Abstract: Microwell biochips (11) are formed from a thin flat plate (13) of polymeric material having a plurality of regularly spaced holes (15) that extend completely therethrough and create microwells. The lower end of each hole is closed by a microporous, hydrophobic, polymeric membrane (17) laminated to the undersurface of the plate which retains an aqueous test solution in the wells until a vacuum is applied to the undersurface thereof to effect draining of the solution and of any wash solution that might be subsequently added. A spot of polymerizing isocyanate-functional hydrogel is applied generally centrally to the porous membrane surface at the bottom of each well in a manner so as to cover only a minor portion of the surface and out of contact with the well sidewalls, thus leaving substantial surface area through which drainage can be readily effected. Biological capture agents are associated with the polymerizing hydrogel so as to become immobilized as a part thereof.

Abstract: A reflective substrate is used to amplify the photon signal captured from overlying analyte domains containing photon emitters. The reflective substrate provides substantial desired signal amplification of the photon emissions from each domain via interference effects induced in the incident excitation and/or emission energies. A dielectric is interposed between the domains and the reflective surface, which has a thickness such that substantial destructive interference occurs with respect to emission photons or excitation photons or both at the attachment surface. When analyte domains have a three-dimensional structure such that a significant fraction of their volume extends at least ¼ wavelength above the attachment surface provided by the dielectric, substantial constructive signal amplification can take place of signals generated within the analyte domains.

Abstract: A method for encapsulating biologics within a hydrogel by using an aqueous solution of an isocyanate-functional hydrogel prepolymer which is mixed with an amount of biologics and an aqueous solution containing a dithiol crosslinking agent under physiological pH conditions. An additional bidentate crosslinking agent may be included. The product of such method may be a bioreactor or an assay device having a plurality or different biologics encapsulated at predetermined locations in a substrate.

Abstract: A microflow apparatus for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts. The posts are spaced across the width of a collection region in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, have arcuate cross-sections, and are randomly arranged so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic coating, preferably a hydrogel containing isocyanate moieties or a PEG or polyglycine of substantial length, and are highly effective in capturing cells or other targeted biomolecules as a result of such streamlined flow disruption.

Abstract: A microflow apparatus for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts. The posts are spaced across the width of a collection region in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, have arcuate cross-sections, and are randomly arranged so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic coating, preferably a hydrogel containing isocyanate moieties or a PEG or polyglycine of substantial length, and are highly effective in capturing cells or other targeted biomolecules as a result of such streamlined flow disruption.

Abstract: Spheroidal beads present an exterior surface of a hydrophilic hydrogel, which is an isocyanate-functional polymer that is polymerized by urethane bonds and cross-linked by urethane and urea bonds. Sequestering agents present at the surface are covalently bound to isocyanate groups or to intermediate linkers that are so bound. These beads allow sequestering agents to retain their native three-dimensional configuration, and as a result of such surface characteristics and hydrophilicity, they achieve highly effective capture of very small subpopulations of rare cells from bodily fluids or the like and very effectively deter nonspecific binding of other biomaterials present in such bodily fluid. They may be all-hydrogel spheroids or hydrogel-coated substrates.

Abstract: Methods for making a microarray having minimal background binding of proteins by appropriately coating a substrate surface which is initially derivatized with organic functional groups. A protein-resistant polymeric coating is applied which has hydrophilic backbone polymers that are crosslinked to a substantial degree via polyfunctional isocyanate moieties. Three dimensional hydrogel microspots containing capture agents are affixed at distinct spatial locations across an array region of the surface to form a microarray. The microspots are affixed either to the substrate or to the coating. The polymeric coating preferably comprises isocyanate-capped PEG crosslinked with a polyfunctional isocyanate to form urethane polymers.

Abstract: A post-incubation treatment is employed to effectively remove targets, such as proteins/protein complexes, or other label-bearing moieties that may non-specifically bind to a microarray substrate during a binding assay. Following incubation, a one-step wash is carried out with a liquid containing digester, e.g., a digestive enzyme (protease) or lysosome, which is effective to remove non-specifically bound targets or at least labeled portions of such targets from the substrate. Proteases are bound to or coated onto large molecules or onto solid particles of such a size such that they are prohibited from entering the porous surfaces of 3-D hydrogel microspots and are unable to reach and digest labeled target-probe complexes that are disposed within such porous hydrogel microspots. Digested segments of such protein which contain labels (or of essentially the entire protein) are carried away in the wash liquid and thus are not present to create background noise during imaging.

