Single-Molecule Bioelectronic Sensors with AI-Aided Data Analysis: Convergence and Challenges.

Single-molecule bioelectronic sensing, a groundbreaking domain in biological research, has revolutionized our understanding of molecules by revealing deep insights into fundamental biological processes. The advent of emergent technologies, such as nanogapped electrodes and nanopores, has greatly enhanced this field, providing exceptional sensitivity, resolution, and integration capabilities. However, challenges persist, such as complex data sets with high noise levels and stochastic molecular dynamics. Artificial intelligence (AI) has stepped in to address these issues with its powerful data processing capabilities. AI algorithms effectively extract meaningful features, detect subtle changes, improve signal-to-noise ratios, and uncover hidden patterns in massive data. This review explores the synergy between AI and single-molecule bioelectronic sensing, focusing on how AI enhances signal processing and data analysis to boost accuracy and reliability. We also discuss current limitations and future directions for integrating AI, highlighting its potential to advance biological research and technological innovation.

The J bs-5YP peptide can alleviate dementia in senile mice by restoring the transcription of Slc40a1 to secrete the excessive iron from brain.

INTRODUCTION: With age and ATP decrease in the body, the transcription factors hypophosphorylation weakens the transcription of Slc40a1 and hinders the expression of the iron discharger ferroportin. This may lead to iron accumulation in the brain and the catalysis of free radicals that damage cerebral neurons and eventually lead to Alzheimer's disease (AD). OBJECTIVES: To prevent AD caused by brain iron excretion disorders and reveal the mechanism of J bs-5YP peptide restoring ferroportin. METHODS: We prepared J bs-YP peptide and administered it to the senile mice with dementia. Then, the intelligence of the mice was tested using a Morris Water Maze. The ATP content in the body was detected using the ATP hydrophysis and Phosphate precipitation method. The activation of Slc40a1 transcription was assayed with ATAC seq and the ferroportin, as well as the phosphorylation levels of Ets1 in brain were detected by Western Blot. RESULTS: The phosphorylation level of Ets1in brain was enhanced, and subsequently, the transcription of Slc40a1 was activated and ferroportin was increased in the brain, the levels of iron and free radicals were reduced, with the neurons protection, and the dementia was ultimately alleviated in the senile mice. CONCLUSION: J bs-5YP can recover the expression of ferroportin to excrete excessive iron in the brain of senile mice with dementia by enhancing the transcription of Slc40a1 via phosphorylating Ets1, revealing the potential of J bs-5YP as a drug to alleviate senile dementia.

Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes.

We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.

Fabrication of electron tunneling probes for measuring single-protein conductance.

Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein-electrode contact. We use a biotinylated thiol modification, in which a biotin-streptavidin-biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.

Single-molecular protein-based bioelectronics via electronic transport: fundamentals, devices and applications.

Biomolecular electronics is a rapidly growing multidisciplinary field that combines biology, nanoscience, and engineering to bridge the two important fields of life sciences and molecular electronics. Proteins are remarkable for their ability to recognize molecules and transport electrons, making the integration of proteins into electronic devices a long sought-after goal and leading to the emergence of the field of protein-based bioelectronics, also known as proteotronics. This field seeks to design and create new biomolecular electronic platforms that allow for the understanding and manipulation of protein-mediated electronic charge transport and related functional applications. In recent decades, there have been numerous reports on protein-based bioelectronics using a variety of nano-gapped electrical devices and techniques at the single molecular level, which are not achievable with conventional ensemble approaches. This review focuses on recent advances in physical electron transport mechanisms, device fabrication methodologies, and various applications in protein-based bioelectronics. We discuss the most recent progress of the single or few protein-bridged electrical junction fabrication strategies, summarise the work on fundamental and functional applications of protein bioelectronics that enable high and dynamic electron transport, and highlight future perspectives and challenges that still need to be addressed. We believe that this specific review will stimulate the interdisciplinary research of topics related to protein-related bioelectronics, and open up new possibilities for single-molecule biophysics and biomedicine.

