Heating promoted super sensitive electrochemical detection of p53 gene based on alkaline phosphatase and nicking endonuclease Nt.BstNBI-assisted target recycling amplification strategy at heated gold disk electrode.

An ultrasensitive electrochemical biosensor for detecting p53 gene was fabricated based on heated gold disk electrode coupling with endonuclease Nt.BstNBI-assisted target recycle amplification and alkaline phosphatase (ALP)-based electrocatalytic signal amplification. For biosensor assembling, biotinylated ssDNA capture probes were first immobilized on heated Au disk electrode (HAuDE), then combined with streptavidin-alkaline phosphatase (SA-ALP) by biotin-SA interaction. ALP could catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA). While AA could induce the redox cycling to generate electrocatalytic oxidation current in the presence of ferrocene methanol (FcM). When capture probes hybridized with p53, Nt.BstNBI would recognize and cleave the duplexes and p53 was released for recycling. Meanwhile, the biotin group dropt from the electrode surface and subsequently SA-ALP could not adhere to the electrode. The signal difference before and after cleavage was proportional to the p53 gene concentration. Furthermore, with electrode temperature elevated, the Nt.BstNBI and ALP activities could be increased, greatly improving the sensitivity and efficiency for p53 detection. A detection limit of 9.5 × 10-17 M could be obtained (S/N = 3) with an electrode temperature of 40 °C, ca. four magnitudes lower than that at 25 °C.

Temperature-Controllable Electrodes with a One-Parameter Calibration.

Electrically heated electrodes have been applied for various chemical and biological sensors. However, previous electrically heated electrodes, including microwires and microdiscs, are usually small and often suffer from the requirement of frequent calibrations of the electrode surface temperature ( Ts) at different environment temperatures. Here, we fabricate a temperature-controllable disk electrode (TCDE) with a conventional size (3-5 mm in diameter). A one-parameter temperature calibration is proposed using a temperature transfer coefficient α and a structural model ( Ts = Te + α ( Th - Te)) to estimate Ts ( Th and Te are the temperature of the heating element and environment, respectively). The value of α is unique for a TCDE and mainly dependent on the structure and materials of the electrodes and the solution in nature. Once α is experimentally determined, Ts can be calibrated and found to be applicable to wide fluctuations in room temperature (15.0-33.0 °C) with errors below 1.5% for three types of disk electrodes (gold, glassy carbon, and platinum). The required Ts can be obtained by just setting Th without thermal characterization between the heating power and Ts. A simple relationship for exploring the dependence of α on the height ( H) and radius ( R) of the electrode materials and other constants ( a, b, c, and R0), α = 1 - c - aH - b ( R - R0)2, is revealed by numerical simulations (COMSOL). The impact of the radii of both the insulating materials of the electrode and the electrochemical cells on Ts is also considered. The effect of the solution thermal conductivity on α is studied. TCDEs are expected to be used as a sensor platform with enhanced performance.

Heating enhanced sensitive and selective electrochemical detection of Hg2+ based on T-Hg2+-T structure and exonuclease III-assisted target recycling amplification strategy at heated gold disk electrode.

A sensitive and selective electrochemical Hg2+ sensor was developed based on T-Hg2+-T structure and exonuclease (Exo) III -assisted target recycling amplification at heated gold disk electrode (HAuDE). First, a DNA signal probe P1 was for the first time designed and labeled with ferrocene (Fc) near the attached SH-5'-end, so as to shorten the distance between Fc and the electrode and enhance the initial current of Fc compared with that labeled at the 3'-end far from the electrode. Then the signal amplification was achieved by Exo III-assisted Hg2+ recycling. Briefly, the P1 was complementary to the assistant DNA P2 except the T-T mismatches. In the presence of Hg2+, the P1 self-assembled on the HAuDE could hybridize with P2 and form DNA duplex with blunt end at the 3'- terminus, triggering Exo III to stepwise digest mononucleotides from the 3'-terminus of P1, ultimately liberating Hg2+ and P2, which could be "recycled", resulting in the digestion of a large amount of P1 and significantly decrease the amount of Fc. The electrochemical signal difference before and after digestion was proportional to the Hg2+ concentration. Furthermore, during the digestion period, the Exo III activity could be significantly increased by elevating the electrode temperature, great improving the sensitivity and efficiency for Hg2+ detection. A detection limit of 6.2 pM (S/N = 3) could be obtained with an electrode temperature of 40°C during 60min digestion period, which was lower ca. two magnitudes than that at 0°C and one magnitude than that at 25°C.