Electroanalysis of Enzyme Activity in Small Biological Samples: Alkaline Phosphatase.

The discovery of sulfite-stabilized anodic current of hydroquinone (HQ) at high pH was used to develop two new methods for measuring the activity of the key biomarker alkaline phosphatase (ALP). Both approaches relied on the monitoring of ALP-triggered release of HQ from a substrate hydroquinone diphosphate (HQDP) into a pH 10.00 solution. One detected the released HQ via the internally calibrated electrochemical continuous enzyme assay (ICECEA) at a glassy carbon (GC) electrode with no sample incubation. The other used sample incubation with HQDP and quantified the released HQ via a coulometric assay at a commercial glucose test strip (GTS). The assay solution was optimized by investigating the ALP/HQDP/HQ system at a GC electrode. The ICECEA revealed high affinity of ALP for HQDP (Kmapp, 87 µM; Vmax, 0.36 µM min-1) and detected ALP down to 0.022 U L-1. At GTS, ALP was detected down to 0.064 U L-1 in a 1 µL sample of human serum after a 20 min incubation at room temperature. The linear range (R2, 0.994) extended at least up to 1.7 U L-1 ALP, which covered more than the clinical range for ALP in serum. The interferences from the sample matrix including those from indigenous glucose were eliminated using a charge difference ΔQ (=Qtotal - Qsample matrix) as a signal for ALP. Both advances proposed here are direct (no auxiliary enzymes or labels required), accurate (98 ± 3% ALP signal recovery), and precise (relative standard deviation (RSD), <7%). The HQDP-GTS-based assay advances the analysis of ALP activity in microsized real-life samples.

Oxidases, carbon nanotubes, and direct electron transfer: A cautionary tale.

The case study of four FAD-dependent oxidase enzymes is presented in the context of the often-claimed direct electron transfer (DET) to glucose oxidase at carbon nanotubes (CNT). The selected enzymes included d-amino acid (AAOx), alcohol (AOx), pyranose (PyOx), and choline oxidase (ChOx). Each enzyme (E) was mixed with chitosan and CNT (either multi- or single-walled) to form a CNT/E film on the surface of glassy carbon electrode. All eight CNT/E films displayed redox activity depicted by voltammetric current peaks near -0.4 V. However, no DET was observed for any of the films as indicated by the absence of expected substrate- and oxygen-induced asymmetry in the anodic-to-cathodic charge ratio. The peaks are suggested to be due to the redox of either a dissociated FAD cofactor, in the case of AAOx and AOx, or denatured enzyme in the case of PyOx and ChOx. The amperometric assays of the films revealed the lowering of enzymatic activity of all four oxidases by CNT. The results are consistent with the hypothesis of oxidase molecules displaying a spectrum of enzymatic activity in CNT/E films ranging from voltammetrically untraceable (for molecules adsorbed on CNT) to amperometrically measurable (for molecules remote from CNT). The kinetic studies showed that enzyme molecules with no net charge leached at the slowest rate from CNT/E films. This work adds to a growing number of reports challenging the fallacy of DET to FAD-dependent native oxidases.

Synthesis and Characterization of Pyridine Compounds for Amperometric Measurements of Leukocyte Esterase.

We introduce a new class of substrates (compounds I-III) for leukocyte esterase (LE) that react with LE yielding anodic current in direct proportion to LE activity. The kinetic constants Km and kcat for the enzymatic reactions were determined by amperometry at a glassy carbon electrode. The binding affinity of I-III for LE was two orders of magnitude better than that of existing optical LE substrates. The specificity constant kcat /Km was equal to 2.7, 3.8, and 5.8×105 m-1 s-1 for compounds containing the pyridine (I), methoxypyridine (II), and (methoxycarbonyl)pyridine (III), respectively, thus showing an increase in catalytic efficiency in this order. Compound III had the lowest octanol/water partition coefficient (log p=0.33) along with the highest topological surface area (tPSA=222 Å2 ) and the best aqueous solubility (4.0 mg mL-1 ). The average enzymatic activity of LE released from a single leukocyte was equal to 4.5 nU when measured with compound III.