference SBR ratio determined using a reference probe identical to the tethered molecular beacon probe, except that the reference probe is in a solution and not tethered.
In some embodiments, the conjugate described above can further involve a substrate to which the conjugate is attached. In some embodiments, the invention also provides an array containing (i) a support (the substrate) having a plurality of unique locations and (ii) a plurality of conjugates as described above for different targets, where each conjugate is immobilized to a unique location of the support corresponding to a target. Location on the support is one way in which the specific target associated with a particular conjugate can be identified. In some embodiments, the density of biospecific probes and corresponding primers at one of the plurality of unique locations is at least 1,000 pre, at least 10,000 pre, or at least 20,000 pre. In some embodiments, each microgel on a unique location has a hydrated height in the range of about 50 to about 500 nm, much higher density of probe molecules and corresponding primers projected onto the two-dimensional surface of the substrate/support as compared to probes tethered to the substrate by spacer molecules and/or tethering moieties that are short (about the same size as the probe/primer molecule itself). In some embodiments of the present invention, a microgel having a diameter of 400 nm and a height of 100 nm has about 11,800 probes per microgel, which is equivalent to a density of over 20,000 probes/μm2 when referenced to the area of the substrate/support. In some embodiments, one or more primers are tethered to specific conjugates on the array, reducing restrictions on multiplexing due to primer-primer interactions such as dimerization, and, in some cases, entirely eliminating such restrictions.
In a third aspect of the present invention, the device, one or more conjugates, or an array described above can be used in a method for detecting the presence of one or more targets in a biological sample. Embodiments of methods according to the present invention include the steps of (a) providing a device, one or more conjugates, or an array described above; (b) contacting the device, the one or more conjugates, or the array with the biological sample for a period of time under conditions permitting binding between the one or more targets and the corresponding molecular beacon/hairpin probes or to the corresponding amplification primers; and (c) determining the presence of the binding between the one or more targets and the corresponding probes by fluorescence imaging thereby detecting the presence of the one or more targets. Because of the small size of the conjugate and the high density of the probes and primers, the method can be carried out for high-throughput detection of multiple targets in a sample simultaneously.
A fourth aspect of the present invention provides methods for making a conjugate described above. Methods according to the present invention include the steps of: (i) providing a gel that contains a first member of an affinity pair; (ii) contacting the gel with molecular beacon probes specific for a target, each molecular beacon probe comprising a second member of the affinity pair, for a period of time under conditions permitting binding between the first member and second member; (iii) removing molecular beacon probes that do not bind to the first member; (iv) contacting the gel with amplification primers that produce targets specific to the molecular beacon probes, each primer comprising another member of the affinity pair, for a period of time under conditions permitting binding between the first member and second member; and (v) removing primers that do not bind to the first member. The providing step can be carried out by a process having the steps of: (i) obtaining a substrate; (ii) depositing a precursor of the gel on the substrate a layer; and (iii) exposing the substrate and the layer of precursor to an electron radiation for a period of time under conditions permitting cross-linking within the gel precursor and cross-linking between the resulting gel and substrate.
A fifth aspect of the present invention provides methods for making arrays of the types discussed above. Such methods include the steps of: (i) obtaining a plurality of conjugates described above, which are specific for a plurality of targets, respectively; (ii) obtaining a support that has a plurality of unique locations; and (iii) depositing said plurality of conjugates on the plurality of unique locations, respectively.
The description of the present invention includes a number of non-limiting examples (i.e., Examples 1-7) to illustrate various features of certain embodiments of the present invention, as presented below.
Example 1 illustrates control of the concentration of tethering sites on and near the surface of a biotinylated PEG (B-PEG) microgel according to an embodiment of the present invention. In embodiments of the present invention, the spacing between adjacent tethered molecules may be controlled so as to maximize the amplification efficiency and/or maximize the signal from fluorescing molecular beacon probes.
