Active Probe Research Articles

Synthesis of AIEE active triazine based new fluorescent and colorimetric probes: A reversible mechanochromism and sequential detection of picric acid and ciprofloxacin

Two new triazine based multifunctional fluorescent probes T1 and T2 were synthesized that displayed excellent aggregation induced emission enhancement (AIEE) and reversible mechanochromic character. The AIEE active probes were used for selective fluorescence detection of picric acid (PA) based on fluorescence quenching response. The sensitive detection of PA was achieved through electron and charge transfer between them resulting in the formation of the probe–PA complex. The limits of detection of T1 and T2 for PA were calculated as 1.94 and 2.24 nM, respectively. Subsequently, probe–PA complexes acted as fluorescence enhancement base probes for sequential detection of an antibacterial drug, ciprofloxacin (CIP). The sensitivity (detection limit) of the probe–PA complexes for the recognition of ciprofloxacin was determined as 3.16 and 4.46 nM. Hence, a new strategy was successfully developed for the sensing of ciprofloxacin (CIP). The proposed mechanism for the sensing of PA and ciprofloxacin was evaluated through 1H NMR titration, DLS, and DFT analyses. Meanwhile, probes manifested colorimetric identification of PA and CIP. Advantageously, probe–PA complexes were applied in urine samples for the quantification of ciprofloxacin. Moreover, application in real water samples revealed that the spiked recoveries of PA were attained as > 90%. To the best of our knowledge, it is the first report where triazine based fluorescent probes showed excellent simultaneous responses towards force stimuli (mechanochromism), picric acid, and ciprofloxacin.

Esterase-Activated Precipitating Strategy to Achieve Highly Specific Detection and Long-Term Imaging of Calcium Ions by Aggregation-Induced Phosphorescence Probe.

Spatial and temporal monitoring of bioactive targets such as calcium ions is vitally significant for their essential roles in physiological and biochemical functions. Herein, we proposed an esterase-activated precipitating strategy to achieve highly specific identification and long-term bioimaging of calcium ions via lighting up the calcium ions by precipitation using a water-soluble aggregation-induced phosphorescence (AIP) probe. The designed probe CaP2 has an AIP behavior and can be efficiently aggregated by calcium ions through the coupling coordination of carboxylic acid and cyanide groups, which enables it to light up Ca2+ by precipitating-triggered phosphorescence. Four hydrophilic groups of tetraethylene glycol were introduced to endow the resulting probe CaP3 with extraordinary water solubility as well as excellent cellular penetration. Only when the probe CaP3 penetrates inside the live cells the existing esterase in cells can activate the probe to be transformed active CaP2 probe selectively binding with calcium ion in the surroundings. The probe was used to further evaluate the imaging of intracellular calcium ions in model organisms. The excellent imaging performance of CaP3 in Arabidopsis thaliana seedling roots demonstrates that CaP3 has the excellent capability of monitoring calcium ions in live-cell imaging, and furthermore CaP3 exhibits much better photostability and thereby greater potential in long-term imaging. This work established a general esterase-activated precipitating strategy to achieve specific detection and bioimaging in situ triggered by esterase in live cells, and established a water-soluble aggregation-induced phosphorescence probe with high selectivity to achieve specific sensing and long-term imaging of calcium ions in live cells.

A Self-Assembling Probe for Imaging the States of Golgi Apparatus in Live Single Cells.

Despite the enormous progress in genomics and proteomics, it is still challenging to assess the states of organelles in living cells with high spatiotemporal resolution. Based on our recent finding of enzyme-instructed self-assembly of a thiophosphopeptide that targets the Golgi Apparatus (GA) instantly, we use the thiophosphopeptide, which is enzymatically responsive and redox active, as an integrative probe for revealing the state of the GA of live cells at the single cell level. By imaging the probe in the GA of live cells over time, our results show that the accumulation of the probe at the GA depends on cell types. By comparison to a conventional Golgi probe, this self-assembling probe accumulates at the GA much faster and are sensitive to the expression of alkaline phosphatases. In addition, subtle changes of the fluorophore results in slightly different GA responses. This work illustrates a novel class of active molecular probes that combine enzyme-instructed self-assembly and redox reaction for high-resolution imaging of the states of subcellular organelles over a large area and extended times.

