Owing to the currently overburdened healthcare system there is a demand the development of accurate and reliable self-monitoring systems to reduce hospital exposure of the high-risk groups and medical costs. Urinary Tract Infection (UTI) is one condition which significantly affects patient quality of life and requires frequent intervention especially among post-menopausal women due to high recurrence rates. UTI affects around 150 million people worldwide and costs $3.5 billion, annually. Easy and reliable home-based monitoring will allow for detection of the infection at the early stages. This will ensure timely administration of appropriate antimicrobial drugs and will improve treatment success rates. While a number of UTI biosensors have been developed to overcome high diagnostic errors and delays of traditional lab urine culture tests, these are often label-based and require multistep sample preparation. Often these are culture based (associated with long wait times for pathogen to grow in suitable numbers for reliable sensing) or rely on a single biomarker for analysis. Further, they give semi-quantitative or “yes/no” readouts and, thus, exhibit poor resolution and low sensitivity. Based on prior research(1,2), we propose the development of a label-free combinatorial electrochemical urine dipstick biosensor to measure levels of three key urinary biomarkers- Prostaglandin E2 (PGE-2), Interleukin-6 (IL-6) and C-Reactive Protein (CRP)- for early detection and prediction of UTI onset and relapse.

A standard three-electrode electrochemical system was developed. Affinity based biosensing was used to detect PGE2, IL-6 and CRP expressed in urine using monoclonal antibodies, bound to the working electrode using a thiol crosslinker. Antigen-antibody binding resulted in the modulation of electrical properties at electrode-urine buffer interface. Leveraging this, calibration dose response curves were developed as a function of the biomarker concentration expressed in urine for each biomarker (see figure 1). Electrochemical Impedance Spectroscopy was used to assess sensor performance metrics for a dynamic range of 500-5000 pg/ml for PGE2, 1-500 pg/ml for IL-6 and 10-500 ng/ml for CRP the entire physiological urine pH range (5-8).

As shown in figure 1, linear response was obtained for all 3 biomarkers. For IL-6, the sensor exhibits a wide dynamic range of 1-500 pg/ml with a lower limit of detection of 1 pg/ml. For CRP, the sensor exhibits a wide dynamic range of 10-500 ng/ml with a lower limit of detection of 10 ng/ml. For PGE2, the sensor exhibits a wide dynamic range of 500-5000 pg/ml with a lower limit of detection of 500 pg/ml. Crosslinker-antibody binding was validated using ATR-FTIR spectroscopy. Zeta potential measurements validated antibody stability, indicating stable and reliable biosensing across pH (5 to 8). Figure 2 shows that the sensor was capable of differentiating between low and elevated levels for all 3 biomarkers (p<0.0001) in pooled human urine samples.

The analyte (IL-6, CRP and PGE2) expressed in the urine preferentially binds to its respective monoclonal antibody capture probe with high specificity. This binding activity induces a modulation of the electrical properties (capacitive and resistive changes) at the electrode-urine buffer interface. ATR-FTIR spectroscopy was used to identify peaks corresponding to (i) formation of self-assembled monolayer of crosslinker and (ii) cleavage of NHS ester bond due to antibody-crosslinker binding. This was done to validate the binding chemistry between the different components of the biosensing stack prior to sensing. A dose dependent decrease in impedance behavior, measured as the modulus of impedance, was observed with dosing (n=3 sensors). The sensor response for the low and elevated doses in pooled human urine showed significant difference (unpaired t-test, p<0.0001) for all 3 biomarkers. This can be leveraged to develop multiplexed panel for UTI diagnosis.  Electrochemical Impedance Spectroscopy was used to achieve highly sensitive, label free, rapid and highly specific biosensing in low sample volumes (<100 microliters) sensing in within 5 minutes.

An easy-to-use and cost-effective dipstick sensor was developed to reliably detect IL-6, CRP and PGE2 levels in <100 microliters of urine samples within 5 minutes. This biosensing scheme can be further extended to include more multiple inflammatory biomarkers towards early detection and prediction of UTI at home.

De Nisco NJ, Neugent M, Mull J, Chen L, Kuprasertkul A, de Souza Santos M, et al. J Mol Biol. 2019;
Kamakoti V, Kinnamon D, Choi KH, Jagannath B, Prasad S. Futur Sci OA. 2018;