RESUMEN
Electrochemical aptamer-based (EAB) sensors represent the first molecular measurement technology that is both (1) independent of the chemical reactivity of the target, and thus generalizable to many targets and (2) able to function in an accurate, drift-corrected manner in situ in the living body. Signaling in EAB sensors is generated when an electrode-bound aptamer binds to its target ligand, altering the rate of electron transfer from an attached redox reporter and producing an easily detectable change in peak current when the sensor is interrogated using square wave voltammetry. Due to differences in the microscopic surface area of the interrogating electrodes, the baseline peak currents obtained from EAB sensors, however, can be highly variable. To overcome this, we have historically performed single-point calibration using measurements performed in a single sample of known target concentration. Here, however, we explore approaches to EAB sensor operation that negate the need to perform even single-point calibration of individual sensors. These are a ratiometric approach employing the ratio of the peak currents observed at two distinct square wave frequencies, and a kinetic differential measurement approach that employs the difference between peak currents seen at the two frequencies. Using in vivo measurements of vancomycin and phenylalanine as our test bed, we compared the output of these methods with that of the same sensor when single-point calibration was employed. Doing so we find that both methods support accurately drift-corrected measurements in vivo in live rats, even when employing rather crudely handmade devices. By removing the need to calibrate each individual sensor in a sample of known target concentration, these interrogation methods should significantly simplify the use of EAB sensors for in vivo applications.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Electroquímicas , Animales , Aptámeros de Nucleótidos/química , Ratas , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Técnicas Biosensibles/métodos , Calibración , Electrodos , Ratas Sprague-Dawley , VancomicinaRESUMEN
Electrochemical, aptamer-based (EAB) sensors are the first molecular monitoring technology that is (1) based on receptor binding and not the reactivity of the target, rendering it fairly general, and (2) able to support high-frequency, real-time measurements in situ in the living body. To date, EAB-derived in vivo measurements have largely been performed using three electrodes (working, reference, counter) bundled together within a catheter for insertion into the rat jugular. Exploring this architecture, here we show that the placement of these electrodes inside or outside of the lumen of the catheter significantly impacts sensor performance. Specifically, we find that retaining the counter electrode within the catheter increases the resistance between it and the working electrode, increasing the capacitive background. In contrast, extending the counter electrode outside the lumen of the catheter reduces this effect, significantly enhancing the signal-to-noise of intravenous molecular measurements. Exploring counter electrode geometries further, we find that they need not be larger than the working electrode. Putting these observations together, we have developed a new intravenous EAB architecture that achieves improved performance while remaining short enough to safely emplace in the rat jugular. These findings, though explored here with EAB sensors may prove important for the design of many electrochemical biosensors.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Ratas , Animales , Aptámeros de Nucleótidos/química , Técnicas Electroquímicas , ElectrodosRESUMEN
Disposable single-use electrochemical sensor strips were used for quantitative detection of small concentrations of morphine in untreated capillary whole blood. Single-walled carbon nanotube (SWCNT) networks were fabricated on a polymer substrate to produce flexible, reproducible sensor strips with integrated reference and counter electrodes, compatible with industrial-scale processes. A thin Nafion coating was used on top of the sensors to enable direct electrochemical detection in whole blood. These sensors were shown to detect clinically relevant concentrations of morphine both in buffer and in whole blood samples. Small 38 µL finger-prick blood samples were spiked with 2 µL of morphine solution of several concentrations and measured without precipitation of proteins or any other further pretreatment. A linear range of 0.5-10 µM was achieved in both matrices and a detection limit of 0.48 µM in buffer. In addition, to demonstrate the applicability of the sensor in a point-of-care device, single-determination measurements were done with capillary samples from three subjects. An average recovery of 60% was found, suggesting that the sensor only measures the free, unbound fraction of the drug. An interference study with other opioids and possible interferents showed the selectivity of the sensor. This study clearly indicates that these Nafion/SWCNT sensor strips show great promise as a point-of-care rapid test for morphine in blood.