Biosensor for rapid determination of 3-hydroxybutyrate using bi-enzyme system.
A bi-enzyme-based Clark electrode was developed for the determination of 3-hydroxybutyrate. This sensor is based on the specific dehydrogenation by 3-hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30) in combination with salicylate hydroxylase (SHL E.C. 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, HBDH, catalyses the specific dehydrogenation of 3-hydroxybutyrate consuming NAD(+). The products, NADH, initiate the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. SHL forces the equilibrium of dehydrogenation of 3-hydroxybutyrate by HBDH to the product side by consuming NADH. Dissolved oxygen acts as an essential material for SHL during its enzymatic reactions. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of 3-hydroxybutyrate. Interferences from different amino acids and electroactive substances were found to be minimal due to the specificity of HBDH and the application of a Teflon membrane. The sensor has a fast response (2s) and short recovery time (2 min) with a linear range between 8 and 800 microM 3-hydroxybutyrate and a detection limit of 3.9 microM. A good agreement (R(2)=0.9925) with theoretical calculation was obtained in spiked serum sample measurements.
Kwan
Roger C H
RC
Sino-German Nano-Analytical Lab (SiGNAL), Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China. chroger@ust.hk
Hon
Phoebe Y T
PY
Mak
W C
WC
Law
L Y
LY
Hu
Jamie
J
Renneberg
Reinhard
R
D020155:3-Hydroxybutyric Acid / Q000097:blood* / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014159:Transducers
D006899:Mixed Function Oxygenases; D006884:Hydroxybutyrate Dehydrogenase; C022619:salicylate 1-monooxygenase; D020155:3-Hydroxybutyric Acid