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Protein Microarrays on Carboxymethylated Dextran Hydrogels: Immobilization, Characterization and Application
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
Biacore AB, Rapsgatan 7, S-754 50, Uppsala, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
2004 (English)In: Microchimica Acta, ISSN 0026-3672, E-ISSN 1436-5073, Vol. 147, no 1-2, 21-30 p.Article in journal (Refereed) Published
Abstract [en]

Tetraoctadecylammonium bromide (TOAB, (CH3(CH2)17)4N+Br) has been used to print temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels to create a generic platform for protein microarray applications. The primary reason for printing temporary hydrophobic barriers is to prevent cross-contamination and overflow during microdrop dispensing. Equally important is to eliminate the risk for non-specific binding to the barriers during analyte exposure. This has been accomplished by introducing a regeneration step that removes the barriers after ligand immobilization. The overall fabrication process was characterized by microscopic wetting, atomic force microscopy, imaging ellipsometry, fluorescence microscopy, surface plasmon microscopy and biospecific interaction analysis. A series of model proteins including transferrin, Protein A, anti-myoglobin and bovine serum albumin was spotted into the TOAB-defined areas under different experimental conditions, e.g. at increased humidity and reduced substrate temperature or with glycerol as an additive in the protein solution. Much emphasis was devoted to studies aiming at exploring the homogeneity and activity of the immobilized proteins. The printed barriers were removed after protein immobilization using tert-n-butyl alcohol (TBA). TBA was found to be a very efficient agent as compared to previously used salt regeneration solutions, and the regeneration time could be reduced from 30 to 10 minutes. Finally, the potential of using the well established CMD hydrogel chemistry as a platform for protein microarrays was exploited using surface plasmon microscopy.

Place, publisher, year, edition, pages
2004. Vol. 147, no 1-2, 21-30 p.
Keyword [en]
Reversible hydrophobic barrier, microcontact printing, piezodispensing, protein microarrays, surface plasmon microscopy
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-14919DOI: 10.1007/s00604-004-0223-5OAI: oai:DiVA.org:liu-14919DiVA: diva2:25571
Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2017-12-13
In thesis
1. Imaging surface plasmon resonance
Open this publication in new window or tab >>Imaging surface plasmon resonance
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The central theme of this thesis is the use of imaging Surface Plasmon Resonance (iSPR) as a tool in the characterization of surfaces with laterally varying properties. Within the scope of this work, an instrument for iSPR analysis was designed and built. SPR is a very sensitive technique for monitoring changes in optical properties in the immediate vicinity of a sensor surface, which is very useful in biosensing and surface science research. We have employed SPR in the Kretschmann configuration, wherein surface plasmons are excited by means of an evanescent field arising from total internal reflection from the backside of the sensor surface. In iSPR, the signal is the reflectivity of TM-polarized light which is measured using an imaging detector, typically a CCD camera. Advantages of this technique include extreme surface sensitivity and, because detection is done from the backside, compatibility with complex samples. In addition, SPR is a non-labeling technique, and in imaging mode, a lateral resolution in the µm range can be attained.

The imaging SPR instrument could be operated in either wavelength interrogation mode or in intensity mode. In the former case, the objective is to find the SPR wave-length, λSPR, which is the wavelength at which the reflected intensity is at a minimum. In intensity mode, a snapshot of the intensity reflectance is taken at a fixed wavelength hand incidence angle.

In biosensor science, the use of an imaging technique offers a major advantage by enabling parallelization and thereby increasing throughput. We have, for example, used iSPR in biochemical interaction analysis to monitor immobilization and specific binding to protein and synthetic polypeptide micro arrays. The primary interest has been the study of soft matter surfaces that possess properties interesting in the field of biomimetics or for applications in biosensing. Specifically, the surfaces studied in this thesis include patterned self-assembled monolayers of thiolates on gold, a graft polymerized poly(ethylene glycol) (PEG) based hydrogel, a dextran hydrogel, and a polyelectrolyte charge gradient. Our results show that the PEG-based hydrogel is very well suited for use as a platform in protein immobilization in an array format, owing to the very low unspecific binding. In addition, well defined microarray templates were designed by patterning of hydrophobic barriers on dextran and monolayer surfaces. A polypeptide affinity microarray was further designed and immobilized on such a patterned monolayer substrate, in order to demonstrate the potential of analyte quantification with high sensitivity over a large dynamic range.

Furthermore, iSPR was combined with electrochemistry to enable laterally resolved studies of electrochemical surface reactions. Using this combination, the electrochemical properties of surfaces patterned with self assembled monolayers can be studied in parallel, with a spatial resolution in the µm regime. We have also employed electrochemistry and iSPR for the investigation of potential and current density gradients on bipolar electrodes.

The imaging SPR instrument could be operated in either wavelength interrogation mode or in intensity mode. In the former case, the objective is to find the SPR wave-length, λSPR, which is the wavelength at which the reflected intensity is at a minimum. In intensity mode, a snapshot of the intensity reflectance is taken at a fixed wavelength hand incidence angle.In biosensor science, the use of an imaging technique offers a major advantage by enabling parallelization and thereby increasing throughput. We have, for example, used iSPR in biochemical interaction analysis to monitor immobilization and specific binding to protein and synthetic polypeptide micro arrays. The primary interest has been the study of soft matter surfaces that possess properties interesting in the field of biomimetics or for applications in biosensing. Specifically, the surfaces studied in this thesis include patterned self-assembled monolayers of thiolates on gold, a graft polymerized poly(ethylene glycol) (PEG) based hydrogel, a dextran hydrogel, and a polyelectrolyte charge gradient. Our results show that the PEG-based hydrogel is very well suited for use as a platform in protein immobilization in an array format, owing to the very low unspecific binding. In addition, well defined microarray templates were designed by patterning of hydrophobic barriers on dextran and monolayer surfaces. A polypeptide affinity microarray was further designed and immobilized on such a patterned monolayer substrate, in order to demonstrate the potential of analyte quantification with high sensitivity over a large dynamic range.Furthermore, iSPR was combined with electrochemistry to enable laterally resolved studies of electrochemical surface reactions. Using this combination, the electrochemical properties of surfaces patterned with self assembled monolayers can be studied in parallel, with a spatial resolution in the µm regime. We have also employed electrochemistry and iSPR for the investigation of potential and current density gradients on bipolar electrodes.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 68 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1205
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-14923 (URN)978-91-7393-820-4 (ISBN)
Public defence
2008-09-26, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2017-01-11Bibliographically approved

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Zhou, Ye Andersson, Olof Liedberg, Bo

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