title: Enhancing Protein Capture Using a Combination of Nanoyeast Single-Chain Fragment Affinity Reagents and Alternating Current Electrohydrodynamic Forces.
abstract: New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection of Entamoeba histolytica antigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD of n = 3) < 17.2%, and in buffer down to 5.88 fM with an interassay reproducibility (% RSD, n = 3) of 8.4%. Furthermore, antigen detection using this immunosensor was 10 times more sensitive than previously obtained with the same nanoyeast-scFv reagents in a microfluidic device employing surface-enhanced Raman scattering (SERS) detection in buffer and at least 200 times more sensitive than methods using screen printed gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices.
authors.affiliation: Centre for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Vaidyanathan
authors.affiliation: Centre for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Sakandar
authors.initials: S
authors.lastname: Rauf
authors.affiliation: Centre for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Yadveer S
authors.initials: YS
authors.lastname: Grewal
authors.affiliation: School of Public Health, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Spadafora
authors.affiliation: Centre for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Muhammad J A
authors.initials: MJ
authors.lastname: Shiddiky
authors.affiliation: School of Public Health, University of Washington , Seattle, Washington 98195, United States.
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Cangelosi
authors.affiliation: Centre for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Roads (Bldg 75), The University of Queensland , Brisbane, Queensland 4072, Australia.|School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
mesh_terms: D000953:Antigens, Protozoan / Q000032:analysis / Q000302:isolation & purification*; D015415:Biomarkers / Q000032:analysis; D055664:Electrochemical Techniques; D004748:Entamoeba histolytica / Q000737:chemistry; D057446:Hydrodynamics; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D000953:Antigens, Protozoan; D015415:Biomarkers; D057127:Single-Chain Antibodies