Electrochemical Detection of Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Simulated Urine Using Au@Si/Ni-Porphyrin Nanocomposite for the Prediction of Kidney Injury

Abstract

Kidney Injury is multifactorial out of which ureteropelvic junction obstruction is one of the major causes of concern. There are a multitude of methods for monitoring the deterioration of renal function like serum creatinine levels, ultrasound scans, renograms, and nuclear scans like radiolabelled MAG3 and DMSA which require repeat evaluation or sedation during the process. Many of the biomarkers utilized for the assessment of kidney injury are expressed at the later stages. Urine NGAL is a promising biomarker for risk stratification which is reported to be expressed at the onset of ureteropelvic junction obstruction (UPJO) and is a gold standard for the estimation of tubular injury. Urinary NGAL is also reported to be a specific marker for the prediction of hydronephrosis in children who require surgical intervention. The kidney injury cut-off for NGAL in urine is 185ng/mL and 48 ng/mL in the adult and pediatric population groups respectively. Electrochemical detection of NGAL in patient urine is a fast, reliable, non-invasive, accurate, and cost-effective method for monitoring kidney health to segregate patient groups that require surgical intervention.In this study, an electrochemical sensor was designed with Au(150nm) electrodes fabricated using thermal evaporation on a flexible substrate (PET; 250μm) by using Cr(20nm) as the seed layer. The electrode was immobilized with Au@Si core-shell decorated on Ni-porphyrin nanosheets functionalized with anti-NGAL using covalent chemistry. The nanocomposite was synthesized through a bottom-up approach where the Ni-tetrakis(4-carboxyphenyl) porphyrin nanosheets were synthesized using the solvothermal method in an organic phase (DMF) followed by purification and suspension in ethanol medium. The Si nanocrystals were synthesized in a citrate medium with APTMS as the precursor followed by purification via dialysis. The purified Si nanocrystals (60ppm) and Ni-porphyrin nanosheets (880ppm) were treated in chloroauric acid (0.25mM) and the reducing agent trisodium citrate (34mM) to form Au shell. The physicochemical and electronic properties of the synthesized nanocomposite were evaluated through XRD, XPS, microscopic analysis(TEM, SEM), AFM, UV-Vis, FTIR, and Raman spectroscopy. The resultant nanocomposite was purified and linked to anti-NGAL (100μg/mL) through EDC (0.4M)/NHS (0.1M) chemistry. The antibody-nanocomposite was immobilized on the fabricated Au electrodes (working electrode surface area: 0.35mm2) through BSA(1.2mM)/glutaraldehyde (2.5% v/v) crosslinking. The quantitative electrochemical estimation of NGAL antigen spiked in 0.1M PBS (pH=7.2±0.2) and simulated urine was performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The shelf life of the fabricated sensors was evaluated for 30 days (stored at 4˚ C until use) over the concentration range of 0-5000 ng/mL. The amount of test solution required per measurement for the miniaturized electrodes was 6μL.The sensing mechanism of the electrochemical platform was established through CV by varying the scan rate (0.05 V/s - 2.5 V/s) for 500ng/mL in 0.1M PBS and urine. With increasing scan rate a linear relationship between log ip and log ν was observed. For 0.1M PBS, the process was largely capacitive at various detected potentials (b=0.94 @ 0.5V; b=0.86 @ 0.3V; b=0.85 @ 0.07V). However, in the case of simulated urine, the electrochemical process was diffusion-limited at 0.6V (b=0.49). Therefore, to obtain higher current sensitivity in the diffusion-limited regime, DPV was adopted. With increasing concentration of antigen NGAL, a monotonic decrease in the redox current at 0.6V and 0.45V potential was observed. This signifies that the presence of NGAL antigen leads to the formation of antigen-antibody complex immobilized on the surface of the nanocomposite translating into an increase in the resistance at the electrode surface leading to a decrease in the redox current. The decrease in the current was proportional to the increase in the amount of the test antigen. The sensitivity of the sensor at 0.45V and 0.6V was 2.22 nA/ng mL-1/mm2 and 0.51 nA/ng mL-1/mm2 respectively with the lowest limit of NGAL detection as 12.5ng/mL in simulated urine by using DPV. Similarly, for CV the sensitivity of the sensor at 0.56V was 3.48 nA/ng mL-1/mm2 with 22ng/mL as the limit of detection. The sensor performance was evaluated without the use of nanocomposite (only antibody) as a control for comparison.Therefore, this study is a proof of concept for an effective electrochemical sensor platform design for urinary NGAL detection using a novel nanocomposite. It was established that the use of nanocomposite provides a better surface area to volume ratio through the core-shell nanostructures decorated on the nanosheets translating into higher electrochemical activity. The sensing platform was found to be specific, sensitive, and stable making it a perfect candidate for a point-of-care-testing(POCT) platform. Figure 1