Electrochemical N-Acetyl-β-D-glucosaminidase Urinalysis - Toward Sensor Chip-Based Diagnostics of Kidney Malfunction
At a Glance
Section titled “At a Glance”| Metadata | Details |
|---|---|
| Publication Date | 2021-09-30 |
| Journal | Biomolecules |
| Authors | Piyanuch Vibulcharoenkitja, Wipa Suginta, Albert Schulte |
| Institutions | Vidyasirimedhi Institute of Science and Technology |
| Citations | 11 |
| Analysis | Full AI Review Included |
Executive Summary
Section titled “Executive Summary”This analysis focuses on the development and validation of an electrochemical assay for N-acetyl-β-D-glucosaminidase (GlcNAcase) in urine, targeting early diagnostics for renal tubular damage.
- Core Value Proposition: Introduction of a novel, scalable electrochemical method for GlcNAcase urinalysis, a critical biomarker for acute and chronic kidney malfunction, offering a faster and more specific alternative to serum creatinine tests.
- Sensor Technology: Utilizes Boron-Doped Diamond (BDD) disk electrodes, chosen for their superior resistance to fouling from phenolic oxidation products, ensuring reliable and reproducible measurements in complex urine matrices.
- Detection Methodology: The assay employs Differential Pulse Voltammetry (DPV) to detect the anodic oxidation of 4-nitrophenol (4NP), the redox-active product released by GlcNAcase hydrolysis of the GlcNAc-4NP substrate. DPV minimizes capacitive charging currents, improving signal quality.
- Performance Validation: The method successfully differentiated GlcNAcase concentrations corresponding to healthy (5 U/L), concerning (20 U/L), and critically high (100 U/L) kidney states in both model solutions and spiked human urine samples.
- Scalability and Portability: Proof-of-concept was established using mass-producible BDD Screen-Printed Electrodes (SPEs), demonstrating the feasibility of adapting the assay for use in portable, handheld devices and single-use sensor strips.
- Reaction Kinetics: Linear regression analysis confirmed concentration-dependent reaction rates, with the 100 U/L sample showing a 10-fold higher 4NP generation rate (2.22 µM/min) compared to the 5 U/L sample (0.22 µM/min).
Technical Specifications
Section titled “Technical Specifications”| Parameter | Value | Unit | Context |
|---|---|---|---|
| Working Electrode (WE) Material | Boron-Doped Diamond (BDD) | N/A | Low fouling, wide potential window |
| Standard WE Diameter | 3.0 | mm | BDD disk electrode |
| SPE WE Diameter | 3.6 | mm | Screen-Printed Electrode platform |
| Detection Technique | Differential Pulse Voltammetry (DPV) | N/A | Used for 4NP anodic oxidation |
| Supporting Electrolyte | 0.1 M PBS | pH 7.0 | Standard assay buffer |
| Substrate Concentration | 0.5 | mM | GlcNAc-4NP (4NP-labeled substrate) |
| Practical Limit of Detection (4NP) | ~5 | µM | Lowest concentration for analyzable DPV peaks |
| GlcNAcase Concentration Range | 5, 20, 100 | U/L | Simulating healthy, concerning, and critical states |
| DPV Scan Speed | 0.05 | V s-1 | Voltammetry acquisition parameter |
| DPV Pulse Amplitude | 0.2 | V | Voltammetry acquisition parameter |
| DPV Peak Potential (BDD Disk) | ~1.05 | V | Anodic oxidation of 4NP vs. Ag/AgCl (3 M KCl) |
| DPV Peak Potential (BDD SPE) | ~1.11 | V | Anodic shift (60 mV) observed on SPEs |
| 4NP Generation Rate (100 U/L) | 2.22 | µM/min | Highest GlcNAcase activity slope |
| Incubation Time Tested | 20 to 60 | min | Time required for sufficient 4NP release |
| Average Standard Deviation (Urine) | 10 ± 6 | % | Worst-case scenario for triplicate measurements |
Key Methodologies
Section titled “Key Methodologies”The electrochemical GlcNAcase assay relies on optimizing the BDD electrode surface and utilizing DPV for selective detection of the enzymatic product, 4NP, in diluted urine samples.
