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Researchers Submit Patent Application, "Detection and Assay Devices and Methods of Making and Using the Same", for Approval

September 4, 2014



By a News Reporter-Staff News Editor at Politics & Government Week -- From Washington, D.C., VerticalNews journalists report that a patent application by the inventors CHILKOTI, Ashutosh (Durham, NC); HUCKNALL, Angus (Durham, NC), filed on April 1, 2014, was made available online on August 21, 2014.

The patent's assignee is Sentilus, Inc.

News editors obtained the following quote from the background information supplied by the inventors: "Microarrays are a powerful and established tool in genomics..sup.1 In contrast, the development of antibody (Ab) microarrays into an equivalent tool for proteomics has been limited by: (1) the availability of high affinity and specificity antibodies for capture and detection of protein biomarkers; (2) the susceptibility of proteins to denaturation; and (3) the propensity of Ab's and protein biomarkers to avidly adsorb to surfaces (commonly referred to as the 'non-specific adsorption' problem), which can severely limit the ultimate sensitivity of protein microarrays, especially from complex protein mixtures such as plasma and serum..sup.2 One of the primary factors (others include the intrinsic affinity of the capture antibody and the diffusion of target to the microspot.sup.2-3) that controls the limit-of-detection (LOD) of protein microarrays is the adventitious adsorption of proteins (protein biomarkers and antibodies used for detection)."

As a supplement to the background information on this patent application, VerticalNews correspondents also obtained the inventors' summary information for this patent application: "A first aspect of the present invention is an article (preferably a biomolecular detector or biosensor such as a microarray) having a nonfouling surface thereon, the article comprising:

"(a) a substrate having a surface portion;

"(b) a linking layer on the surface portion; and

" a polymer layer formed on the linking layer (e.g., by the process of surface-initiated polymerization (SIP) of monomeric units thereon). Preferably, each of the monomeric units comprises a monomer (for example, a vinyl monomer) core group having at least one protein-resistant head group coupled thereto, to thereby form a brush molecule on the surface portion. The brush molecule preferably comprises a stem formed from the polymerization of the monomer core groups, and a plurality of branches formed from the head group projecting from the stem; and

"(d) a first member of a specific binding pair (e.g., a protein, peptide, antibody, nucleic acid, etc.) non-covalently bound to the polymer layer.

"A second aspect of the present invention is a method of making an article (preferably a biomolecular detector such as a microarray) having a nonfouling surface thereon, the method comprising: (a) providing a substrate having a surface portion; (b) depositing a linking layer on the surface portion; and forming a polymer layer on the linking layer by the process of surface-initiated polymerization of monomeric units thereon, with each of the monomeric units comprising a monomer (for example, a vinyl monomer) core group having at least one protein-resistant head group coupled thereto, to thereby form a brush molecule on the surface portion; the brush molecule comprising a stem formed from the polymerization of the monomer core groups, and a plurality of branches formed from the hydrophilic head group projecting from the stem; and then (d) non-covalently binding a member of a specific binding pair to the polymer layer.

"In some embodiments the polymer comprises a homopolymer of hydroxy-terminated OEGMA. In another embodiment the polymer comprises of a copolymer of methoxy-terminated OEGMA and hydroxy-terminated OEGMA. In other embodiments the polymer comprises of vinyl monomer bearing other head groups such as hydroxyl (OH), glycerol, or groups known in the art as kosmotropes (see, e.g., Kane et al., infra).

"In some embodiments of the invention, the surface portion comprises a material selected from the group consisting of metals, metal oxides, semiconductors, polymers, silicon, silicon oxide, and composites thereof.

"In some embodiments of the invention the linking layer is continuous; in some embodiments of the invention the linking layer is patterned. In some embodiments of the invention the linking layer is a self-assembled monolayer (SAM). In some embodiments of the invention the linking layer comprises an initiator-terminated silane or an initiator-terminated alkanethiol. In other embodiments the linking layer comprises of the deposition of two layers in separate steps. In the first step, an alkylsilane or alkanethiol is deposited on a surface such as silicon dioxide or glass or gold, and presents a terminal reactive functional group (e.g., amine). In the next step, a bifunctional molecule, which comprises a first functional group reactive towards the terminal group presented by the first linking layer is reacted with the first linking layer deposited in the first step. The second linker molecule contains a second moiety group that acts as an ATRP or free radical initiator.

