Adenosine detection in serum using a surface plasmon resonance biosensor with molecularly imprinted polymers incorporating modified thymidine monomers

Abstract

Stress is a response to stimuli which disrupt the homeostasis of a cell or organism. Adenosine is a purine nucleoside which functions as an immunomodulator and signalling molecule, with elevated levels present in tissues exposed to stress. Current methods used to determine adenosine levels within the body involve chromatography coupled with mass spectrometry, which while sensitive is time consuming and costly, highlighting the need for a quicker and more cost-effective detection method. Six nanoMIPs were produced using solid-phase synthesis targeting adenosine: a plain nano-MIP, an acrylamide-dT nano-MIP (bearing an acrylamide-modified thymidine molecule), and a carboxy-dT nanoMIP (bearing a carboxy-modified thymidine molecule) were made using two different methods. The first involved glutaraldehyde as the linker molecule connecting the template to the solid phase, whilst the second used EDC/NHS coupling chemistry. This allowed us to alter the orientation of the template to present either the base or sugar outwards. SPR was used to test the nanoMIP binding affinities and selectivity against adenosine, thymidine, deoxyguanosine and deoxycytidine. It was found the binding affinities of the nanoMIPs increased with use of the modified thymidine monomers, with equilibrium dissociation constants (K_D) values of the plain nanoMIP, acrylamide-dT nanoMIP and carboxy-dT nanoMIP being 221 nM, 9.35 nM, and 2.11 nM respectively for the glutaraldehyde method. The following K_D values were obtained for the EDC/NHS method: 212 nM, 5430 nM, and 111 nM for the plain nanoMIP, acrylamide-dT nanoMIP and carboxy-dT nano-MIP respectively. This illustrated the glutaraldehyde method produced more effective nanoMIPs than using EDC/NHS. This is surprising as it is counter-intuitive to the imagined Watson–Crick pairing. When challenged with the other nucleosides, excellent selectivity was observed. Fetal bovine serum was used to test the capability of the nanoMIPs in complex matrixes with consistent results produced throughout.