IndoLabel: Predicting Indoor Location Class by Discovering Location-Specific Sensor Data Motifs

This study presents a method for predicting location classes of a room such as a kitchen, and restroom, where a user is located by discovering location-specific sensor data motifs in sensor data observed by user’s sensor devices, such as smartwatch, without requiring labeled training data collected in a target environment. For example, we can observe similar waveforms corresponding to kitchen knife chopping actions using body-worn accelerometers in kitchens and can also observe similar sound features by active sound probing in bathrooms because of their water-resistant walls. This indicates that such location-specific sensor data motifs can be inherent information for location class prediction in almost every environment. This study proposes a novel method that automatically detects location-specific motifs from time series sensor data by calculating a score that represents the “location specificity” of each motif in a time series. Previous studies on location class prediction assume that location-specific sensor data are always observed in a room or use handcrafted rules and templates to detect location-specific sensor data resulting in difficulties in applying them to several realistic environments. In contrast, our method, named IndoLabel, can automatically discover short sensor data motifs, specific to a location class, and can automatically build an environment-independent location classifier without requiring handcrafted rules and templates. The proposed method was evaluated in real house environments using leave-one-environment-out cross-validation and achieved a state-of-the-art performance although labeled training data in the target environment was unavailable.

A Water Soluble 2-Phenyl-5-(pyridin-3-yl)-1,3,4-oxadiazole Based Probe: Antimicrobial Activity and Colorimetric/Fluorescence pH Response.

The growing demand of responsive tools for biological and biomedical applications pushes towards new low-cost probes easy to synthesize and versatile. Current optical probes are theranostic tools simultaneously responsive to biological parameters/analyte and therapeutically operating. Among the optical methods for pH monitoring, simple small organic molecules including multifunctional probes for simultaneous biological activity being highly desired by scientists and technicians. Here, we present a novel pH-responsive probe with a three-ring heteroaromatic pattern and a flexible cationic chain. The novel molecule shows real-time naked-eye colorimetric and fluorescence response in the slightly acidic pH range besides its excellent solubility both in the organic phase and in water. In addition, the small probe shows significant antibacterial activity, particularly against Escherichia coli. Single-crystal X-ray study and density functional theory (DFT) calculations rationalize the molecule spectroscopic response. Finally, molecular dynamics (MD) elucidate the interactions between the probe and a model cell membrane.

Multi-channel detection of Au(III) ions by a novel rhodamine based probe

Ionic gold species display remarkable features in chemical and biological activities. However, high amount of gold ions, especially Au3+ ions (200 µM), seriously threaten living organisms and vital human organs. Therefore, accurate detection of Au3+ ions is highly desirable. In this work, a novel rhodamine based optical and electrochemical active probe, integrated with triphenylamine and thiophene moiety, exhibited highly selective detection of Au(III) ions compared to the other metal species, such as Ag+, K+, Li+, Na+, Hg2+, Ba2+, Ca2+, Cd2+, Cu2+, Co2+, Mg2+, Mn2+, Ni2+, Pb2+, Zn2+ Pd2+, Al3+, Ce3+, Cr3+, Fe3+ and Cr6+. Exploiting hydrolysis and ring-opening reaction, the probe offers privileged properties such as naked-eye detection (colorimetric), both “turn-on” and “turn-off” fluorescence responses, voltammetric read-out with an anodic shift, low detection limit (340 and 315 nM for “turn-on” and “turn-off”, respectively), rapid response, and applicability in determining Au(III) in complex water samples.

Recent Advances in the Fluorescent Probes for Flavinase Activity: Design and Applications.