With respect to Example 1,
In order to fluoresce, the gel-tethered molecular beacon probes 10 must hybridize and open. As discussed with respect to
The concentration of biotins within a microgel 30 can be controlled. In the case of microgels formed by electron-beam patterning of PEG thin films, for example, the biotin concentration can be controlled by varying the molecular weight of the B-PEG precursor polymer or by blending B-PEG homopolymer with hydroxyl-terminated PEG homopolymer. Bifunctional biotinylated PEG homopolymer is commercially available with molecular weights ranging from 1 kDa-10 kDa, and monofunctional biotinylated PEG is available over an even greater range of molecular weights. Other functionalized polymeric precursors can be custom synthesized. To further control the inter-oligonucleotide spacing, one can either use homopolymer precursor of higher/lower molecular weight, which will affect the electron dose required for crosslinking, or by blending biotinylated PEG with hydroxyl-PEG of the same, or different, molecular weight. Alternatively, after microgel patterning, the concentration of SA-activated biotins actually functionalized can be controlled by varying the oligonucleotide concentration in the functionalizing solution or adjust the time allowed for these oligonucleotides to tether to the SA-activated biotin sites.
The average number of molecular beacon probes, or other tethered molecules, on a microgel 30 can be determined using an established streptavidin-release assay. As a substrate, one can use, for example, a 5 mm×7 mm Si substrate with microgels patterned on it in a square array with 1 μm inter-gel spacings. Such a surface thus contains enough microgels (e.g., on the order of 106) to yield detectable and reliable signals, and, since the number of microgels on the surface is known, the average signal per microgel can be determined. One can, for example, expose such a surface to 500 μL 95% of formamide solution with 10 mM EDTA (pH 8.2) for 1 hr at 65° C. The SA-biotin bonds break because of this treatment and release fluorescently labeled reagents into solution, the concentration of which can be determined spectro-photometrically.
Example 2 provides an example of the modification of molecular beacons suitable for use untethered in aqueous solution in order to make them compatible with tethering to B-PEG microgels.
Much is already known about the design of molecular beacon probes for use in untethered assay applications. These can be modified, or new ones designed if necessary using established design techniques, to render them compatible with a gel-tethering approach. Similarly, many primers which create amplicons specific to corresponding probes have been identified or can be designed using established methods, and these, too, can be modified to render them compatible with a gel-tethering approach. The amplification primers are typically linear oligonucleotides. They can be synthesized with biotin, a spacer of oligo ethylene glycol (e.g. SP9), and an oligonucleotide spacer of controllable length (e.g., 10 T) at their 5′ end. Such molecules can be tethered to microgels via the SA-B interaction.
Table 1 summarizes molecular beacon probes, NASBA amplification primers, and complementary targets and templates appropriate for simplex or multiplex assays of bloodstream infection, according to an embodiment of the present invention, and is adapted from: Zhao et al. J. Clinical Microbiology, 2009. 47(7): pp. 2067-2078, the disclosure of which is incorporated by reference herein. The molecular beacon probes and NASBA amplification primers listed in Table 1, once modified with appropriate spacers and tethering moieties, are appropriate for use in applications involving gel-tethered amplification and/or gel-tethered detection according to embodiment of the present invention. Assays such as this one, or others which can be developed with appropriate design of molecular beacon probes and amplification primers, can be used in a gel-tethered integrated diagnostic platform according to an embodiment of the present invention. Embodiments of the present invention are also applicable to RNA/DNA amplification and detection applications, such as cancer detection or cancer identification to which gel-tethered molecular diagnostic approaches can be applied.
GTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAGTTAAT
Candida
Aspergillus
AAGTTCCGTTTGAAAGGCGCCCTCGTGCGCCGTGTGCCGAA
GGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTTAG-
Example 3 illustrates the case of solution NASBA coupled with gel-tethered molecular beacon probes according to an embodiment of the present invention. In the present invention, copies of antisense counterparts to the biological targets may be amplified in solution using the existing NASBA protocols and interact favorably with gel-tethered probes.
In contrast to the vast majority of molecular diagnostics (MDx) tests that involve end-point labeling to induce fluorescence, the fluorescence emitted by microgel-tethered beacons increases continuously with time during the amplification process, realizing the concept of real-time detection.
Example 4 illustrates the case of tethered molecular beacon probes together with one type of amplification primer being tethered to the same microgel, accordingly to an embodiment of the present invention. In the present invention, amplification and detection may occur when both a primer and a probe are gel-tethered.
Embodiments of the present invention provide a partial gel-tethered NASBA process. For example, biotinylated primer P1 66 and biotinylated molecular beacons 10 can both be tethered to the same microgel spots 30. This places the amplifying primer 66 and the detecting molecular beacon probe 10 in immediate proximity to each other. When template DNA+ 64, AMV-RT (not shown), and the T7 polymerase (not shown) are added to the surrounding solution, the NASBA process produces RNA− amplicons 70, which subsequently hybridize with the molecular beacon probes 10 causing them to fluoresce. While untethered amplification primers (not shown) can be included in the surrounding solution, these are not required for every embodiment of the present invention.