- Electrode Regeneration: The BDD disk working electrode surface was polished between measurements using a thick suspension of 0.4 µm alumina powder to remove any polymeric residue (from 4NP oxidation) and prevent electrode fouling, ensuring consistent performance.
- Electrochemical Setup: Experiments utilized a portable PalmSens4 mini-potentiostat in a three-electrode configuration: BDD disk (WE), Platinum sheet (CE), and Ag/AgCl (3 M KCl) (RE).
- Assay Buffer and Substrate: The assay was conducted in 0.1 M Phosphate Buffered Saline (PBS) at pH 7.0, supplemented with 0.5 mM of the synthetic substrate, GlcNAc-4NP.
- Sample Preparation: Urine samples were diluted 10-fold into the buffered electrolyte to minimize matrix effects and background currents from complex urinary constituents.
- Enzymatic Reaction: GlcNAcase (either added to PBS models or present in urine) was allowed to act on GlcNAc-4NP at room temperature (25 °C) for incubation times ranging from 20 to 60 minutes.
- Signal Acquisition: Differential Pulse Voltammetry (DPV) was performed anodically (typically 0.6 V to 1.2 V) to measure the peak current corresponding to the oxidation of the enzymatically released 4NP.
- Scalability Demonstration: The procedure was successfully transferred to mass-producible BDD Screen-Printed Electrode (SPE) platforms, confirming their suitability for commercial strip test development, despite a minor 60 mV anodic shift in peak potential compared to the standard BDD disk.
Commercial Applications
Section titled “Commercial Applications”The developed electrochemical GlcNAcase urinalysis technology is highly relevant for decentralized diagnostics and sensor manufacturing, leveraging the unique properties of BDD electrodes.
- Point-of-Care (POC) Diagnostics:
- Development of handheld, portable potentiostats coupled with single-use BDD sensor strips for rapid, non-invasive kidney health checks.
- Enables convenient personal home health care monitoring for individuals at high risk (e.g., those with diabetes, hypertension, or obesity).
- Clinical and Professional Use:
- Quick prescreening tool for physicians’ offices and clinical laboratories to provide early warning of Acute Kidney Injury (AKI) or Chronic Kidney Disease (CKD).
- Monitoring patient response to treatment or exposure to nephrotoxic compounds.
- Sensor Manufacturing (BDD SPEs):
- Mass production of BDD Screen-Printed Electrodes (SPEs) for diagnostic strips, capitalizing on BDD’s stability and anti-fouling characteristics in biological fluids.
- Enzyme Assay Platforms:
- The methodology can be adapted for other enzyme activity assays where the product is a redox-active phenol, utilizing BDD’s robustness against phenol electro-oxidation fouling.
View Original Abstract
N-Acetyl-β-D-glucosaminidase (GlcNAcase) is a valuable biomarker for kidney health, as an increased urinary level of the enzyme indicates cell damage within the renal tubular filtration system from acute or chronic organ injury or exposure to nephrotoxic compounds. Effective renal function is vital for physiological homeostasis, and early detection of acute or chronic renal malfunction is critically important for timely treatment decisions. Here, we introduce a novel option for electrochemical urinalysis of GlcNAcase, based on anodic differential pulse voltammetry at boron-doped diamond disk sensors of the oxidizable product 4-nitrophenol (4NP), which is released by the action of GlcNAcase on the synthetic substrate 4NP-N-acetyl-β-D-glucosaminide (GlcNAc-4NP), added to the test solution as a reporter molecule. The proposed voltammetric enzyme activity screen accurately distinguishes urine samples of normal, slightly elevated and critically high urinary GlcNAcase content without interference from other urinary constituents. Moreover, this practice has the potential to be adapted for use in a hand-held device for application in clinical laboratories by physicians or in personal home health care. Evidence is also presented for the effective management of the procedure with mass-producible screen-printed sensor chip platforms.
Tech Support
Section titled “Tech Support”Original Source
Section titled “Original Source”References
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