Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection.
Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.
Yakovleva
Julia
J
Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119899, Russia.
Davidsson
Richard
R
Bengtsson
Martin
M
Laurell
Thomas
T
Emnéus
Jenny
J
D000327:Adsorption; D001280:Atrazine / Q000032:analysis* / Q000737:chemistry; D001628:Beverages / Q000032:analysis; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation* / Q000379:methods; D005504:Food Analysis / Q000295:instrumentation / Q000379:methods; D005506:Food Contamination / Q000032:analysis; D005638:Fruit / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000032:analysis / Q000737:chemistry*; D007124:Immunoenzyme Techniques / Q000295:instrumentation* / Q000379:methods; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis / Q000737:chemistry*; D014874:Water Pollutants, Chemical / Q000032:analysis
C029152:G-substrate; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A; D014874:Water Pollutants, Chemical; D006735:Horseradish Peroxidase; D001280:Atrazine