Example 5 illustrates the case of a hybrid amplification and detection process according to an embodiment of the present invention.
In the illustrated process, RNA+ 44 can be amplified using primers 60 in solution and using a primer 66 that is microgel tethered. This is a variation of combined amplification and detection, according to embodiments of the present invention, which involves a partial gel-tethered NASBA process together with a solution NASBA process. Such an embodiment can be used, for example, for the real-time detection of RNA. For example, biotinylated primer P2 60 and biotinylated molecular beacons 10 can both be tethered to the same microgel spots 30. This again places an amplifying primer 60 and the detecting molecular beacon probe 10 in immediate proximity to each other. When biological target RNA+ 44, AMV-RT (not shown), and the T7 polymerase (not shown) are added to the solution, the NASBA process produces RNA-amplicons 70, which can either subsequently hybridize with the beacons 10 causing them to fluoresce, or, hybridize with the gel-tethered P2 60. The hybridized, gel-tethered P2 60 is converted to a ds-DNA structure 68, tethered at one end, by exposure to and reaction with AMV-RT and RNase H. RNA− amplicons 70 generated by this ds-DNA structure 56 are in close proximity to the molecular beacon probes, accelerating the detection process.
Example 6 illustrates the case of RNA− amplicons created from double-stranded DNA that is tethered to a microgel spot. Such amplicons can hybridize to molecular beacon probes also tethered to that same spot.
In this exemplary embodiment, double-stranded DNA with a biotin group at each end are created by mixing solutions containing two complementary single-stranded DNA molecules (not shown), each biotinylated at one end. After mixing, this solution is heated to 65° C. for 3 minutes to melt any hybridizations and then slowly cooled to room temperature during which time biotinylated ds-DNA forms 68. When exposed to T7 polymerase, RNA-amplicons 70 are created from the ds-DNA 68. Microgel spots can be functionalized with biotinylated ds-DNA 68 and biotinylated molecular beacon probes 10, which are complementary to the RNA− amplicons 70. For example, a solution of ds-DNA 68 and molecular beacon probes 10 can be exposed to streptavidin-activated biotinylated microgel spots 30. After about 60 minutes incubation, both ds-DNA 68 and molecular beacons 10 are tethered to the microgels 30 (see
The microgel spots were each functionalized with both ds-DNA and molecular-beacon probes. The positive control microgel spot was exposed to a NASBA amplification solution that contained no enzymes, but was spiked with ss-DNA complementary to the gel-tethered molecular beacon probes. In this case, the ss-DNA hybridizes to the molecular-beacon probes.
Example 7 illustrates the case of two amplification primers and one corresponding molecular beacon probe tethered to the same microgel, according to an embodiment of the present invention. In the present invention, gel-tethered NASBA amplification may be coupled with gel-tethered molecular beacon detection.
Such embodiments of the present invention provide arrayed microgel spots which enable the capture, amplification, and detection of biospecific oligonucleotide targets.
When multiple spots 30 such as these are arrayed on a surface of, for example, glass or silicon, according to embodiments of the present invention, each spot 30 may be functionalized with a set of molecular beacon probes 10 and corresponding amplification primers 60, 66 specific to a particular target 44, 70 (i.e., the various microgels 30 in an array may be modified such that each modified microgel 30 becomes specific to a different target). The number of biological or complementary targets 44, 70 that may be probed by the array thus depends on the number of spots 30 in the array. Since the amplification primers are all tethered to microgels within each spot, this embodiment of the invention eliminates the need to have primers in solution. Consequently, the complexation of primers free in solution is eliminated. An array according to this embodiment of the present invention can thus become highly multiplexed while still enabling all of the spots in the array to be exposed to the same, single solution that will contain one or more targets, and eliminates the need to create individual reaction chambers, each of which contains only one primer set and the corresponding probe and all of which contain a sample of the parent target-containing solution.
Further to the exemplary embodiments presented above, the present invention further includes, but is not necessarily limited to, the following embodiments, each of which operates according to the processes discussed above.
