A pH-independent electrochemical aptamer-based biosensor supports quantitative, real-time measurement in vivo

Abstract

The development of biosensors capable of achieving accurate and precise molecular measurements in the living body in pH-variable biological environments (e.g. subcellular organelles, biological fluids and organs) plays a significant role in personalized medicine. Because they recapitulate the conformation-linked signaling mechanisms, electrochemical aptamer-based (E-AB) sensors are good candidates to fill this role. However, this class of sensors suffers from a lack of a stable and pH-independent redox reporter to support their utility under pH-variable conditions. Here, in response, we demonstrate the efficiency of an electron donor π-extended tetrathiafulvalene (exTTF) as an excellent candidate (due to its good electrochemical stability and no proton participation in its redox reaction) of pH-independent redox reporters. Its use has allowed improvement of E-AB sensing performance in biological fluids under different pH conditions, achieving high-frequency, real-time molecular measurements in biological samples both in vitro and in the bladders of living rats.

Fig. 1. Electrochemical aptamer-based (E-AB) sensors employing the exTTF derivative as a pH-independent redox reporter. (A) The DNA aptamer was modified at the 5′-terminus with a self-assembly monolayer as an anchor group to the Au electrode and modified at the 3′-terminus with exTTF as a redox reporter. (B) We fabricated E-AB sensors from exTTF-modified DNA probes and implanted the sensors in the bladder of a living rat, which undergoes pH changes. This sensor platform achieved quantitative, real-time monitoring of analytes directly in vivo.

Fig. 2. The synthetic route and characterization of the exTTF-aptamer conjugate. (A) Synthesis scheme of exTTF-COOH and the conjugates (Scheme S1†). (B) RP-HPLC trace of the amine-modified cocaine aptamer (cocaine-apt-NH₂) and kanamycin aptamer (kanamycin-apt-NH₂), and their conjugates. (C) Mass spectrometry analysis of both conjugates confirms the success of the modification reaction between the DNA aptamer and exTTF derivative.

Fig. 3. E-AB sensors employing the exTTF redox reporter exhibited pH-independent performances in McIlvaine buffer under a wide range of pH conditions. (A) and (B) show the voltammograms recorded from MB- and exTTF-based kanamycin-detecting sensors under pH-variable conditions. (C) and (D) Both cocaine- and kanamycin-detecting sensors exhibited excellent stability under high-frequency measurement conditions (>1200 scans) over a duration of 12 hours. (E) and (F) When challenging our sensors with their targets under various pH conditions, we observed a pH-independent signal change. The error bars here and in the following figures represent the standard deviation of at least three independently fabricated sensors.

Fig. 4. The pH-independent performance likewise holds for exTTF-based sensors deployed in biological systems, greatly improved in comparison to that observed from MB-based sensors. Here we take kanamycin-detecting sensors as an example. (A) The titration plots of MB-based sensors under various pH conditions. (B) These sensors exhibited a variation in K_D ranging from 10 μM to 200 μM, over the pH range from 4.5 to 8.5. (C) and (D) In contrast, exTTF-based sensors exhibited pH-independent target-dose responses, with similar results achieved for cocaine-detecting sensors (Fig. S18†). (E) We then deployed exTTF-based sensors in a real urine sample with a series of target concentrations, achieving a target-dose response curve in good accordance with those ranges determined in artificial biological samples. (F) By applying this titration calibration plot to estimate the recovery rate of kanamycin in real urine samples from three healthy individuals, we achieved a good recovery rate of 85–90% over a 50-fold concentration span (the solid line represents 100% recovery, and the dashed lines represent ±20% accuracy bands).

Fig. 5. The pH-independent E-AB sensor supports real-time, continuous measurements in vivo. (A) When deploying our sensor in the bladder of a living rat (pH: 5.55), we achieved a micromolar precision and observed an excretion of kanamycin within the duration of half an hour. (B) When deploying our sensors in the bladder of a living rat with pH adjustment, we observed negligible difference in its performance, indicating that our sensors are pH independent. Kanamycin-detecting sensors exhibited a consistent pH-independent performance, with results in good accordance when deploying them in several individual rats (see the raw current and converted concentration information in Fig. S25†). The pH values indicated in B panel were immediately obtained from urine samples collected from the bladder during the in vivo measurements using a pH meter.