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Optimizing immobilization conditions on a two dimensional carboxylbiosensor surface: pH dependence of antibody orientation andantigen binding capacity
Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden/The Ångström Laboratory, Solid State Electronics, Uppsala University, P.O. Box 534, SE-751 21 Uppsala, Sweden.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan.
Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden.
Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden.
2009 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309Artikel i tidskrift (Övrigt vetenskapligt) Submitted
Abstract [en]

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We have shown that successful immobilization is highly dependent on surface pKa, antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Generic immobilization conditions were demonstrated on a panel of antibodies which resulted in an average coefficient of variation of 4% for the immobilization of these antibodies.

Antigen binding capacity as a function of immobilization pH was studied. In most cases the antigen binding capacity followed the immobilization response. However, the antigen to antibody binding ratio differed between the antibodies investigated, and for one of the antibodies, the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab antibodies on different antibody surfaces showed that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Ort, förlag, år, upplaga, sidor
2009.
Nationell ämneskategori
Naturvetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-19690OAI: oai:DiVA.org:liu-19690DiVA, id: diva2:227489
Tillgänglig från: 2009-07-14 Skapad: 2009-07-14 Senast uppdaterad: 2017-12-13Bibliografiskt granskad
Ingår i avhandling
1. Self Assembled Monolayers for Quartz Crystal Microbalance based Biosensing
Öppna denna publikation i ny flik eller fönster >>Self Assembled Monolayers for Quartz Crystal Microbalance based Biosensing
2009 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The work in this thesis has been focused on developing surfaces for use in biosensor systems, especially for quartz crystal microbalances. The surfaces were prepared by adsorption of organosulfur molecules onto gold substrates, so called self assembled monolayers (SAMs). By chemical synthesis these thiols can be specifically tailored to provide surfaces with desired properties. The investigated surfaces were all based on thiols terminated with carboxylic acid groups to render hydrophilic surfaces onto which desired proteins can be covalently attached.

In order to increase the performance of two dimensional carboxyl surfaces, a method for improving the immobilization of proteins to the surface was investigated. The immobilization levels of antibodies were increased by using N-hydroxysulfo-succinimide (sulfo-NHS), instead of N-hydroxy-succinimide (NHS), as stabilizer of the amine reactive intermediate formed by reaction of the carboxyl groups with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The negatively charged sulfo-NHS intermediate promotes the attraction of overall positively charged proteins and enables immobilization also at low pH. In addition, the orientation of the immobilized antibodies was shown to be dependent on the pI of the antibody and to have a profound effect on the subsequent interaction with the antigen.

The organization of carboxyl terminated SAMs can be poor due to the repellation between the polar terminal groups. By using acidified ethanol as solvent during the assembly step of monolayer formation, the organization in carboxyl terminated alkyl and oligo(ethylene glycol) SAMs was improved. However, the carboxyl groups were found to be converted to ethyl esters, the rate being related to the acid strength. Furthermore, the long-term stability of carboxyl oligo(ethylene glycol) SAMs was investigated. Here, the effect of alkyl chain length on the storage stability was of interest. A short alkyl chain was shown to have a profound negative effect on the storage stability of the SAM, resulting in decomposition and loss of functionality over time compared to when thiols with longer alkyl chains were studied.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2009. s. 31
Serie
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1408
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:liu:diva-19679 (URN)LiU-TEK-LIC-2009:16 (Lokalt ID)9789173935876 (ISBN)LiU-TEK-LIC-2009:16 (Arkivnummer)LiU-TEK-LIC-2009:16 (OAI)
Presentation
2009-08-17, Planck, Fysikhuset, Campus Valla, Linköpings Universitet, Linköping, 13:15 (Engelska)
Opponent
Handledare
Anmärkning

In publication incorrect ISBN: 9879173935876

Tillgänglig från: 2009-07-14 Skapad: 2009-07-10 Senast uppdaterad: 2020-04-20Bibliografiskt granskad

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