Flavinases, including monoamine oxidase (MAO-A/MAO-B), quinone oxidoreductase (NQO1), thioredoxin reductase (TrxR), nitroreductase (NTR) and so on, are important redox enzymes in organisms. They are considered as biomarkers of cell energy metabolism and cell vitality. Importantly, their aberrant expression is related to various disease processes. Therefore, the accurate measurement of flavinase is useful for the early diagnosis of diseases, which has aroused great concern in the scientific community. Various methods are also available for the detection of flavinases, fluorescence probes are considered to be one of the best detection methods due to their easy and accurate sensing capability. This review aims to introduce the advances in the design and application of flavinase probes in the last five years. This study focuses on analyzing the design strategies and reaction mechanisms of flavinases fluorescent probes and discusses the current challenges, which will further advance the development of diagnostic and therapeutic approaches for flavinase-related diseases.

APPROACHES OF PROBING SIGNAL TYPE DETERMINATION OF MEANS LOCAL PROBING FOR DISTRIBUTED POWER GRIDS TECHNICAL CONDITION DIAGNOSTIC

The paper presents the modes of ice detection on a 110 kV overhead line. Methods of detecting damage during ice of power transmission wires are analyzed. Proposals for improving the approaches to determining the damage caused by ice on the wires of overhead power lines have been developed. The article focuses on the method of location probing, which consists in applying a pulse signal to the line and determining the total time spent on its propagation along the wire in the forward and reverse directions after reflection from the end of the line or from high-frequency barrier. In the locational method of detecting ice, information about the appearance of ice is carried by pulses reflected from any inhomogeneity of the wave resistance of the line on it. All negative factors, including ice, change the wave resistance of a long line, creating inhomogeneities in overhead power lines and increases the travel time of the reflected pulse of a given section of the line in the presence of ice. They can be determined by active probing. The problem is that these inhomogeneities can be small in amplitude and active sounding is unable to detect the reflected signal from the homogeneity as the power of noise and interference can be greater, and most importantly they can be on branched power lines. The paper shows that increasing the duration of the probing pulse with the use of different types of modulation allows to increase the energy of the probing signal, to provide the required spectrum width and, accordingly, the resolution of the device at a distance. The article proposes a method of taking into account the “blind zone”, in which it is not possible to determine the reflected signals from the place of heterogeneity of overhead power lines. At the time of emission of the probing signal, the input of the receiver is closed and therefore reflected from the homogeneity of the signals do not arrive at the receiver. In FMICW radar, the measurement process to determine the range is to measure the difference between the current frequency of the emitted signal and the frequency of the reflected signal in the ion probe. It is proposed to introduce an algorithm and a method of ionosonde operation for the development of a device for diagnosing the technical condition of electrical distribution networks.

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.

Enhanced oxidation of antibiotic residuals (Levofloxacin) using a green composite of ZnO@polyaniline/bentonite (Zn@PA/BE) as multifunctional photocatalyst under visible light

ABSTRACT Green nanocomposite of ZnO supported into polyaniline/bentonite hybrid structure (Zn@/PA/BE) was synthesised and characterised and assessed as a multifunctional photocatalyst of 1.86 eV as bandgap energy. The Zn@/PA/BE catalyst was applied in the oxidation of the levofloxacin (LV) residuals in water in the presence of visible light. The recognised oxidation results demonstrate significant enhancement in photocatalytic activity by using the polyaniline/bentonite composite as a carrier for ZnO catalyst. Using the Zn@/PA/BE at 0.5 g/L as dosage resulted in complete oxidation for 10, 20, 30, 40, and 50 mg/L after illumination intervals of 25, 40, 60, 70, and 85 min, respectively. This was confirmed by the significant declination in the TOC content of the treated sample (10 mg/L) until the complete removal after 45 min reflecting the complete degradation and mineralisation. The formed secondary organic compounds during the incomplete oxidation reactions were identified. The intermediate compounds suggested oxidation pathways of carboxylation/de-carboxylation, hydroxylation, de-methylation, de-piperazinylation, and defluorination mechanisms. The hydroxyl radicals were detected as the most effective oxidising species during the reactions based on the active trapping tests and probe molecule investigation. The recyclability experiments reflected the significant stability and reusability of Zn@/PA/BE as a photocatalyst during the oxidation of LV molecules.