Single-Molecule Surface-Enhanced Raman Spectroscopy.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) has the potential to detect single molecules in a non-invasive, label-free manner with high-throughput. SM-SERS can detect chemical information of single molecules without statistical averaging and has wide application in chemical analysis, nanoelectronics, biochemical sensing, etc. Recently, a series of unprecedented advances have been realized in science and application by SM-SERS, which has attracted the interest of various fields. In this review, we first elucidate the key concepts of SM-SERS, including enhancement factor (EF), spectral fluctuation, and experimental evidence of single-molecule events. Next, we systematically discuss advanced implementations of SM-SERS, including substrates with ultra-high EF and reproducibility, strategies to improve the probability of molecules being localized in hotspots, and nonmetallic and hybrid substrates. Then, several examples for the application of SM-SERS are proposed, including catalysis, nanoelectronics, and sensing. Finally, we summarize the challenges and future of SM-SERS. We hope this literature review will inspire the interest of researchers in more fields.

Sub-5 nm nanogap electrodes towards single-molecular biosensing.

Nanogap electrodes (NGEs) with sub-5 nm gap has been widely used in single-molecule sensing and sequencing, with the characteristics of label-free, high sensitivity, rapid detection and low-cost. However, the fabrication of sub-5 nm gap electrodes with high controllability and reproducibility still remains a great challenge that impedes the experimental research and the commercialization of the nanogap device. Here, we review the common currently used fabrication methods of nanogap electrodes, such as gap narrowing deposition, mechanical controllable break junctions and the fabrication methods combined with nanopore or nanochannel. We then highlight the typical applications of nanogap electrodes in biological/chemical sensing fields, including single molecule recognition, single molecule sequencing and chemical kinetics analysis. Finally, the challenges of nanogap electrodes in single molecule sensing/sequencing are outlined and the future directions for sensing perspectives are suggested.

Measuring conductance switching in single proteins using quantum tunneling.

Interpreting the electrical signatures of single proteins in electronic junctions has facilitated a better understanding of the intrinsic properties of proteins that are fundamental to chemical and biological processes. Often, this information is not accessible using ensemble and even single-molecule approaches. In addition, the fabrication of nanoscale single-protein junctions remains challenging as they often require sophisticated methods. We report on the fabrication of tunneling probes, direct measurement, and active control (switching) of single-protein conductance with an external field in solution. The probes allowed us to bridge a single streptavidin molecule to two independently addressable, biotin-terminated electrodes and measure single-protein tunneling response over long periods. We show that charge transport through the protein has multiple conductive pathways that depend on the magnitude of the applied bias. These findings open the door for the reliable fabrication of protein-based junctions and can enable their use in future protein-embedded bioelectronics applications.

DNA-encoded bimetallic Au-Pt dumbbell nanozyme for high-performance detection and eradication of Escherichia coli O157:H7.

Infectious Escherichia coli O157:H7 threatens the health of millions people each year. Thus, it is important to establish a simple and sensitive method for bacterial detection and eradication. Herein, a DNA-programming strategy is explored to synthesize anisotropic dumbbell-like Au-Pt nanoparticles with excellent catalytic and anti-bacterial activities, which were applied in the simultaneous detection and eradication of pathogenic bacteria. The DNA sequence-dependent growth of bimetallic nanoparticles is firstly studied and polyT20 has the tendency to form dumbbell-like Au-Pt bimetallic structures based on gold nanorods seeds. PolyA20 and polyC20 can also form similar structures but only at much lower DNA concentrations, which can be explained by their much higher affinity to the metal surfaces than T20. The as-prepared nanoparticles exhibit high nanozyme catalytic activity resulting from the synergistic effect of Au and Pt. Under light irradiation, the Au-Pt nanoparticles show high photothermal conversion efficiency and enhanced catalytic activity, which can be applied for the eradication and detection of E. coli O157:H7 with a robust efficacy (95%) in 5 min and provides excellent linear detection (10-107 CFU/mL), with a detection limit of 2 CFU/mL. This study offered new insights into DNA-directed synthesis of nanomaterials with excellent biosensing and antibiotic applications.

Combined quantum tunnelling and dielectrophoretic trapping for molecular analysis at ultra-low analyte concentrations.