Embodiments of the present invention provide a device for capturing, amplifying, detecting, and identifying one or more biological target molecules. In some embodiments, the device comprises an array of isolated areas on a surface, each area having quenched probes and amplification primers tethered thereto. In some embodiments, each of the aforesaid probes and primers is tethered to a structure that is patterned on one of the isolated areas. In some embodiments, each of the isolated areas includes one or more patterned microgels. In some embodiments, the aforesaid probes and primers are tethered to the structure through one or more tethering moieties that are chemically or physically incorporated into the structure. In such embodiments, the aforesaid probes are molecules that undergo a conformational change when they bind to a complementary molecule (i.e., a complementary target), such that the conformational change leads to a detectable signal. In some embodiments, the structure is hydrophilic and, except for the tethering moieties, interacts weakly with the probes, the amplification primers, the enzymes used for amplification, the complementary targets, and the probe-tethering molecules, such that the structure does not interfere with construction or use of the device. In some embodiments, the structure, when hydrated, provides a gradient in crosslink density which is lowest at the surface of the structure, and is such that, at some position in the structure, the target molecule is unable to penetrate further towards the center of the structure. In some embodiments, the complementary target is uniquely identified with the biological target.
In some embodiments of the device, the array contains at least five areas each containing a structure tethered to a set of probes and primers specific to a particular complementary and/or biological target molecule. In some embodiments of the device, the array contains at least ten areas each containing a structure tethered to a set of probes and primers specific to a particular target molecule. In some embodiments of the device, the array contains at least twenty areas each containing a structure tethered to a set of probes and primers specific to a particular target molecule. In some embodiments of the device, the array contains more than twenty areas each containing a structure tethered to a set of probes and primers specific to a particular target molecule. In some embodiments of the device, each of the sets of probes and primers in at least some of the areas corresponds to a different target molecule, and all of the areas are simultaneously exposed to a primer-free aqueous solution to test whether or not that solution contains any of the target molecules corresponding to any of the areas within the array.
In some embodiments of the device, the structure is a patterned microgel. In some such devices, the structure is an electron-beam-patterned microgel. In some such devices, the microgel includes poly(ethylene) glycol. In some such devices, the structure is a biotinylated, e-beam patterned poly(ethylene) microgel.
In some embodiments of the device, the tethered probes are hairpin oligonucleotides labeled with a fluorophore and a non-fluorescent quencher, the combination of which fluoresces when the hairpin binds to its complementary target. In some embodiments, the tethered primer or tethered primers are oligonucleotides, which can create targets complementary to the probes by a process including an operation on the biological target molecule. In some embodiments of the device, the biological target molecule includes an oligonucleotide. In some embodiments, the oligonucleotide includes non-natural nucleotides, nucleotide analogs, or non-natural inter-nucleotide linkages.
In some embodiments of the device, the tethered probes include linear DNA, and are not necessarily in a hairpin configuration. The linear DNA is arranged such that it hybridizes to the complementary target. The hybridized linear DNA is detected using a post-hybridization fluorescent labeling method, examples of which are known in the art.
Some embodiments of the device include a tethering moiety based on the streptavidin-biotin interaction.
In some embodiments of the device, the probes are such that, when untethered in solution and bound to their complementary targets, they have a signal-to-background ratio that is not more than ten times as large as the signal-to-background ratio that they have in the device. In some embodiments of the device, the probes are such that, when untethered in solution and bound to their complementary targets, they have a signal-to-background ratio that is not more than five times as large as the signal-to-background ratio that they have in the device.
Embodiments of the present invention further include methods of making the aforesaid devices of the present invention, as enabled by the present disclosure, including the disclosures of the documents incorporated by reference herein. Embodiments of the present invention further include methods of capturing, amplifying, detecting, and/or identifying one or more target biological molecules using the aforesaid devices of the present invention, as enabled by the present disclosure, including the disclosures of the documents incorporated by reference herein.
It should be understood that the embodiments described herein are merely exemplary in nature and that a person skilled in the art may make many variations and modifications thereto without departing from the scope and spirit of the present invention. All such variations and modifications, including those discussed above, are intended to be included within the scope and spirit of the invention, as defined by the appended claims.
The present application claims the benefit of U.S. Provisional Patent Application No. 61/932,559, filed on Jan. 28, 2014, which is incorporated by reference herein in its entirety.
This project was partially supported by the U.S. Army Research Office via Grant No. W911NF-12-0331 and by the National Science Foundation via Grant No. IIP 1262903 and Grant No. CBET-1402706. The Government of the United States of America may have certain rights with regard to the disclosed subject matter.
| Number | Date | Country | |
|---|---|---|---|
| 61932559 | Jan 2014 | US |