Miniature Active Differential Magnetic Field Probe With High Sensitivity for Near-Field Measurements

In this communication, a magnetic field probe with high common-mode rejection ratio (CMRR) and high sensitivity for the near-field measurement is proposed, fabricated, and calibrated. The proposed magnetic field probe consists of a differential dual loop (DDL) to improve the CMRR to the electric field, a balun to convert the differential end to the single end, and a two-stage amplifier achieving proper gain to further enhance the sensitivity. The sensitivity of the probe is raised by more than 50 dB at 30 MHz compared with the reference probes. The measured CMRR is more than 40 dB from 200 kHz to 1 GHz. The measurements of the passive circuits are conducted, and validating that the sensitivity of the proposed probe is significantly improved.

Cyclic Voltammetry as a Probe of Selective Ion Transport within Layered, Electrode-Supported Ion-Exchange Membrane Materials

Cyclic voltammetry was applied to investigate the permselective properties of electrode-supported ion-exchange polymer films intended for use in future molecular-scale spectroscopic studies of bipolar membranes. The ability of thin ionomer film assemblies to exclude mobile ions charged similarly to the polymer (co-ions) and accumulate ions charged opposite to the polymer (counterions) was scrutinized through use of the diffusible redox probe molecules [Ru(NH3)6]3+ and [IrCl6]2−. With the anion exchange membrane (AEM) phase supported on a carbon disk electrode, bipolar junctions formed by addition of a cation exchange membrane (CEM) overlayer demonstrated high selectivity toward redox ion extraction and exclusion. For junctions formed using a Fumion® AEM phase and a Nafion® overlayer, [IrCl6]2− ions exchanged into Fumion® prior to Nafion® overcoating remained entrapped and the Fumion® excluded [Ru(NH3)6]3+ ions for durability testing periods of more than 20 h under conditions of interest for eventual in situ spectral measurements. Experiments with the Sustainion® anion exchange ionomer uncovered evidence for [IrCl6]2− ion coordination to pendant imidazolium groups on the polymer. A cyclic voltammetric method for estimation of the effective diffusion coefficient and equilibrium extraction constant for redox active probe ions within inert, uniform density electrode-supported thin films was applied to examine charge transport mechanisms.

Accelerating paint superresolution imaging by active probe control

Many biological processes occur in 3D across large volumes. In order to get an accurate representation of the underlying structure with single molecule localisation microscopy (SMLM) stringent labelling requirements must be met. This critical limit is defined by the Nyquist criterion where, for a 200 × 200 × 600 nm volume ∼40,000 molecules are required. This constitutes a lower threshold, and stochastic sampling typical of SMLM techniques means this is often insufficient. Even if an appropriate density were to be achieved, it is still necessary to be able to contrast the localisation from the background.

Erianin suppresses proliferation and migration of cancer cells in a pyruvate carboxylase-dependent manner

Erianin is a natural small molecule dibenzyl compound extracted from Dendrobium officinale or Dendrobium chrysotoxum. Studies show erianin has many pharmacological functions such as antioxidant, antibacterial, antiviral, improving diabetic nephropathy, relaxing bronchial smooth muscle and anti-tumor. However, the erianin-mediated molecular mechanism is elusive, and the target protein of erianin is not clear yet. Here, we screened and identified that the target protein of erianin in human hepatoma HepG2 cells is human pyruvate carboxylase, and explored the anti-tumor signal pathway regulated by erianin in several cell lines. Firstly, the interaction between human pyruvate carboxylase and erianin was studied by bioinformatics and biochemical methods. Secondly, in vitro, erianin can specifically inhibit the activity of human pyruvate carboxylase, and the purified human pyruvate carboxylase can specifically bind to the activity probe of erianin. Thirdly, human pyruvate carboxylase is highly expressed in a variety of malignant tumors, and the inhibitory effect of erianin on tumor cells is positively correlated with the expression of human pyruvate carboxylase, and erianin can selectively inhibit the activity of pyruvate carboxylase. Finally, erianin can regulate the pyruvate carboxylase-mediated Wnt/ β- Catenin pathway. All of which provide important data for the further study of the anticancer mechanism of erianin, and lay a solid foundation for the further development and utilization of erianin.

