Helixohm

Helixohm is stable for years at room temperature

PCR reaction

Electrochemical PCR experiments were performed in 1X LEOx buffer for PCR, 1X LEOx (0.5µM), 200 µM of each dNTPs, 1.25 U HotStarTaq Plus DNA polymerase (0.025 U.µL^-1), 200 nM of each primer, and different amount of DNA target. Real-time electrochemical monitoring of PCR reactions was performed in a 50 µL electrochemical cell under controlled temperature conditions. The PCR reactions were run at 93°C for 15 min, followed by 50 cycles at 93°C for 90 s, 60°C for 150 s, and 72°C for 30 s. The electrochemical wells was scanned by square wave voltammetry (SWV) at the end of the elongation step (i.e., at 72°C) of each PCR cycle at a maximal sampling rate of 0.3 Hz. The SWV parameters were 50 Hz frequency, 40 mV half wave amplitude and 1 mV potential step, while the scanned potential windows were from 400 to 900 mV (vs. silver pseudo reference electrode). The SWV peak current of each scan was baseline-corrected and integrated, and the peak charge reported as a function of the PCR cycle number. The resulting amplification curves were corrected from the signal drift observed in the absence of specific signal decrease by, for example, subtracting a least-square fitted line across the first cycles and then offsetting the data sets to align the value of these corrected cycles. Melt curve analysis was run immediately after the PCR amplification, starting with a denaturation step at 93°C for 90 s followed then by a cooling step at 50°C for 5 min, and next 1°C.min^-1 linear ramp of temperature (from 60°C to 92°C) during which SWV scans were recorded every 0.5°C (using the same SWV parameters than during PCR). The resulting SWV peak charges were reported as a function of the temperature.

LAMP reaction

Electrochemical LAMP experiments were performed in 1X LEOx buffer for LAMP, 1X LEOx (0.5µM), 400 µM of each dNTPs, 1.6 μM of each of the BIP and FIP primers, 0.8 μM of each of the LB and LF primers, 0.4 μM of each of the B3 and F3 primers, 0.32 U of Bst 2 WarmStart DNA polymerase, and different amount of DNA target. Real-time electrochemical monitoring of LAMP reactions was performed in a 50 µL electrochemical cell under controlled temperature conditions. LAMP reaction mixes were maintained at 65 °C while the square wave voltammetric (SWV) responses of cell were recorded at a suitable sampling rate (every 40 s) to get a sufficiently well-resolved evolution of the signal. Melt curve analysis was performed immediately following each LAMP reaction by applying a linear ramp of temperature to electrochemical cell, starting from a cooling step at 50 °C for 1 min followed by a continuous rise of temperature up to 91 °C at a ramp rate of a 1.1 °C min. During the continuous increase of temperature, SWV signals were acquired every 13 s. Each SWV peak was automatically baseline- corrected and integrated, and the resulting peak charge then reported as a function of either time or temperature.

General electrochemical protocol

Any potentiostat suitable for voltammetry (range nA to µA) and ideally for square wave voltammetry is appropriate. It is recommended to use screen-printed carbon electrode (see list above) to obtain (i) the best signal-to-noise ratio and (ii) to work at low volumes (<50µL). It is recommended to use the LEOx probe in a concentration between 0.5µM and 5µM in aqueous solution with an electrolytic support (KCl, Phosphate, Trizma buffer). The LEOx probe is naturally in oxidized state with a standard potential of 0.65 V/SCE.