Full counting statistics of single electron transfer events in electrochemical processes as a new approach for ultimate biosensing – SIBI

The partner LEM assembled on ultra-thin gold electrodes a first model redox DNA system, made of short strands of DNA with ferrocene (Fc) head, of dT20 and dT50 sequences. He measured the electrochemical response, at the ensemble scale by fast cyclic voltametry, and also at the scale of a few individual strands by atomic force electrochemical microscopy (AFM-SECM). The modulation of the electrochemical response by hybridization of the strands anchored by complementary DNA chains in solution has been demonstrated and studied. These experimental data are now compared to theoretical predictions from models and numerical simulations developed in parallel by LIMMS. This study should allow a thorough understanding of the dynamic response of the Fc-DNA chains anchored on electrode. Beyond its own interest, this result constitutes a prerequisite for the continuation of the project.
For the simulation part carried out at LIMMS, a software (Q-Biol) has been developed. It allows, by combining molecular dynamics with a realistic large grain model and DNA-dependent sequence validated by numerous experiments, and quantum charge transport, to numerically simulate electron counting experiments. This allows not only to explore electron counting approaches for electrochemistry, but also to provide an important support for the experiments performed at LEM.
For the fabrication part carried out at LIMMS, several wafers of gold nanodots of different diameters (around 10 nm), thickness (from 4 to 10 nm) have been fabricated. These different parameters will facilitate the experimental conditions for the Mt-AFM-SECM measurements and also for the assembly of single molecules per pad. Considering the cost of e-beam for writing large surfaces and object sizes, this required a complete study to optimize the use of the machines for «high speed« writing.
A method to suppress part of the noise in the nanotransistors, very important for the project, has been validated and published.
3 wafers of nanotransistors (20 nm) were fabricated. Then, tests were performed to successfully align 20 nm nanodots precisely on top of nanotransistors of equivalent size. The yield is quite low for the moment, but sufficient.
The nanotransistors have been tested at LIMMS. They are operational with an efficiency in line with expectations (50%), taking into account the variation of parameters to optimize the chances of detection of single elementary charges. They have also been tested in fluidic environment (with/without gold nanodots). We encountered a difficulty of leakage through the contacts, the chips being enlarged for AFM measurements. New technological steps are being tested to solve this problem.

* First studies in fast cyclic voltametry (100 000 V/s) and in atomic force electrochemical microscopy, of the effect of the chain length on the response of DNA strands with redox head anchored on electrode surfaces.
* Approach to suppress noise in nanotransistor sensors by exploiting a stochastic resonance effect (Sci.Rep. 2020)
* Application of the theory of «full counting statistics of single-electron transport« historically developed by the mesoscopic physics community to electrochemistry. Associated experiments (mean value and noise) have been performed. A publication is in progress.

From the experimental point of view, the assembly and electrochemical interrogation of Fc-DNA systems, as well as the constraints related to their handling and storage are now mastered by the LEM. The effect of the crucial parameter of redox DNA strand length on the dynamics of electron transport can now be studied in detail by fast cyclic voltammetry and AFM-SECM. The results will be compared to the predictions of the Q-Biol software developed in parallel at LIMMS. This knowledge will then be transposed to the decoration by Fc-DNA chains of gold nanoplots, organized in arrays on silicon surface, and their electrochemical interrogation. The LEM has now these surfaces, recently fabricated by the LIMMS.

Communications (conferences): I. Madrid et al. Jahn-Teller Distortion in confined DNA (oral), Solid-state devices and Materials conference.

International journals: Y. Kutovyii et al, Noise suppression beyond the thermal limit with nanotransistor biosensors Sci.Rep. 10, 12678 (2020)

The first and main objective of the SIBI project, is to develop a fully original “all-electrical” sensing scheme enabling the detection and counting of single electron transfer events, a so far elusive goal in electrochemistry. The innovative methodology proposed here exploits the few millivolts voltage shift induced by a single electron stored across an attofarad range nanocapacitor. We intend to show that this electron counting technology could be the basis of ultimate sensing in bioelectrochemistry. We will therefore implement it in a configuration mimicking an electrochemical conformational DNA sensor (E-DNA). A redox-labelled single stranded DNA chain, used as a capturing (probe) strand, will be end-attached to a 10 nm size gold nanodot, fabricated by e-beam lithography, and aligned on top of a nanocapacitor. For proof of concept experiments, the nanoelectrode-tip of an atomic force electrochemical microscope, operated in molecular touching mode (Mt/AFM-SECM), will be used to address electrochemically the redox-reporter of the DNA probe strand. Discrete charging steps of the nanocapacitor, corresponding to the cycling motions of the redox label being oxidized at the tip and reduced at the nanodot, will be detected using state of the art silicon transistor nanotechnology that provides stability and elementary charge sensitivity. Observation of these steps will be the first ever demonstration of single electron counting in electrochemistry. Beyond this unprecedented detection, measurement of the frequency of the charging steps will yield unique access to quantitative information regarding the conformational dynamics of the end-anchored DNA chain. These information will be deciphered by designing cutting edge numerical simulations of the dynamics and electrochemical behaviour of end-grafted redox DNA chains. More precisely, as the second objective of this project, we propose to mimic theoretically the full biomolecular system using a realistic molecular dynamics model for DNA end-attached to electrode surfaces. Quantum transport master equations based on the Quantum Marcus theory that accounts for redox-molecule/electrode distance dependence of the electronic coupling and reorganization energy will be computed based on the position of the redox label to compute the probability of transferring an electron every picosecond. The experimental and theoretical results will be directly compared, and the numerical code adjusted. In addition, as the recent progress in machine learning for signal processing are perfectly adapted for the signal to be measured, we propose to use recurrent neural networks (RNN), to optimize the analysis of the single-electron transfer traces, in the perspective of analysing a large amount of data from sensor arrays. We could for example envision the automatic extraction of the sizes of a population of DNA strands, with single-molecule resolution Beyond its fundamental interest, our modelling approach will help gaining a quantitative understanding in the transduction mechanism of E-DNA sensors, which is still lacking to date.
As a last goal of the project, the experimental and theoretical knowledge acquired will be invested in developing an actual conformational electrochemical E-DNA nano-biosensor, capable of single analyte molecule sensitivity, thanks to single electron counting and miniaturization. A sensing platform, consisting in an array of individually addressable nanocapacitors, forming an open nanogap with patterned nanoelectrodes, will be developed. The sensing chip will be employed to assay the Epithelial cell adhesion molecule (EpCAM), a prognostic marker in cancer, by making use of an EpCAM-specific redox-labeled aptamer as the capture DNA probe. We thus expect to demonstrate actual single molecule sensing, made possible by electron counting, for an assay of actual biological relevance.