Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase

Abstract

We report an amperometric biosensor for the urinary disease biomarker para-hydroxyphenylacetate (p-HPA) in which the allosteric reductase component of a bacterial hydroxylase, C1-hpah, is electrically wired to glassy carbon electrodes through incorporation into a low-potential Os-complex modified redox polymer. The proposed biosensing strategy depends on allosteric modulation of C1-hpah by the binding of the enzyme activator and analyte p-HPA, stimulating oxidation of the cofactor NADH. The pronounced concentration-dependence of allosteric C1-hpah modulation in the presence of a constant concentration of NADH allowed sensitive quantification of the target, p-HPA. The specific design of the immobilizing redox polymer with suitably low working potential allowed biosensor operation without the risk of co-oxidation of potentially interfering substances, such as uric acid or ascorbic acid. Optimized sensors were successfully applied for p-HPA determination in artificial urine, with good recovery rates and reproducibility and sub-micromolar detection limits. The proposed application of the allosteric enzyme C1-hpah for p-HPA trace electroanalysis is the first successful example of simple amperometric redox enzyme/redox polymer biosensing in which the analyte acts as an effector, modulating the activity of an immobilized biocatalyst. A general advantage of the concept of allosterically modulated biosensing is its ability to broaden the range of approachable analytes, through the move from substrate to effector detection.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacter baumannii reductase C1 subunit

“…The lower LOD for the electrochemical bioassay with dissolved C1 was likely to be because NADH had freer access to C1 in solution than with the reductase immobilized within the redox polymer. It is worth mentioning that use of C1 as an immobilized element of redox polymer-based biosensors, as proposed in earlier published work (20,21), could form the basis of a personal urinary test device with screen-printed electrode strips modified with the enzyme-polymer layer. For urinary 4-HPA analysis in clinical laboratories and as an analytical tool for inspection of libraries of effector candidates and/or genetically engineered redox enzyme variants, work with C1 in solution is more practical because of its simplicity and speed and because it avoids the extra steps needed to prepare biosensors modified with a redox polymer.…”

“…The dependence of the allosteric effect on modulator concentration also permits assay of the allosteric modulator, widening the range of analytical applicability. Despite this useful practical feature, electrochemical biosensing with allosteric enzymes remains an underdeveloped practice, and the few published examples either work with rather complex designs of the sensor readout or require specially synthesized nanomaterials as components of the immobilization matrix in order to generate an adequate signal (16)(17)(18)(19)(20)(21). Thus, the utilization of allosteric enzymes on electrode surfaces and the design of simple and sustainable strategies for allosteric electrochemical assays of substrate or effector is a topical issue, with many methodological opportunities.…”

Engineering Protein Switches for Rapid Diagnostic Tests

“…Protein switches have thus far been largely developed for healthcare applications, where there is great potential to improve disease prediction, speed up diagnosis, and monitor drug dosing, to give better patient outcomes. There are wide opportunities for protein switch antibody tests: those developed for infectious disease serology could be widened to autoimmune, allergy, and broader immunology applications, ,,,, and those for therapeutic drug monitoring can guide dosing in the ever-growing biologics market. ,,, Small molecule tests have also been developed for drug monitoring, , as well as metabolic , and disease biomarker assays. , Tests for a range of proteins have also been reported, and the scope expands as new biomarkers are discovered. ,,, Human health applications dominate the literature, but the significant opportunities for quantitative on-site tests should be explored in broader sectors. Some protein switch assays have been developed for food standards, agricultural disease monitoring, and veterinary immunology applications, , but there are much wider prospects in these settings and others, including environmental monitoring, illicit drug detection, and sports medicine …”

“…25,30,31,40 Small molecule tests have also been developed for drug monitoring, 26,43 as well as metabolic 27,62 and disease biomarker assays. 33,100 Tests for a range of proteins have also been reported, and the scope expands as new biomarkers are discovered. 29,30,41,44 Human health applications dominate the literature, but the significant opportunities for quantitative on-site tests should be explored in broader sectors.…”

Enzymatic Cycle-Inspired Dynamic Biosensors Affording No False-Positive Identification

“…Allosteric regulation of biomolecules and chemical events is an intriguing natural phenomenon. Its significance is not just crucial for dynamic control of biological processes, but it also inspires toolboxes for programmable synthetic nanodevice engineering. − Over the past two decades, there has been considerable progress in the design of controllable biosensor components, − precision switches, − and genetic regulators − based on the principle that the activity of the synthetic structure corresponds to a varied free-energy state regulated by an allosteric effector. These efforts have focused on exploring new allosteric toolboxes to construct synthetic allosteric systems. − However, designing synthetic allosteric systems that can reversibly switch their conformational states is more challenging because multiple states relative to positive/negative activities must be flexible enough that switching can be reversibly toggled by allosteric effectors.…”