Quantum tunnelling offers a unique opportunity to study nanoscale objects with atomic resolution using electrical readout. However, practical implementation is impeded by the lack of simple, stable probes, that are required for successful operation. Existing platforms offer low throughput and operate in a limited range of analyte concentrations, as there is no active control to transport molecules to the sensor. We report on a standalone tunnelling probe based on double-barrelled capillary nanoelectrodes that do not require a conductive substrate to operate unlike other techniques, such as scanning tunnelling microscopy. These probes can be used to efficiently operate in solution environments and detect single molecules, including mononucleotides, oligonucleotides, and proteins. The probes are simple to fabricate, exhibit remarkable stability, and can be combined with dielectrophoretic trapping, enabling active analyte transport to the tunnelling sensor. The latter allows for up to 5-orders of magnitude increase in event detection rates and sub-femtomolar sensitivity.

Instrument response effects on the retrieval of oceanic lidar.

Seawater properties can be retrieved from oceanic lidar returns. However, the actual returns include the ideal returns convolved by the instrument response, which inevitably introduces retrieval error. In this paper, instrument response effects on the retrieval of oceanic lidar are analyzed from different aspects. The results demonstrate that the retrieval of the lidar attenuation coefficient near the water surface is affected by the instrument response in homogeneous water. Considering the ratio of the signal distortion region (relative error of attenuation >10%) to the maximum detection depth (three dynamic ranges) is less than 20%, the pulse width of the instrument response should be less than 10-0.042(Kd)-2+0.709(Kd)-1+1.136ns. In addition, an average relative error of 55% will be introduced to the retrieval of phytoplankton layer thickness in the stratified water, which can be reduced to 6% after correcting for the influence of the instrument response. However, a relative error greater than 20% still exists when the instrument response length is two times larger than the layer thickness. These conclusions provide guidelines to a future design of oceanic lidar.

Ultrasensitive monitoring of DNA damage associated with free radicals exposure using dynamic carbon nanotubes bridged interdigitated electrode array.

There are currently increasingly concerns over DNA damage related to free radicals due to their vital roles in human health, especially high-performance detection method. Herein, we report an ultra- sensitive monitoring of DNA damage associated with free radicals exposure using interdigitated electrode (IDE) array for the first time. The proposed IDE array was equipped with DNA-wrapped carbon nanotube-based bridges, which utilized the DNA damage mechanism due to the free radicals' attack and the efficient electrical detection nature of the interdigitated electrode. Experiments have been performed, and the results showed the device's capability for detecting DNA damage induced by multiple free radicals generated from different sources, including the Fenton reaction, UV radiation and cigarette smoke, showing the promising ability for DNA damage detection. In addition, the carbon nanotubes bridge-based interdigitated electrode sensor enabled different levels of sensing of DNA damage with great sensitivity and a wide detection range. It was illustrated that the ultrasensitive detection of free radicals generated from ultraviolet radiation (15 min - 125 min), cigarette smoke tar (1 µg/mL to 10 µg/mL) and Fenton reaction under different concentration of H2O2 (2.5 pM - 100 pM), have been detected successfully. Typically, the IDE array supports further performance improvement for the electrochemical detection in an ultrasensitive and high throughput route.

Gold Nanobones Enhanced Ultrasensitive Surface-Enhanced Raman Scattering Aptasensor for Detecting Escherichia coli O157:H7.

Sensitive, robust, and highly specific detection of Escherichia coli O157:H7, one of the most hazardous foodborne pathogens and the cause of numerous diseases, is needed to ensure public health. Herein, a one-pot step method is reported for the preparation of multifunctional gold nanobones (NBs) (GNRApt-1+RhB) from gold nanorods (GNRs) comediated by an aptamer (Apt-1) and the signal molecule rhodamine B (RhB) for surface-enhanced Raman scattering detection of E. coli O157:H7. The characterized result showed that Apt-1 and RhB were embedded in the gold NBs, and then, this combination exhibited good recognition, excellent stability, and significant Raman signal intensity enhancement. The Raman enhancement derived from a strong electromagnetic field distribution with the locations at the apex of both ends of the GNRApt-1+RhB and the signal stability was because of the firm embedment of Apt-1 (poly A20 + E. coli O157:H7 aptamers) and RhB on the surface of the GNRApt-1+RhB. Optimization experiments established that surface-enhanced Raman-scattered RhB absorption at 1350 cm-1 had a strong linear relationship (y = 180.30x - 61.49; R2 = 0.9982) with E. coli O157:H7 concentration over the range of 10-10,000 cfu/mL with a limit of detection of 3 cfu/mL. This novel aptasensor sensitively detects E. coli O157:H7 and has great promise for food pathogenic bacteria detection.

