# US 8,148,170 B2 · Immunosensors: scFv-linker design for surface immobilization > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,148,170 B2 - Document ID: US8148170B2 - Status: Granted - Kind: B2 - Application number: 12/931,801 - Filed: 2011-02-10 - Published: 2011-08-18 - Granted: 2012-04-03 - Earliest claimed: 2003-06-05 - Assignees: Oakland University; Vanderbilt University - Inventors: Xiangqun Zeng; Raymond L. Mernaugh - Web page: https://patentgrep.com/patent/US8148170B2 ## CPC - C07K 16/4283: {against an allotypic or isotypic determinant on Ig} - B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors - B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites - B82Y 40/00: Manufacture or treatment of nanostructures - G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings} - C07K 2317/622: Single chain antibody (scFv) - Y10S 435/81: Packaged device or kit - Y10S 436/805: Optical property - Y10S 436/808: Automated or kit - Y10S 436/809: Multifield plates or multicontainer arrays - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract An apparatus and methods for binding an analyte of interest in a sample are provided. The apparatus comprises a substrate with an exposed surface with an compound, that is electrostatically charged or capable of forming hydrogen bonds, provided bound to the solid substrate. A recombinant single chain antibody (scFv) molecule specific for the analyte of interest, having one or more amino acids with charged or hydrogen-bond forming sidechains in a linker polypeptide portion, is bound to the layer on the solid substrate. When the analyte of interest is present in the sample the scFv binds the analyte to the solid substrate. The apparatus can be used with an immunoglobulin layer to detect Fc receptors, so as to detect microorganisms such as Staphylococcus aureus having protein A or protein G. ## Claims ### Claim 1 An apparatus comprising: (a) a solid substrate with an exposed surface; (b) a compound provided as a layer, bound to the solid substrate; and (c) a plurality of recombinant single chain antibodies (scFv's) specific for a target molecule and bound to the compound on the solid substrate, each scFv comprising: (i) an antibody variable light chain (VL) polypeptide specific for the target molecule, (ii) an antibody variable heavy chain (VH) polypeptide specific for the target molecule, and (iii) a linker polypeptide covalently linking the antibody variable light chain (VL) polypeptide to the antibody variable heavy chain (VH) polypeptide, the linker polypeptide having an amino acid sequence comprising one or more amino acids with sidechains that bind to the compound, wherein the scFv is capable of binding the target molecule to the solid substrate, when provided to the apparatus. ### Claim 2 (depends on 1) The apparatus of claim 1, wherein the recombinant single chain antibodies (scFv's) are specific for immunoglobulins as the target molecules, and the apparatus further comprises a plurality of the immunoglobulins bound to the scFv molecules so that Fc regions of the immunoglobulins are exposed as a binding layer for Fc receptors. ### Claim 3 (depends on 1) The apparatus of claim 1, wherein the target molecules are analytes of interest in a sample and the apparatus detects whether the analyte of interest is present in the sample. ### Claim 4 (depends on 1) The apparatus of claim 1, wherein the compound and the amino acid sidechains form electrostatic interactions. ### Claim 5 (depends on 1) The apparatus of claim 1, wherein the compound and the amino acid sidechains form hydrogen bonds. ### Claim 6 (depends on 1) The apparatus of claim 1, wherein the amino acids with sidechains are arginine or tyrosine. ### Claim 7 (depends on 1) The apparatus of claim 1, wherein the compound is electrostatically charged. ### Claim 8 (depends on 1) The apparatus of claim 1, wherein the compound is an anionic polyelectrolyte or 2-mercaptoethanol. ### Claim 9 (depends on 8) The apparatus of claim 8, wherein the compound is the anionic polyelectrolyte and the anionic polyelectrolyte is poly(sodium 4-styrenesulfonate) (PSS) or 11-mercaptoundecanoic acid (MUA). ### Claim 10 (depends on 1) The apparatus of claim 1, wherein the substrate is gold. ### Claim 11 (depends on 1) The apparatus of claim 1, wherein the apparatus is provided as a binding component of an immunosensor. ### Claim 12 (depends on 11) The apparatus of claim 11, wherein the immunosensor is a quartz crystal microbalance (QCM) device, a surface plasmon resonance (SPR) device, or an electrochemical impedance analyzer device. ### Claim 13 (depends on 1) The apparatus of claim 1, wherein the apparatus is provided as a microtiter plate for an ELISA assay or as an affinity matrix for immunopurification. ### Claim 14 (depends on 1) The apparatus of claim 1, wherein the amino acids with sidechains are separated by one or more spacer amino acids. ### Claim 15 (depends on 14) The apparatus of claim 14, wherein the spacer amino acids are selected from the group consisting of glycine and serine. ### Claim 16 (depends on 1) The apparatus of claim 1, wherein the amino acid sequence of the linker polypeptide comprises a series of two or more arginine-glycine (RG) repeats. ### Claim 17 (depends on 1) The apparatus of claim 1, wherein the amino acid sequence of the linker polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:12. ### Claim 18 (depends on 1) The apparatus of claim 1, wherein the compound and the amino acid sidechains form hydrophobic interactions. ### Claim 19 (depends on 1) The apparatus of claim 1, wherein the compound comprises negatively charged amino acids. ### Claim 20 (depends on 1) A method of determining whether an analyte with an Fc receptor is present in a sample, the method comprising: (a) providing the sample; (b) providing an immunosensor device having a component are apparatus according to claim 1, wherein: (i) the scFv is specific for an immunoglobulin as the target molecule, (ii) the VL polypeptide is specific for the immunoglobulin, (iii) the VH polypeptide is specific for the immunoglobulin, (iv) the scFv is capable of binding the immunoglobulin to the solid substrate of the apparatus, and (v) the apparatus further comprises a plurality of the immunoglobulins bound to the scFv molecules so that Fc regions of the immunoglobulins are exposed as a binding layer for the Fc receptor; (c) applying the sample to the apparatus for a time sufficient for the scFv on the substrate to bind to the Fc receptor if present in the sample; (d) detecting the Fc receptors in the sample bound to the scFv on the solid substrate with the immunosensor device; and (e) determining whether the analyte is present in the sample by the result in step (d). ### Claim 21 (depends on 20) The method of claim 20, wherein the immunosensor device is a quartz crystal microbalance (QCM) device or a surface plasmon resonance (SPR) device. ### Claim 22 (depends on 20) The method of claim 20, wherein the Fc receptor is Protein A or Protein G. ### Claim 23 (depends on 20) The method of claim 20, wherein the analyte with the Fc receptor is Staphylococcus aureus. ### Claim 24 (depends on 20) The method of claim 20, wherein the immunoglobulin is IgG.