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.

Determination of sorbitol dehydrogenase in microsamples of human serum.

The enzyme sorbitol dehydrogenase (SDH) is an emerging biomarker of drug-induced liver injury (DILI). This paper introduces determination of SDH in microliter samples of human serum at commercial glucose test strips. The determination relies on the oxidation of NADH cofactor, which is used by SDH reacting with its substrates. The strips could detect NADH down to 5.0 µM (5 pmol), which was two orders of magnitude better than the prior relevant limit of detection. The concentration of cofactors (NADH, NAD+) and substrates (fructose, sorbitol) for SDH determination at a strip was optimized via internally-calibrated amperometric assays at a chitosan/nitrogen-doped carbon nanotube electrode. Such an electrode provided reliable assay data for over 3 months with no need for its reactivation. The assays yielded kinetic parameters Km and kcat and demonstrated higher apparent affinity of SDH for NADH and fructose than NAD+ and sorbitol. The glucose strips detected SDH down to 98 pM (98 amol) in buffers and 200 pM (200 amol) in human serum after 20-min incubation with an optimized (c ≥ 10Km) mixture of cofactor + substrate. The charge ΔQ flowing through a strip was linear (R2, 0.994) up to 6.0 nM SDH, which covered enzyme's clinical range. The ΔQ was selective for SDH, independent of sample matrix, and free of interferences from indigenous glucose. The use of glucose strip as an electrolytic microcell to detect picomoles of NADH and attomoles of SDH is a step toward a point-of-care monitoring of DILI.

Electroanalysis of Infection with Methyl Pyruvate.

The discovery of infection enzyme leukocyte esterase (LE) hydrolyzing a mitochondrial substrate methyl pyruvate (MP) was explored in the development of electroanalytical methods for LE in human biofluids. The LE + MP reaction was coupled with alcohol oxidase to produce hydrogen peroxide, which was then reduced at a nitrogen-doped carbon nanotube electrode at -0.20 V, yielding current proportional to the LE content in a sample. The kinetic assays revealed a fast turnover (kcat = 15 s-1) and high specificity constant (kcatKm-1 = 2.3 × 106 M-1 s-1) for the LE-triggered hydrolysis of MP. The analytical assays were short (5 min) and the quantified LE was in the clinically relevant range of 22-300 µg L-1 (R2, 0.985). The immuno-electroanalysis could detect the picomole quantity of LE and yielded linear calibration plots up to 150 µg L-1 of LE with the same slope regardless of the sample matrix (urine, saliva, and phosphate buffer). The spike-and-recovery experiments displayed an LE recovery of 99-104%. The amperometric immunoassay of LE was less laborious than traditional enzyme-linked immunosorbent assay (ELISA) for LE and reduced the required sample incubation time from 4 h (sandwich ELISA) to 30 min (immuno-electroanalysis). The proposed combination of immunosorption with internally calibrated amperometry can also be used for a selective determination of other enzymes, which form enzymatically active immune complexes.

Infection Screening in Biofluids with Glucose Test Strips.

The four glucosyl esters were synthesized and tested for the determination of infection enzyme leukocyte esterase (LE) in human synovial (joint) fluid and urine. The esters acted as LE substrates releasing glucose in a direct proportion to the activity of LE in a sample. The freed glucose was then detected by a coupled-enzyme assay at either a nitrogen-doped carbon nanotube (N-CNT) electrode or a commercial glucose test strip. The assays at the N-CNT electrode detected LE down to 0.81 nM (25 µg L-1) and showed the fastest kinetics (2.1 × 105 M-1 s-1) for esters with the least crowded space around their carbonyl group. When used with glucose strips, the esters discerned clinically relevant levels of LE up to at least 26 nM (800 µg L-1) in the microliter-sized samples of bodily fluids. The reading of glucose strips with a potentiostat, instead of a personal glucose meter (blood glucometer), shortened the time of required sample incubation from 3 h to 5 min. Correcting the signal of incubated sample for that of original sample eliminated matrix effects and accounted for the presence of native glucose. The new esters have a potential to extend the use of glucose strips (already used by millions for diabetes monitoring) to the quantification of the severity of urinary tract and periprosthetic joint infections.

Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN-/H2O2 System.

Strategies to detect and characterize myeloperoxidase (MPO) are needed, given that this "split personality" enzyme kills harmful microorganisms but also damages a host tissue. Here, we describe electrochemical approaches to measure MPO by using the pseudohalogenation (MPO/SCN-/H2O2) and catalase-like (MPO/H2O2) cycles. Their kinetics were determined by monitoring the consumption of H2O2 with a nitrogen-doped carbon nanotubes (N-CNT) electrode, which could detect 0.50 µM H2O2 at -0.20 V. The unique design of internally calibrated electrochemical continuous enzyme assay (ICECEA) and electrode stability allowed use of one N-CNT electrode for over half a year to reliably determine MPO. The kinetic measurements showed that (a) SCN- did not affect the affinity of MPO for H2O2, (b) catalase-like cycle was slower, and (c) MPO retained enzymatically active conformation after complexation with its antibody Ab both in a solution and on the surface of an antibody dipstick (d/Ab). The homogeneous assays could detect 5.2 µg L-1 MPO (35 pM) via a faster cycle. The heterogeneous immunoassays with the capture of MPO on d/Ab could detect 60 µg L-1, which was suitable for the accurate detection of MPO in human saliva (101% recovery). Replacing a detection antibody of ELISA with ICECEA as a signal transducer for immunoassays offers a rapid method for the selective determination of enzymes; for example, time of MPO quantification was cut from 3-4 h (sandwich ELISA) to ∼20 min (ICECEA-dipstick).

Determination of omega-3 fatty acids in fish oil supplements using vibrational spectroscopy and chemometric methods.

The potential of Fourier transform infrared (FT-IR), near-infrared (NIR), and Raman spectroscopic techniques combined with partial least squares (PLS) regression (PLSR) to predict concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total omega-3 fatty acids (n-3 FAs) in fish oil supplements was investigated. FT-IR spectroscopy predicted EPA (coefficient of determination (R(2)) of 0.994, standard error of cross-validation (SECV) of 2.90%, and standard error of prediction (SEP) of 2.49%) and DHA (R(2) = 0.983, SECV = 2.89%, and SEP = 2.55%) with six to seven PLS factors, whereas a simpler PLS model with two factors was obtained for total n-3 FAs (R(2) = 0.985, SECV = 2.73%, and SEP = 2.75%). Selected regions in the NIR spectra gave models with good performances and predicted EPA (R(2) = 0.979, SECV = 2.43%, and SEP = 3.11%) and DHA (R(2) = 0.972, SECV = 2.34%, and SEP = 2.60%) with four to six PLS factors. Both the whole and selected NIR regions gave simple models (two PLS factors) with similar results (R(2) = 0.997, SECV = 2.18%, and SEP = 1.60%) for total n-3 FAs. The whole and selected regions of Raman spectra provided models with comparable results and predicted EPA (R(2) = 0.977, SECV = 3.18%, and SEP = 2.73%) and DHA (R(2) = 0.966, SECV = 3.31%, and SEP = 2.56%) with seven to eight PLS factors, whereas a simpler model (three PLS factors) with R(2) = 0.993, SECV = 2.82%, and SEP = 3.27% was obtained for total n-3 FAs. The results demonstrated that FT-IR, NIR, and Raman spectroscopy combined with PLSR can be used as simple, fast, and nondestructive methods for quantitative analysis of EPA, DHA, and total n-3 FAs. FT-IR and NIR spectroscopy, in particular, have the potential to be applied in process industries during production of fish oil supplements.