Evaluation of non-specific binding suppression schemes for neutravidin and alkaline phosphatase at the surface of reticulated vitreous carbon electrodes.
Non-specific binding (NSB) of high-molecular-weight proteins onto electrode surfaces can complicate the application of electroanalytical techniques to clinical and environmental research, particularly in biosensor applications. We present herein various strategies to modify the surface of reticulated vitreous carbon (RVC) electrodes to suppress non-specific binding of biomolecules onto its surface. Non-specific binding and specific binding (SB) of two enzyme conjugates, neutravidin-alkaline phosphatase (NA-ALP) and biotinylated alkaline phosphatase (B-ALP), and also neutravidin itself, were studied using hydroquinone diphosphate (HQDP) as an enzyme substrate for ALP inside the pores of RVC electrodes that had been subjected to various modification schemes. The extent of NSB and SB of these biomolecules inside RVC pores was assessed by measuring the initial rate of generation of an electroactive product, hydroquinone (HQ), of the enzyme-catalyzed reaction, using linear scan voltammetry (LSV) for HQ detection. Electrodes functionalized with phenylacetic acid and poly(ethylene glycol) (PEG) showed low NSB and high SB (when biotin capture ligands were included in the modification scheme) in comparison with unmodified electrodes and RVC electrodes modified in other ways. A simple sandwich bioassay for neutravidin was performed on the RVC electrode with the lowest NSB. A concentration detection limit of 52+/-2 ng mL(-1) and an absolute detection limit of 5.2+/-0.2 ng were achieved for neutravidin when this assay was performed using a 100 microL sample size.
Shedge
Hemangi Y
HY
Hunter Laboratory, Department of Chemistry, Clemson University, Clemson, SC 29634, USA.
Creager
Stephen E
SE
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001360:Avidin / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002244:Carbon / Q000737:chemistry*; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D011485:Protein Binding
D004800:Enzymes, Immobilized; D006873:Hydroquinones; D010755:Organophosphates; C105856:hydroquinone 1,4-bisphosphate; C093483:neutravidin; D001360:Avidin; D001710:Biotin; D002244:Carbon; D000469:Alkaline Phosphatase