The application of polythiol molecules for protein immobilisation on sensor surfaces.
The immobilisation of bio-receptors on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The development of a protocol for biomolecule immobilisation onto a surface plasmon resonance (SPR) sensor surface using inexpensive polythiol compounds is presented here. The method used here is based on the reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. The self-assembled thiol monolayers were characterised using contact angle and XPS. The possibility to immobilise proteins on monolayers was assessed by employing BSA as a model protein. For the polythiol layers exhibiting the best performance, a general protocol was optimised suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti Salmonella typhimurium. The kinetic data was obtained for PSA binding to anti-PSA and for S. typhimurium cells with a detection limit of 5x10(6) cells mL(-1) with minimal non-specific binding of other biomolecules. These findings make this technique a very promising alternative for amine coupling compared to peptide bond formation. Additionally, it offers opportunity for immobilising proteins (even those with low isoelectric point) on neutral polythiol layers without any activation step.
Kyprianou
Dimitris
D
Cranfield Health, Cranfield University, Cranfield, Bedfordshire, UK.
Guerreiro
Antonio R
AR
Nirschl
Martin
M
Chianella
Iva
I
Subrahmanyam
Sreenath
S
Turner
Anthony P F
AP
Piletsky
Sergey
S
D015374:Biosensing Techniques / Q000295:instrumentation; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D017430:Prostate-Specific Antigen / Q000032:analysis; D011485:Protein Binding; D015203:Reproducibility of Results; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation*; D013499:Surface Properties
D004800:Enzymes, Immobilized; D017430:Prostate-Specific Antigen