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Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection

Yakovleva, J; Davidsson, Richard LU ; Bengtsson, Martin LU ; Laurell, Thomas LU and Emnéus, Jenny LU (2003) In Biosensors & Bioelectronics 19(1). p.21-34
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
immobilisation, hydrophilic polymers, protein G and A, microfluidics, immunosensor, chemiluminescence
in
Biosensors & Bioelectronics
volume
19
issue
1
pages
21 - 34
publisher
Elsevier
external identifiers
  • wos:000186242000003
  • pmid:14558995
  • scopus:1542404812
ISSN
1873-4235
DOI
10.1016/S0956-5663(03)00126-X
language
English
LU publication?
yes
id
72f9a2c4-6050-4435-a184-76eb61b397a8 (old id 297359)
date added to LUP
2007-09-24 13:09:32
date last changed
2017-05-28 04:25:34
@article{72f9a2c4-6050-4435-a184-76eb61b397a8,
  abstract     = {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.},
  author       = {Yakovleva, J and Davidsson, Richard and Bengtsson, Martin and Laurell, Thomas and Emnéus, Jenny},
  issn         = {1873-4235},
  keyword      = {immobilisation,hydrophilic polymers,protein G and A,microfluidics,immunosensor,chemiluminescence},
  language     = {eng},
  number       = {1},
  pages        = {21--34},
  publisher    = {Elsevier},
  series       = {Biosensors & Bioelectronics},
  title        = {Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection},
  url          = {http://dx.doi.org/10.1016/S0956-5663(03)00126-X},
  volume       = {19},
  year         = {2003},
}