"In some embodiments of the invention the surface-initiated polymerization is carried out by atom transfer radical polymerization (ATRP); in some embodiments of the invention the surface-initiated polymerization is carried out by free radical polymerization.

"In some embodiments, the article further comprises a protein, peptide, oligonucleotide or peptide nucleic acid non-covalently bound to the polymer layer. In some embodiments the protein, peptide, oligonucleotide or peptide nucleic acid coupled to the polymer layer or to the surface consists of or consist essentially of a single preselected molecule (this is, one such molecule is coupled to the surface portion via the brush molecule, to the exclusion of other different molecules). The preselected molecule may be a member of a specific binding pair, such as a receptor.

"A further aspect of the invention is a method of detecting a second member of a specific binding pair in a sample, comprising the steps of: (a) providing a detector as described herein; (b) contacting a sample (e.g., an aqueous sample or biological fluid) suspected of containing the second member(s) to the detector; and then determining the presence or absence of binding of the second member to the first member, the presence of binding indicating the presence of the second member in the sample. The determining step can be carried out by any suitable technique, such as by sandwich assay, as discussed further below.

"Arrays.

"In some embodiments of the foregoing methods and devices, useful for the detection of multiple different analytes, the first member of said specific binding pair is non-covalently bound to said polymer at a discrete probe location, and the biomolecular detector further comprises: (e) a plurality of additional first members of a specific binding pairs non-covalently bound to said polymer layer at a plurality of additional discrete probe locations to thereby form an array thereon. In some embodiments the array has a density of 5 to 10,000 discrete probe locations per cm.sup.2 thereon; in some embodiments the array has a density of 10,000 to 1 million discrete probe locations per cm.sup.2 thereon; and in some embodiments the array has a density of 1 million to 1 billion discrete probe locations per cm.sup.2 thereon.

"An advantage of the foregoing methods is the variety of techniques by which the detecting step can be carried out. For example, the detecting step may be carried out by: (a) ellipsometry; (b) surface plasmon resonance (SPR); localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; (d) surface acoustic wave (SAW) devices; (e) quartz-crystal microbalance with dissipation (QCM-D) (e) atomic force microscopy, (f) fluorescence spectroscopy or imaging; (g) autoradiography, (h) chemiluminescent imaging; and (i) optical detection of metal nanoparticles either by extinction or scattering. etc.

"Still other aspects of the present invention are explained in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1. A schematic diagram of an array of the present invention.

"FIG. 2. Synthesis of POEGMA brushes on glass via SI-ATRP. Cleaned slides were functionalized with APTES in step 1, and modified to present an ATRP initiator in step 2. Slides were then immersed in a polymerization solution in step 3 to synthesize surface tethered brushes of POEGMA.

"FIG. 3. (A) Example of signal and background intensities in an array used for generation of IL-6 dose response curves (B) Dose response curves of OPG in buffer and serum on POEGMA. (C) Dose response curves of IL-6 in serum on POEGMA and nitrocellulose. In B and C, the Y-axis shows the average background subtracted fluorescence intensity in printed spots and the X-axis shows analyte concentration in solution. Error bars represent one standard deviation.

"FIG. 4. Cy-5 labeled goat anti-rabbit IgG (Jackson) printed on POEGMA substrates prepared for both covalent and non-covalent attached to produce arrays.

"FIG. 5. Incubation of the arrays of FIG. 4 with Cy5 labeled goat anti-rabbit IgG yielded similar spot intensities for both immobilization methods, however, background levels on the activated slides (covalently coupled arrays) increased dramatically."

For additional information on this patent application, see: CHILKOTI, Ashutosh; HUCKNALL, Angus. Detection and Assay Devices and Methods of Making and Using the Same. Filed April 1, 2014 and posted August 21, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1865&p=38&f=G&l=50&d=PG01&S1=20140814.PD.&OS=PD/20140814&RS=PD/20140814

Keywords for this news article include: Antibodies, Immunology, Nanoparticle, Sentilus Inc, Blood Proteins, Nanotechnology, Immunoglobulins, Emerging Technologies.

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Source: Politics & Government Week


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