Plasmon-activated nanozymes with enhanced catalytic activity by near-infrared light irradiation.

Nanozymes have attracted extensive attention due to their great potential as alternatives to natural enzymes. Optical control as an external stimulus has become the most attractive method because of its high spatial and temporal resolution. Under the action of excitation light, free electrons on the surface of gold nanorods (GNRs) will collectively oscillate, which is called localized surface plasmon resonance (LSPR). This unique LSPR effect is promising in the application of plasmon-accelerated enzyme-like catalytic reactions. Pt-tipped gold nanorod-based nanozymes (Pt-GNRs) were synthesized by the modification of Pt nanoclusters onto the tips of GNRs. The as-prepared Pt-GNRs exhibited excellent enzyme-like catalytic activity toward hydrogen peroxide. Furthermore, it was found that the enzyme-like catalytic activity of Pt-GNRs could be notably enhanced using near-infrared (NIR) light irradiation, because of the photothermal effect and hot electron effect produced by the LSPR of GNRs. Finally, the catalytic activity and cytotoxicity of Pt-GNRs were evaluated in 4T1 cells, which further demonstrated that the Pt-GNR-based nanozymes possess great potential in cancer treatment.

Janus γ-Fe2O3/SiO2-based nanotheranostics for dual-modal imaging and enhanced synergistic cancer starvation/chemodynamic therapy.

Multimodal cancer synergistic therapy exhibited remarkable advantages over monotherapy in producing an improved therapeutic efficacy. In this work, Janus-type γ-Fe2O3/SiO2 nanoparticles (JFSNs) are conjugated with glucose oxidase (GOx) for synergistic cancer starvation/chemodynamic therapy. The γ-Fe2O3 hemisphere of JFSNs can perform photoacoustic/T2 magnetic resonance dual-modal imaging of tumors. GOx on the surface of JFSNs catalyzes the decomposition of glucose and produces H2O2 for cancer starvation therapy. Subsequently, the γ-Fe2O3 hemisphere catalyzes the disproportionation of H2O2 to generate highly reactive hydroxyl radicals in an acidic tumor microenvironment. The close distance between GOx and JFSNs ensures adequate contact between the γ-Fe2O3 hemisphere and its substrate H2O2, thus enhancing the catalytic efficiency. This synergy of glucose depletion, biotoxic H2O2 and hydroxyl radicals significantly suppresses 4T1 mammary tumor growth with minimal adverse effects.

Auxetic Thermoresponsive Nanoplasmonic Optical Switch.

Development and use of metamaterials have been gaining prominence in large part due to the possibility of creating platforms with "disruptive" and unique optical properties. However, to date, the majority of such systems produced using micro or nanotechnology are static and can only perform certain target functions. Next-generation multifunctional smart optical metamaterials are expected to have tunable elements with the possibility of controlling the optical properties in real time via variation in parameters such as pressure, mechanical stress, and voltage or through nonlinear optical effects. Here, we address this challenge by developing a thermally controlled optical switch, based on the self-assembly of poly( N-isopropylacrylamide)-functionalized gold nanoparticles on a planar macroscale gold substrate. We show that such meta-surfaces can be tuned to exhibit substantial changes in the optical properties in terms of both wavelength and intensity, through the temperature-controlled variation of the interparticle distance within the nanoparticle monolayer as well as its separation from the substrate. This change is based on temperature-induced auxetic expansion and contraction of the functional ligands. Such a system has potential for numerous applications, ranging from thermal sensors to regulated light harnessing.

Plasmon-Based Colorimetric Nanosensors for Ultrasensitive Molecular Diagnostics.

