title: Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.
abstract: Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.
authors.affiliation: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN) and ‡School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Wang
authors.forename: Sakandar
authors.initials: S
authors.lastname: Rauf
authors.forename: Yadveer S
authors.initials: YS
authors.lastname: Grewal
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Spadafora
authors.forename: Muhammad J A
authors.initials: MJ
authors.lastname: Shiddiky
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Cangelosi
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schlücker
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
mesh_terms: D000942:Antigens, Bacterial / Q000302:isolation & purification*; D001710:Biotin / Q000737:chemistry; D004748:Entamoeba histolytica / Q000737:chemistry* / Q000472:pathogenicity; D007118:Immunoassay; D057230:Limit of Detection; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013059:Spectrum Analysis, Raman; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties
chemical_list: D000942:Antigens, Bacterial; D057127:Single-Chain Antibodies; D001710:Biotin; D019809:Streptavidin