Real-Time Monitoring of a Protein Biomarker

Abstract

The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, life-threatening conditions. In response, here we present an electrochemical aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, we have developed an electrochemical, aptamer-based (EAB) sensor against Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clinically relevant NGAL concentrations over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how we detect, monitor, and treat many important diseases.

Keywords: acute kidney injury; aptamer; electrochemical sensor; neutrophil gelatinase-associated lipocalin; real-time protein monitoring.

Figure 1.

EAB sensors employ the binding-induced folding of a redox-reporter-modified aptamer to generate an easily measurable, rapidly reversible electrochemical signal (the changes in conformation affects the rate of electron transfer thus changing the intensity of the electrochemical signal) without the need for exogenous reagents or wash steps, thus enabling continuous, real-time molecular monitoring. The platform’s binding-induced folding signal transduction mechanism is highly selective, allowing EAB sensors to perform even when challenged in complex sample streams.

Figure 2.

Real-time monitoring of clinically relevant threshold levels of NGAL in urine. (A) Threshold NGAL level indicative of diseases has variously been reported to be from 2 to 32 nM. The useful dynamic range of our sensor spans these concentrations in both artificial urine and authentic human urine. The data represent the average signals and the standard deviations of at least three independently fabricated sensors, illustrating the excellent sensor-to-sensor reproducibility we achieve even with these hand-held devices. (B) The sensor responds promptly to the addition (blue arrows) and removal (red arrows) of NGAL. Shown are three cycles of addition (32 nM) and removal of NGAL over 2 h. (C) The sensor easily monitors a continuous rise in (spiked) NGAL that mimics the profile expected for acute renal injury.

Figure 3.

EAB sensors can be used to monitor NGAL levels in situ in a urinary catheter. (A) In order to mimic a real-life situation, we created a system that passes artificial urine through a 14fr Foley catheter that contains two micron-scale EAB sensors. In particular, using a pump we increased the level of NGAL in the reservoir at the bottom (yellow lines), while a second pump circulates the artificial urine through an IV bag (representing an artificial bladder) and then through the catheter (blue lines). Using this, we monitored NGAL concentrations with 3 min resolution over the course of 3 h. (B) At 1 h, we started introducing NGAL, linearly increasing its concentration over the course of 1 h to 32 nM. As shown, both sensors responded to the addition in real-time and with closely similar responses. The few minutes of lag time between the addition of NGAL and the first sensor response is associated with the time required for NGAL to pass through the pump and the artificial bladder before reaching the sensors.