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Microfluidic Methods for Protein Microarrays
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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: urn:nbn:se:kth:diva-26083ISBN: 978-91-7415-761-1 (print)OAI: oai:DiVA.org:kth-26083DiVA: diva2:369849
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
List of papers
1. Increasing robustness and sensitivity of protein microarrays through microagitation and automation
Open this publication in new window or tab >>Increasing robustness and sensitivity of protein microarrays through microagitation and automation
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2006 (English)In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 564, no 1, 66-73 p.Article in journal (Refereed) Published
Abstract [en]

Assay systems that employ protein microarrays for the analysis of complex samples are powerful tools to generate a high amount of data from a limiting amount of sample. Due to miniaturization, these systems are susceptible to fluctuations during signal generation and the use of uniform conditions for sample incubation and during the assay procedure is required to get reproducible results. Diffusion limits may prevent constant conditions on all parts of the array and can lead to the decease of the sensitivity of the array. Therefore, we set-up an automated assay system integrating a novel microagitation device using surface acoustic wave (SAW) technology. Multiplexed assays for the detection of autoantibodies from human serum and sandwich immunoassay for the detection of matrix metalloproteases (MMPs) were used to evaluate the system. Diffusion-rate limited solid phase reactions were enhanced by microagitation using the SAW technology resulting in up to three-fold higher signals.

Keyword
automation, surface acoustic waves (SAW), microagitation, protein microarrays, diffusion limitation
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-25837 (URN)10.1016/j.aca.2006.01.041 (DOI)000236640100009 ()
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2017-12-12Bibliographically approved
2. Expanding assay dynamics: A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
Open this publication in new window or tab >>Expanding assay dynamics: A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
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2008 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 6, 956-963 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-17572 (URN)10.1373/clinchem.2007.099812 (DOI)000256325800005 ()2-s2.0-44849128262 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. Non-contact protein microarray fabrication using a procedure based on liquid bridge formation
Open this publication in new window or tab >>Non-contact protein microarray fabrication using a procedure based on liquid bridge formation
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2009 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 393, no 2, 591-598 p.Article in journal (Refereed) Published
Abstract [en]

Contemporary microarrayers of contact or noncontact format used in protein microarray fabrication still suffer from a number of problems, e. g. generation of satellite spots, inhomogeneous spots, misplaced or even absent spots, and sample carryover. In this paper, a new concept of non-contact sample deposition that reduces such problems is introduced. To show the potential and robustness of this pressure-assisted deposition technique, different sample solutions known to cause severe problems or to be even impossible to print with conventional microarrayers were accurately printed. The samples included 200 mg mL(-1) human serum albumin, highly concentrated sticky cell adhesion proteins, pure high-salt cell-lysis buffer, pure DMSO, and a suspension of 5-mu m polystyrene beads. Additionally, a water-immiscible liquid fluorocarbon, which was shown not to affect the functionality of the capture molecules, was employed as a lid to reduce evaporation during microarray printing. The fluorocarbon liquid lid was shown to circumvent hydrolysis of water-sensitive activated surfaces during long-term deposition procedures.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-18066 (URN)10.1007/s00216-008-2509-7 (DOI)000261936700022 ()2-s2.0-58049213792 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
4. Magnetic bead-based detection of autoimmune responses using protein microarrays.
Open this publication in new window or tab >>Magnetic bead-based detection of autoimmune responses using protein microarrays.
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2009 (English)In: New biotechnology, ISSN 1871-6784, Vol. 26, 269-276 p.Article in journal (Refereed) Published
Abstract [en]

In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.

Keyword
TECHNOLOGY; DIAGNOSTICS; DISEASES; ASSAY
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11220 (URN)10.1016/j.nbt.2009.07.011 (DOI)000273519000003 ()19664732 (PubMedID)2-s2.0-77952310841 (Scopus ID)
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-01-20Bibliographically approved
5. mu FBI: A Microfluidic Bead-Based Immunoassay for Multiplexed Detection of Proteins from a mu L Sample Volume
Open this publication in new window or tab >>mu FBI: A Microfluidic Bead-Based Immunoassay for Multiplexed Detection of Proteins from a mu L Sample Volume
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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.

Keyword
BREAST-CANCER, MICROARRAY ANALYSIS, IMMUNOAFFINITY CE, PHOSPHORYLATION, BIOPSIES, ARRAYS, CELLS, TECHNOLOGY, ACTIVATION, PREDICTION
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-25840 (URN)10.1371/journal.pone.0013125 (DOI)000282359300019 ()2-s2.0-78049283378 (Scopus ID)
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-12Bibliographically approved

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