Abstract: A method of making a microarray by coating a flat substrate with a polymerizable hydrogel layer which contains anchoring moieties dispersed uniformly therethroughout. Following curing, a continuous layer of uniform thickness is securely attached to the upper surface of the substrate through an array region thereof. A plurality of different probes are then attached to create microspots at distinct spatial locations on the surface of this slab layer by linking the probes to the anchoring moieties in the cured hydrogel. Such anchoring moieties may employ linking systems such as organic chelators, that are activated by copper or some other metal, and complementary pairs such as avidin-biotin.

Abstract: Methods for detecting in a single assay any one of multiple chromosomal disorders that result from aneuploidy or certain mutations, particularly microdeletions, and kits for use therein. A polymerase chain reaction (PCR) is carried out to amplify eukaryotic genomic DNA using a plurality of primer oligonucleotide pairs wherein one primer of each pair has a detectable label attached 5? thereto. A plurality of the primer pairs are targeted to DNA segments of different chromosomes of interest which are indicative of potential chromosomal disorders, and one pair is targeted for a control gene. The amplified PCR products are purified, and single-stranded DNA having the detectable labels is obtained therefrom and hybridized with spots on a microarray that each contain DNA oligonucleotide probes having nucleotide sequences complementary to a nucleotide sequence of one strand of each segment.

Abstract: Methods for detecting a short tandem repeat polymorphism (STRP), such as fragile X syndrome, wherein PCR is used to amplify nucleic acid along the chromosome in the genomic DNA which includes all of the STRs of interest plus a substantial contiguous segment of the nucleic acid adjacent to the STRs. Single-stranded product is then obtained, and colorimetric-labeled oligonucleotides which target for (i) STRs and (ii) the contiguous DNA segment are hybridized with this single-stranded product which is then bound to a solid phase and separated from the remainder of the target material. The labeled oligonucleotide target material is recovered by treatment with base and then hybridized to a microarray having a plurality of spots containing suitable oligonucleotide probes complementary thereto.

Abstract: A biochip is formed with a plurality of optically clear hydrogel microspots attached to the top surface of a solid substrate in the form of an array. Each of the microspots is formed of a hydrogel of polyethylene glycol, polypropylene glycol or a copolymer thereof having reactive isocyanate groups. Binding entities or probes are immobilized in these microspots, which entities are effective to selectively hybridize to or sequester target biomolecules, such as target cells. Different binding entities are immobilized in microspots in different regions of an array to create a biochip that can be used to assay for or separate a number of target biomolecules, such as cells from maternal blood.

Abstract: Quantification in a straightforward manner in an mRNA expression analysis is accomplished by using biotinylated oligo d(T) and coupling to a solid support (e.g., streptavidin-derivatized magnetic beads). Labeled targets specific to certain mRNAs of interest that are to be identified and quantified are added to a biological sample containing the mRNAs and biotinylated oligo d(T). The beads are preferably added after hybridization, but may be earlier added. Following attachment to the beads, unbound and non-specifically bound targets and non-mRNA material are removed by successive stringent washings. All the mRNA material is then eluted from the beads by subjection to conditions that separate the poly A and the oligo d(T), and the original mRNAs are then degraded by basic hydrolysis to leave single-strand synthetic targets, which are then hybridized to their specific probes suitably carried on a microarray.

Abstract: Novel and efficient syntheses create novel piperazinone intermediates which facilitate the production and use of PNAs. Such syntheses and the products enhance the feasibility of a system which permits the rapid identification of PNA oligomers useful as therapeutics, diagnostics and/or gene characterization tools. A first component of the system is a universal PNA library that most preferably incorporates one or more universal nucleotide bases into carefully selected positions within each oligomer species thereby providing the library with the screening ability of a much larger library. The second component of the system is a high throughput screening system that includes a number of assays designed to provide information on the binding activities of the different PNAs to a target nucleotide sequence (generally, a DNA or RNA sequence).

Abstract: Methods for preparing a biochip are provided herein wherein the biomolecular probe to be used with the biochip is alternatively bound to a hydrogel prepolymer prior to or simultaneously with polymerization of the prepolymer. In particularly preferred embodiments, a polyurethane-based hydrogel prepolymer is derivatized with an organic solvent soluble biomolecule, such as a peptide nucleic acid probe in aprotic, organic solvent. Following derivatization of the prepolymer, an aqueous solution, for example sodium bicarbonate, preferably buffered to a pH of about 7.2 to about 9.5, is added to the derivatized prepolymer solution to initiate polymerization of the hydrogel. Alternatively, a water soluble biomolecule, such as DNA or other oligonucleotide, is prepared in an aqueous solution and added to the polyurethane-based hydrogel prepolymer such that derivatization and polymerization occur, essentially, simultaneously.