▎ 摘　　要

NOVELTY - Biomarker detection in flowable sample volume involves: (a) fabricating a sensing area of: (i) a periodic array of nanostructures comprising a polymeric nanopost; a metal layer conformed to the shape of and over portion of each nanopost; binding layer that facilitates molecular binding conformed to the shape of and over portion of the metal layer at each nanopost; and receptor molecules over portion of the binding layer at each nanopost specifically functionalized for binding to a biomarker molecule of interest; (b) presenting the flowable sample volume to periodic array of nanostructures at the sensing area to provide a larger surface area, increased loading capacity, and radial or spherical diffusion paths for the flowable sample volume as compared to a planar surface; (c) (i) taking electrochemical measurements; and (ii) taking surface plasmon resonance (SPR) measurements; and (d) evaluating the electrochemical and SPR measurements relative to the biomarker molecule of interest. USE - The methods are useful for biomarker detection in flowable sample volume; and for fabricating biomarker detector. The system is useful for detecting a target biomarker, e.g. ErbB, from sample (all claimed). ADVANTAGE - The system has a small footprint, low sample consumption, and improved detection reliability. The integrated dual-modality sensor offers higher sensitivity (through higher surface area and diffusions from nanoposts for electrochemical measurements), as well as the dynamic measurements of antigen-antibody bindings (through the SPR measurement), while operating simultaneously in a same sensing area using a same sample volume. The methods, systems, and apparatus provide for relatively inexpensive fabrication of high uniformity three-dimensional (3D) nanostructures for presentation of increased surface area and radial diffusion of analyte across the bio-functionalized sensing area; provide for improved reproducibility performance of such sensing surfaces, including in mass production; allow for simultaneous acquisition of dual modality measurements from a smaller footprint, shared sensing area with lower sample consumption; (d) allow for improved detection reliability; (e) can leverage high sensitivity of detection with dynamic tracking of antigen-antibody interactions, enzymatic reactions, or aptamer-based reactions (e.g. aptamer-cleavage reactions) at the sensing surfaces. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for: (1) a system for detecting a target biomarker from a sample, having a small footprint, low sample consumption, and improved detection reliability, comprising: (a) a sensing chip with a sensing area comprising: (i) a periodic array of nanostructures comprising nanoposts covered in metal and a binding layer biofunctionalized with anti-target molecules that bind with target biomarker molecules related to the target biomarker; (b) a microfluidic circuit to provide a volume of sample to the periodic array of nanostructures at the sensing area of the sensing chip; (c) an electrochemical sensing modality comprising an electrode set and a source of electrical power adapted to obtain electrochemical measurements at the periodic array of nanostructures at the sensing area, the array of nanostructures presenting a larger surface area, increased loading capacity, and radial or spherical diffusion paths to a sample volume as compared to a planar surface; (d) SPR sensing modality comprising an illumination source and a spectrometer adapted to obtain SPR measurements at the periodic array of nanostructures at the sensing area, where the SPR sensor comprises: (i) a light source and optics to couple light from the light source light to illuminate the sensing area, and (ii) collection optics and a spectrometer to collect and measure reflectance from the sensing area; and (e) a control circuitry in operative connection to and adapted to: (i) control the microfluidic subsystem to present a sample volume to the periodic array of nanostructures at the sensing area; (ii) simultaneously collect signals from: (1) operation of the source of electrical power and the electrode set in the electrochemical sensing modality; and (2) operation of light source and spectrometer in the SPR sensing modality; (iii) process the collected signals into one or more of (a) an estimate of presence and/or concentration of the biomarker of interest in a sample volume, and (b) another parameter relating to the biomarker of interest; (2) a biomarker detector comprising: (a) a microfluidic chip comprising a sensing area and a microfluidic network to supply a sample volume to the sensing area; (b) the sensing area comprising: (i) a periodic array of nanostructures, each nanostructure comprising: (1) a metal layer over portion of each nanostructure; (2) a binding layer over portion of the metal layer at each nanostructure; and (3) receptor molecules over portion of the binding layer at each nanostructure specifically functionalized for binding to a biomarker of interest; (c) where both electrochemical measurements and SPR measurements can be taken and evaluated for presence of the biomarker of interest at the same sensing area and using the same sample volume; (3) fabricating the biomarker detector which involves: (a) creating a nano stamp of nanoposts; (b) pouring a polymeric solution onto the nano-stamp, curing the polymeric solution on the nano-stamp, and peeling the cured polymeric solution from the nano-stamp to create a nanohole mold; (c) pouring a UV curable polymeric solution onto the mold, and exposing the polymeric solution to UV to produce an array of polymer nanoposts; (d) depositing a metal layer onto the nanoposts; (e) drop coating the metalized nanoposts with nanosheets of graphene oxide (GO); (f) biofunctionalizing the metalized and GO coated nanoposts with anti-molecules to a biomarker of interest to create the nanostructures; and (g) creating microfluidic channels in a photo pattemable polymer substrate to create the microfluidic network; and (4) a biosensor comprising: (a) a microfluidic channel to deliver an analyte sample; and (b) a sensor chip operatively connected to the microfluidic channel, the sensor chip comprising a patterned periodic array of nanostructures comprising nanoposts coated with an electrical conductor and a graphene-based material, and functionalized with specific receptor molecules, the periodic array of nanostructures configured to detect biomarker molecules in a limited volume of an analyte sample with accuracy and precision via electrochemical and SPR signals from a single sensing area at the patterned periodic array of nanostructures by presenting: (i) a spatially well-defined nanostructured working electrode for electrochemical sensing; and (ii) a nanostructured plasmonic crystal for SPR sensing via excitation of surface plasmon polaritons.