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mu FBI: A Microfluidic Bead-Based Immunoassay for Multiplexed Detection of Proteins from a mu L Sample Volume
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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2010 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 10, e13125- p.Article in journal (Refereed) Published
Abstract [en]

Background: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material. Methodology/Principal Findings: A microfluidic bead-based immunoassay, mu FBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 mu L assay volume. In addition, only 1 mu L of detection antibodies and 1 mu L of the reporter molecule Streptavidin-Phycoerythrin were required. The mu FBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. mu FBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30. Conclusions/Significance: The mu FBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections.

Place, publisher, year, edition, pages
2010. Vol. 5, no 10, e13125- p.
Keyword [en]
BREAST-CANCER, MICROARRAY ANALYSIS, IMMUNOAFFINITY CE, PHOSPHORYLATION, BIOPSIES, ARRAYS, CELLS, TECHNOLOGY, ACTIVATION, PREDICTION
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-25840DOI: 10.1371/journal.pone.0013125ISI: 000282359300019Scopus ID: 2-s2.0-78049283378OAI: oai:DiVA.org:kth-25840DiVA: diva2:360090
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-12Bibliographically approved
In thesis
1. Microfluidic Methods for Protein Microarrays
Open this publication in new window or tab >>Microfluidic Methods for Protein Microarrays
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein microarray technology has an enormous potential for in vitro diagnostics (IVD)1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market. However, in order for protein microarrays to become broadly accepted tools in IVD, a number of criteria have to be fulfilled concerning robustness and automation. Robustness and automation are key demands to improve assay performance and reliability of multiplexed assays, and to minimize the time of analysis.

These key demands are addressed in this thesis and novel methods and techniques concerning assay automation, array fabrication as well as performance and detection strategies related to protein microarrays are presented and discussed. In the first paper an automated assay format, based on planar protein microarrays is described and evaluated by the detection of several auto-antibodies from human serum and by quantification of matrix metalloproteases present in plasma. Diffusion-rate limited solid phase reactions were enhanced by microagitation, using the surface acoustic wave technology, resulting in a slightly increased signal-to-noise ratio. In the second paper of the thesis, a novel multiplexed immunoassay system was developed by combining a direct immunoassay with a competitive system. This set-up allows quantification of analytes present in widely varying concentrations within a single multiplex assay. In the third paper, a new concept for sample deposition is introduced, addressing contemporary problems of contact or non-contact microarrayers in protein microarray fabrication.

In the fourth paper, a magnetic bead-based detection method for protein microarrays is described as a cost-effective alternative approach to the commonly used fluorescence-based confocal scanning systems. The magnetic bead-based detection could easily be performed by using an ordinary flatbed scanner. In addition, applying magnetic force to the magnetic bead-based detection approach enables to run the detection step more rapidly. Finally, in paper five, a microfluidic bead-based immunoassay for multiplexed detection of receptor tyrosine kinases in breast cancer tissue is presented. Since the assay is performed inside a capillary, the amounts of sample and reagent material could be reduced by a factor of 30 or more when compared with the current standard protein microarray assay.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. ix, 51 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2010:45
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-26083 (URN)978-91-7415-761-1 (ISBN)
Public defence
2010-11-19, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101112Available from: 2010-11-12 Created: 2010-11-12 Last updated: 2010-11-15Bibliographically approved

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