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Imaging surface plasmon resonance
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-14923ISBN: 978-91-7393-820-4 (print)OAI: oai:DiVA.org:liu-14923DiVA: diva2:25589
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
List of papers
1. Protein Microarrays on Carboxymethylated Dextran Hydrogels: Immobilization, Characterization and Application
Open this publication in new window or tab >>Protein Microarrays on Carboxymethylated Dextran Hydrogels: Immobilization, Characterization and Application
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.

Keyword
Reversible hydrophobic barrier, microcontact printing, piezodispensing, protein microarrays, surface plasmon microscopy
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-14919 (URN)10.1007/s00604-004-0223-5 (DOI)
Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2017-12-13
2. Imaging SPR for detection of local electrochemical processes on patterned surfaces
Open this publication in new window or tab >>Imaging SPR for detection of local electrochemical processes on patterned surfaces
2008 (English)In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 134, no 2, 545-550 p.Article in journal (Refereed) Published
Abstract [en]

Imaging surface plasmon resonance (iSPR) was used in conjunction with voltammetry to investigate the possibility of detecting local electrochemical processes in situ on chemically modified electrodes. More specifically, a pattern of self-assembled monolayers (SAMs) of thiocholesterol and 1-hexadecanethiol was microcontact printed on gold substrates, and the blocking characteristics on different parts of the pattern were investigated. The SPR images reflected the changes in the refractive index over the working electrode due to electrochemical processes, which in the present case showed the ability of the SAMs to impede faradaic reactions. The results show that differences in packing densities or porosity of SAMs in different regions of a patterned surface can be visualized as electrochemical images using iSPR. The strength of utilizing an optical detection method for electrochemical characterization lies in the ability to achieve lateral resolution in real-time. Electrochemical reactions can also be used to enhance the contrast in SPR images of thin layers of components with similar thicknesses and refractive indices.

Keyword
Imaging surface plasmon resonance, Microcontact printing, Local electrochemical analysis, Surface analysis
National Category
Inorganic Chemistry
Identifiers
urn:nbn:se:liu:diva-14884 (URN)10.1016/j.snb.2008.05.042 (DOI)
Available from: 2008-09-29 Created: 2008-09-29 Last updated: 2017-12-13
3. Formation of Molecular Gradients on Bipolar Electrodes
Open this publication in new window or tab >>Formation of Molecular Gradients on Bipolar Electrodes
2008 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 47, no 16, 3034-3036 p.Article in journal (Refereed) Published
Keyword
Bipolar electrodes, electrochemistry, imaging, molecular gradients, surface chemistry
National Category
Inorganic Chemistry
Identifiers
urn:nbn:se:liu:diva-14881 (URN)10.1002/anie.200705824 (DOI)
Available from: 2008-09-29 Created: 2008-09-29 Last updated: 2017-12-13Bibliographically approved
4. Gradient Hydrogel Matrix for Microarray and Biosensor Applications: An Imaging SPR Study
Open this publication in new window or tab >>Gradient Hydrogel Matrix for Microarray and Biosensor Applications: An Imaging SPR Study
2009 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, no 1, 142-148 p.Article in journal (Refereed) Published
Abstract [en]