AIEE active new fluorescent and colorimetric probes for solution and vapor phase detection of Nitrobenzene: A reversible mechanochromism and application of logic gate

Two new triphenylamine (TPA) based probes 1 and 2 were successfully synthesized through the Suzuki-Miyaura coupling reaction. In contrast to the basic core TPA that showed aggregation caused quenching (ACQ) phenomenon, probes presented in this study exhibited unique aggregation-induced emission enhancement (AIEE) and reversible mechanochromic properties due to their unique design of propeller-shaped geometry. AIEE active probes displayed highly sensitive solution and vapor phase detection of nitrobenzene (NB), which is a common constituent of various health hazards and unavoidable environmental threats. In solution, the highly sensitive fluorescence quenching response of probes towards NB was ascribed to a combined effect of its adjustable smaller size and photoinduced electron transfer (PET) process with size being a predominant factor. Fluorescence based detection limits of probes 1 and 2 for NB were determined as low as 2.17 and 3.21 nM, respectively. Advantageously, probes exhibited selective colorimetric detection of NB. Further, probes were found to be quite sensitive for the trace detection of NB in real samples. Additionally, the fluorescence response of probes was successfully used for the construction of logic gates.

Cancer cell-selective aggregation-induced emission probe for long-term plasma membrane imaging

Long-term plasma membrane (PM) tracking is important for studying the membrane function and diagnosis of membrane-related diseases. However, current PM probes can easily diffuse into the cytoplasm, which is undesirable. Here, we report an amphipathic aggregation-induced emission (AIE)-active far-red fluorescent probe, named DENPB, for long-term PM imaging. In live cancer cells, DENPB can be turned on after insertion into the PM and shows long-term membrane retention. It can be used for PM tracking for 6 h without obvious internalization. Interestingly, data show that it selectively stained the PM of live cancer cells (e.g., HeLa, HepG2, MDA-MB-231, MCF-7) but showed no signal in live non-cancer cells (e.g., RAW 264.7, NIH 3T3, HFF). It can also distinguish paraformaldehyde-fixed cancer cells from non-cancer cells by showing different signal patterns. This work provides a rational design strategy for a long-term PM-retention probe and a useful tool for cancer cell-specific staining that may greatly contribute to cancer diagnosis.

Colorimetric and Electrochemical Methods for the Detection of SARS-CoV-2 Main Protease by Peptide-Triggered Assembly of Gold Nanoparticles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been regarded as one of the ideal targets for the development of antiviral drugs. The currently used methods for the probing of Mpro activity and the screening of its inhibitors require the use of a double-labeled peptide substrate. In this work, we suggested that the label-free peptide substrate could induce the aggregation of AuNPs through the electrostatic interactions, and the cleavage of the peptide by the Mpro inhibited the aggregation of AuNPs. This fact allowed for the visual analysis of Mpro activity by observing the color change of the AuNPs suspension. Furthermore, the co-assembly of AuNPs and peptide was achieved on the peptide-covered electrode surface. Cleavage of the peptide substrate by the Mpro limited the formation of AuNPs/peptide assembles, thus allowing for the development of a simple and sensitive electrochemical method for Mpro detection in serum samples. The change of the electrochemical signal was easily monitored by electrochemical impedance spectroscopy (EIS). The detection limits of the colorimetric and electrochemical methods are 10 and 0.1 pM, respectively. This work should be valuable for the development of effective antiviral drugs and the design of novel optical and electrical biosensors.

Magnetic Nanorobots as Maneuverable Immunoassay Probes for Automated and Efficient Enzyme Linked Immunosorbent Assay.

