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 mum depth and 25 pm 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-mug 1(-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 mug 1-1, and IC50 values of 0.096 and 0.130 mug 1(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCI 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 mug 1(-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved.

Subject headings

TEKNIK OCH TEKNOLOGIER  -- Medicinteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Medical Engineering (hsv//eng)

NATURVETENSKAP  -- Kemi -- Analytisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Analytical Chemistry (hsv//eng)

Keyword

immobilisation

hydrophilic polymers

protein G and A

microfluidics

immunosensor

chemiluminescence

Publication and Content Type

art (subject category)

ref (subject category)