Colorimetric detection of target analytes with high specificity and sensitivity is of fundamental importance to clinical and personalized point-of-care diagnostics. Because of their extraordinary optical properties, plasmonic nanomaterials have been introduced into colorimetric sensing systems, which provide significantly improved sensitivity in various biosensing applications. Here we review the recent progress on these plasmonic nanoparticles-based colorimetric nanosensors for ultrasensitive molecular diagnostics. According to their different colorimetric signal generation mechanisms, these plasmonic nanosensors are classified into two categories: (1) interparticle distance-dependent colorimetric assay based on target-induced forming cross-linking assembly/aggregate of plasmonic nanoparticles; and (2) size/morphology-dependent colorimetric assay by target-controlled growth/etching of the plasmonic nanoparticles. The sensing fundamentals and cutting-edge applications will be provided for each of them, particularly focusing on signal generation and/or amplification mechanisms that realize ultrasensitive molecular detection. Finally, we also discuss the challenge and give our future perspective in this emerging field.

Near-infrared photoluminescence biosensing platform with gold nanorods-over-gallium arsenide nanohorn array.

The near-infrared (NIR) optical detection of biomolecules with high sensitivity and reliability have been expected, however, it is still a challenge. In this work, we present a gold nanorods (AuNRs)-over-gallium arsenide nanohorn-like array (GaAs NHA) system that can be used for the ultrasensitive and specific NIR photoluminescence (PL) detection of DNA and proteins. The fabrication of GaAs NHA involved the technique of colloidal lithography and inductively coupled plasma dry etching, yielding large-area and well-defined nanostructural array, and exhibiting an improved PL emission compared to the planar GaAs substrate. Importantly, we found that the DNA-bridged AuNRs attachment on NHA could further improve the PL intensity from GaAs, and thereby provide the basis for the NIR optical sensing of biological analytes. We demonstrated that DNA and thrombin could be sensitively and specifically detected, with the detection limit of 1 pM for target DNA and 10 pM for thrombin. Such ultrasensitive NIR optical platform can extend to the detection of other biomarkers and is promising for clinical diagnostics.

DNAzyme-integrated plasmonic nanosensor for bacterial sample-to-answer detection.

Pathogenic bacteria pose a serious threat to public safety and health, and cause significant losses in the global economy and in lives. The current golden standard, culture-based method, for bacterial detection is often costly, laborious, and time-consuming (even weeks). Thus, there is an urgent need to develop rapid, reliable and easy-to-use approaches for bacterial detection. Herein, we present a new detection strategy termed as 'DNAzyme-Integrated Plasmonic Nanosensor' (DIPNs) that can selectively detect target bacteria in a simple, inexpensive and culture-free process, which combines real-time DNAzyme-based sensor and enzyme-responsive nanoplasmonic biosensor system. The DIPNs platform takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and enzyme-responsive plasmonic nanoparticles' localized surface plasmon resonance (LSPR) as the signal readout. Using Escherichia coli (E. coli) as a model analyte, we demonstrated that the DIPNs system can provide the fast and low concentration (down to 50 bacteria per mL) quantification of E. coli even in the complex fluids (e.g., milk, serum, juice) with naked eyes, which would be expected for the detection of bacterial pathogens in the simple, low-cost and on-site manner.

Flexible and Transparent Surface-Enhanced Raman Scattering (SERS)-Active Metafilm for Visualizing Trace Molecules via Raman Spectral Mapping.

Raman spectral mapping is a powerful tool for directly visualizing the composition, structure, and distribution of molecules on any surface of interest. However, one major limitation of Raman mapping is its overlong imaging time caused by the intrinsic weak Raman signal. Here, we developed a fast Raman imaging approach based on a flexible and transparent surface-enhanced Raman scattering (SERS)-active metafilm. This particular SERS substrate can be conformably attached to a sample surface to enhance the Raman signal of analytes and the good optical transparency allow excitation and collection of signal from the backside of the substrate. Therefore, by simply attaching it to the surface of interest, a fast Raman imaging can be realized. We noticed that the imaging speed can be increased by several orders of magnitude, compared to a conventional Raman mapping approach. Importantly, the proposed approach required little or no sample preparation and exhibited good generalizability that can be performed perfectly on different surfaces. It is believed that the proposed methodology will provide new trends for chemical imaging using Raman microscopy.