As a typical, classical, but powerful biochemical sensing technology in analytical chemistry, enzyme-linked immunosorbent assay (ELISA) shows excellence and wide practicability for quantifying analytes of ultralow concentration. However, long incubation time and burdensome laborious multistep washing processes make it inefficient and labor-intensive for conventional ELISA. Here, we propose rod-like magnetically driven nanorobots (MNRs) for use as maneuverable immunoassay probes that facilitate a strategy for an automated and highly efficient ELISA analysis, termed nanorobots enabled ELISA (nR-ELISA). To prepare the MNRs, the self-assembled chains of Fe3O4 magnetic particles are chemically coated with a thin layer of rigid silica oxide (SiO2), onto which capture antibody (Ab1) is grafted to further achieve magnetically maneuverable immunoassay probes (MNR-Ab1s). We investigate the fluid velocity distribution around the MNRs at microscale using numerical simulation and empirically identify the mixing efficiency of the actively rotating MNRs. To automate the analysis process, we design and fabricate by 3-D printing a detection unit consisting of three function wells. The MNR-Ab1s can be steered into different function wells for required reaction or wishing process. The actively rotating MNR-Ab1s can enhance the binding efficacy with target analytes at microscale and greatly decrease incubation time. The integrated nR-ELISA system can significantly reduce the assay time, more importantly during which process manpower input is greatly minimized. Our simulation of the magnetic field distribution generated by Helmholtz coils demonstrates that our approach can be scaled up, which proves the feasibility of using current strategy to construct high throughput nR-ELISA detection instrument. This work of taking magnetic micro/nanobots as active immunoassay probes for automatic and efficient ELISA not only holds great potential for point-of-care testing (POCT) in future but also extends the practical applications of self-propelled micro/nanorobots into the field of analytical chemistry.

Oxygen vacancy modulated MnO2 bi-electrode system for attomole-level pathogen nucleic acid sequence detection

Electrochemical nucleic acid detection has recently opened a relatively low budget, straightforward and speedy approach of detection with the aid of an electrochemically active redox probe. However, electrochemical sensors of nucleic acid amplification characteristically employ a tri-electrode geometry involving standard reference electrode usually made up of a noble metal that adds up the cost of detection. This work proposes a sensitive and rapid approach for detecting the endpoint of nucleic acid amplification test using a novel bi-electrode sensing geometry. The sensing layer of the electrode was comprised of a transition metal oxide to tune its electronic state for regulating its electrochemical response. The XPS analysis indicates a higher contribution of Mn3+ oxidation state than Mn4+ when annealed at elevated temperature, suggesting the formation of oxygen vacancies into the sensing layer. The formation of oxygen vacancies improved the electrical conduction of the layer as evident from the significant reduction in charge transfer resistance (Rct) value from electrochemical impedance spectroscopy (EIS) measurements. The fabricated device was then used to detect dengue virus sequence DNA (using rolling circle amplification) and Staphylococcus aureus genomic DNA (using polymerase chain reaction) with the limit of detection in the order of 103 target DNA copies

Photoactivated Bacteriorhodopsin/SiNx Nanopore-Based Biological Nanofluidic Generator with Single-Protein Sensitivity.

Nanofluidics is an emerging hot field that explores the unusual behaviors of ions/molecules transporting through nanoscale channels, which possesses a broad application prospect. However, in situ probing bioactivity of functional proteins on a single-molecule level by a nanofluidic device has not been reported, and it is still a big challenge in the field. Herein, we reported a biological nanofluidic device with a single-protein sensitivity, based on natural proton-pumping protein, bacteriorhodopsin (bR), and a single SiNx nanopore. Nanofluidic single-molecule probing of bR proton-pumping activity and its light response were achieved under applied voltage of 0 V, by biologically self-powered steady-state ionic current nanopore sensing. Green-light irradiation of the device led to the monitoring of a steady-state proton current of ∼3.51 pA/per bR trimer, corresponding to charge density of 815 μC/cm2 generated by each bR monomer, which far exceeded the previously reported value of 1.4 μC/cm2. This finding and method would promote the development of artificial biological and hybrid nanofluidic devices in biosensing and energy conversion applications.