In Situ Microarray Fabrication and Analysis Using a Microfluidic Flow Cell Array Integrated with Surface Plasmon Resonance Microscopy

Abstract

Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.

Poly[oligo(ethylene glycol) methacrylate‐co‐glycidyl methacrylate] Brush Substrate for Sensitive Surface Plasmon Resonance Imaging Protein Arrays

“…Covalent attachment of proteins on thiol‐modified gold surface is the conventional method of SPRi detection 5. In comparison to the brush‐based SPRi method, 11‐mercaptoundecanoic acid (MUA) as the supporting matrix was employed to detect CEA, but no differentiable signal from the negative control was detected with 200 ng mL −1 CEA in human serum, of which the Δ R for human serum without target proteins was ca.…”

“…Multiplexed detection of disease biomarkers can provide the advantages of small sample consumption, reduced assay time, low manufacturing cost, and high‐throughput, along with improved diagnostic accuracy due to multiple biomarkers often being associated with a single disease 1–3. Surface plasmon resonance imaging (SPRi) is such a powerful analytical tool, operating by measuring changes of the local refractive index on various addressable spots of an array for high‐throughput, label‐free, and real‐time detections 4–10. However, to date SPRi has not had sufficient sensitivity and specificity for practical diagnostic applications, particularly in human biological samples.…”

Surface chemistry and morphology in single particle optical imaging

“…However, sensitivity is a limiting issue in SPRi compared to the conventional SPR due to the difficulties in sample delivery in real time over the multiplexed spotted sensor surface. The use of microfluidic flow cells and the amplification of the signal with various techniques improve signal-to-noise ratio in many applications [73][74][75][76][77]. The effective thickness of the captured target layer is the layer of analyte on top of the surface probes on the SPR sensor, which has the major effect on the magnitude of SPR response.…”

New trends in instrumental design for surface plasmon resonance-based biosensors

“…Additionally, SPR imaging allows for real-time detection of the reaction occurring inside the microchannel. A number of groups used the microfluidic platform to decrease the nonspecific binding and instrument drift by internal referencing for individual flow cells in a parallel processing microfluidic network (Amarie et al 2010; Eddings et al 2009; Liu et al 2009). One group recently reported the fabrication of a 1 μ m diameter SPR microcavity with a detection limit 10 6 times smaller than classical SPR biosensors (Amarie et al 2010).…”