(Invited) Continuous Monitoring of Small Molecules Using Electrochemical Aptamer-Based Biosensors with CMOS Electronics

The ability to continuously monitor specific “molecules” over a long period of time can provide fundamentally new insights into disease dynamics and the transition from a healthy state and lead to the design of next-generation screening and detection tools. However, currently only a few biomolecules, such as glucose, oxygen, and dopamine, can be continuously and real-time monitored with significant relevance to chronic disease management. The prevalent glucose sensor uses glucose oxidase (GOx), an enzyme designed to oxidize glucose persistently in its proximity, producing an electrochemical current proportional to the glucose concentration. The enzyme's specificity means it cannot be readily adapted to target different biomolecules, thus constraining its versatility. This limitation also extends to many enzyme-based sweat sensors. In contrast, oxygen and dopamine measurements exploit their inherent molecular characteristics. Oxygen saturation (SpO2) in blood is determined via pulse oximetry, which uses the distinct light absorption properties of deoxygenated and oxygenated blood under red and infrared lights. Dopamine, a neurotransmitter integral to our emotional responses, is highly electrochemically active and can undergo reversible oxidation, allowing it to be detected by analytical methods like fast-scan cyclic voltammetry (FSCV). Nevertheless, such distinct properties are not common among biomolecules, making these methods non-transferable to a broader range. To this end, our group and others have previously utilized aptamers to achieve continuous detection of small molecules in vivo . Aptamers are “synthetic antibodies” composed of nucleic acids that can specifically bind to the target analytes in complex samples, such as the whole blood (Fig. 1(a)). Importantly, they can be engineered into “aptamer-switches” that undergo structure-switching upon target binding in a reversible manner. By conjugating electroactive reporters to the aptamers, the changes in their structure (thus, the analyte concentration) can be detected electrochemically. As sample preparation is not needed, aptamer switches are capable of continuous monitoring of biomolecules in vivo . Coupling with structure-switching aptamers, our team has presented the first wireless, electrochemical aptamer-sensing chip in the 65-nm CMOS technology and achieved continuous recording of small-molecule drugs, e.g., antibiotics or chemotherapeutics, from a freely moving rodent (Fig. 1(b) and 1(c)). The chip features a chronoamperometric (CA) sensing circuit that probes the electron transfer (ET) kinetics of the structure-switching aptamers that are correlated to the concentration of target analytes. Notably, we have innovated a sample-and-hold (S/H) circuit technique into our readout front end to overcome the noise/power trade-off commonly experienced in conventional circuit design (Fig. 1(d)). This technique employs two switched-capacitor circuits that maintain the electrode potentials on an open-loop basis, disabling the power-intensive and noise-contributing on-chip amplifiers during the recording phase. The technique achieves simultaneous reductions of 286× in circuit noise (4.36 nA rms to 15.2pA rms ) and 23× in power consumption (5.25 mW to 0.22 mW), along with a 10× increase in throughput compared to the traditional square-wave voltammetry (SWV) readout methods that have been commonly used in interrogating E-AB sensors (5 vs. 0.5 Samples/sec). Fig. 1(d) shows the measured current transients that reflects the changes in ET kinetics at different target concentrations. While previous circuit focuses on lowering the electronics noise, we also develop a second electrochemical readout circuit that aims at boosting the detection signal through redox current integration at the circuit domain. This integration process captures the charges, instead of the current, leading us to term this technique square-wave voltcoulometry (SWVC, Fig. 1(e)). Our analysis demonstrates that the proposed SWVC technique can yield a remarkable signal improvement of 250×. Importantly, this improvement is achieved while maintaining nearly identical levels of input-referred noise (5 pA rms ) at a power consumption of only few mW. Fig. 1(f) compares the measurements obtained using conventional SWV readout and the proposed SWVC technique. The results demonstrate a remarkable 25-fold improvement in the signal-to-noise ratio (SNR), and we anticipate another 4× boosting once we optimize the test setup. We've also addressed issues related to drift and biofouling. We utilize kinetic-differential measurements (KDM) that monitor the rate of current change rather than absolute current values, mitigating the effects of biosensor detachment from the electrode surface. We also develop a dual-aptamer strategy where two aptamer sensors responding differentially to the same analyte targets are utilized to increase the signal while rejecting the slow-moving drifts (Fig. 1(g)). For biofouling, we employ a combinatorial-selected polyacrylamide hydrogel coating to specifically resist platelet adhesion (Fig. 1(h)). By integrating these advancements, we have validated our technology in vivo within whole blood via a probe catheterized into a rat's vein and interstitial fluids (ISF) through whole-system implantation (Fig. 1(i)). This technology also holds promise for the multiplexed monitoring. Figure 1