A biosensor matrix based on UV-initiated graft copolymerized poly(ethylene glycol) methacrylate and 2-hydroxyethyl methacrylate has been studied using imaging surface plasmon resonance (iSPR). By using a photo mask and a programmable shutter to vary the exposure time laterally, a gradient of matrix spots with physical thicknesses ranging from a few to tens of nanometers was generated. To maximize the dynamic range, imaging SPR was employed in wavelength interrogation mode. By finding the minimum in the reflectance spectra from each pixel of an image, SPR wavelength maps were constructed. The shift in SPR wavelength upon biospecific interaction was then measured both as a function of matrix thickness and composition. The performance of the matrix was evaluated in terms of immobilization of human serum albumin, biomolecular interaction with its antibody, and nonspecific binding of human fibrinogen. In addition, a low molecular weight interaction pair based on a synthetic polypeptide and calmodulin was also studied to explore the size selectivity of the hydrogel matrix. Our results show that the gradient matrix exhibits excellent properties for quick evaluation and screening of optimal hydrogel performance. The mixed hydrogel matrices display very low levels of nonspecific binding. It is also evident that the low molecular weight calmodulin is capable of freely diffusing and interacting throughout the entire hydrogel matrix, whereas the much larger albumin and its corresponding antibody, in particular, are partly/completely hindered from penetrating the interior of the matrix. This size-selectivity is attributed to a significant UV-initiated cross-linking or branching of the matrix during fabrication and/or protein mediated multipoint attachment during immobilization.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-16523 (URN)10.1021/bm801029b (DOI)
Available from: 2009-01-30 Created: 2009-01-30 Last updated: 2017-12-14
5. Lateral Control of Protein Adsorption on Charged Polymer Gradients
Open this publication in new window or tab >>Lateral Control of Protein Adsorption on Charged Polymer Gradients
Show others...
2009 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 25, no 6, 3755-3762 p.Article in journal (Refereed) Published
Abstract [en]

This work describes the fabrication, characterization, and protein adsorption behavior of charged polymer gradients. The thin gradient films were fabricated by a two-step technique using UV-initiated free-radical polymerization in a reactor with a moving shutter. A homogeneous layer of cationic poly(2-aminoethyl methacrylate hydrochloride) was first formed, followed by a layer of oppositely charged poly(2-carboxyethyl acrylate) with a continuously increasing thickness. Adsorption from protein solutions as well as human blood plasma was investigated by imaging surface plasmon resonance and infrared microscopy. The results showed excessive protein adsorption in the areas where one of the polymers dominated the composition, while there was a clear minimum at an intermediate position of the gradient. The charge of the surface was estimated by direct force measurements and found to correlate well with the protein adsorption, showing the lowest net charge in the same area as the protein adsorption minimum. We therefore hypothesize that a combination of the charged polymers, in the right proportions, can result in a protein-resistant surface due to balanced charges.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-17501 (URN)10.1021/la803443d (DOI)
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2017-12-13
6. A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays
Open this publication in new window or tab >>A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays
2009 (English)In: Biosensors and bioelectronics, ISSN 0956-5663, Vol. 24, no 8, 2458-2464 p.Article in journal (Refereed) Published
Abstract [en]

This work describes a concept for extending the dynamic range of quantification in an affinity biosensor assay by using a set of ligands with different affinities toward a common analyte. For a demonstration of the principle, three synthetic, biotinylated polypeptides capable of binding a model protein analyte with different affinities (10-9 M ≤ Kd ≤ 10-7 M) were immobilized in a microarray format on a gold slide covered with an oligo(ethylene glycol)-containing alkane thiolate self-assembled monolayer. For controllable immobilization, coupling was mediated by the biotinneutravidin interaction. A five-element affinity array, comprising single-peptide spots as well as spots where peptides were immobilized in mixtures, was realized by means of piezodispensation. Imaging surface plasmon resonance was used to study binding of the analyte to the different spots. The lower limit of quantification was ~3 nM and the corresponding upper limit was increased by more than an order of magnitude compared to if only the highest-affinity ligand would have been used. Affinity array sensors with multiple ligands for each analyte are particularly interesting for omitting dilution steps and providing highly accurate data in assays where several analytes such as disease biomarkers with extremely variable concentrations are quantified in parallel.

Keyword
Imaging surface plasmon resonance, Biosensor, Affinity microarray, Analyte quantification, Synthetic polypeptide
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-14922 (URN)10.1016/j.bios.2008.12.030 (DOI)
Available from: 2008-09-30 Created: 2008-09-30 Last updated: 2015-05-29

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