An Ultrasensitive and Efficient microRNA Nanosensor Empowered by the CRISPR/Cas Confined in a Nanopore.

This work presents a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-nanopipette nano-electrochemistry (Cas = CRISPR-associated proteins) capable of ultrasensitive microRNA detection. Nanoconfinement of the CRISPR/Cas13a within a nanopipette leads to a high catalytic efficacy of ca. 169 times higher than that in bulk electrolyte, contributing to the amplified electrochemical responses. CRISPR/Cas13a-enabled detection of representative microRNA-25 achieves a low limit of detection down to 10 aM. Practical application of this method is further demonstrated for single-cell and real human serum detection. Its general applicability is validated by addressing microRNA-141 and the SARS-CoV-2 RNA gene fragment. This work introduces a new CRISPR/Cas-empowered nanotechnology for ultrasensitive nano-electrochemistry and bioanalysis.

A Bifunctional Wearable Sensor Based on a Nanoporous Membrane for Simultaneous Detection of Sweat Lactate and Temperature.

Wearable sensors for non-invasive, real-time detection of sweat lactate have far-reaching implications in the fields of health care and exercise physiological responses. Here, we propose a wearable electrochemical sensor with gold nanoelectrode arrays fabricated on the nanoporous polycarbonate (PC) membrane by encapsulating lactate oxidase (LOx) in chitosan (CS) hydrogel for detecting body temperature and sweat lactate concurrently. Flexible gold nanoporous electrodes not only enhance electrode area but also offer a nanoconfined space to accelerate the catalytic reaction of LOx and control substrate concentration on the surface of LOx to decrease substrate inhibition. The proposed sensor has a long durability of 13 days and better selectivity for the detection of sweat lactate over a wide linear range (0.01-35 mM) with a low detection limit (0.144 µM). Furthermore, temperature-dependent transmembrane currents passing through the sensor are used to estimate body temperature. We then use multiple linear regression to adjust the effect of temperature on lactate detection and succeed in monitoring lactate molecules in sweat and body temperature during exercise.

Bridging Ionic Current Rectification and Resistive-Pulse Sensing for Reliable Wide-Linearity Detection.

As two mainstream ionic detection techniques, ionic current rectification (ICR) suffers from large fluctuations in trace level detection, while resistive-pulse sensing (RPS) encounters easy clogs in high-concentration detection. By rationally matching the nanopore size with the DNA tetrahedron (TDN), this work bridges the two techniques to achieve reliable detection with wide linearity. As a representative analyte, miRNA-10b could specifically combine with and release TDN from the interior wall, which thus induced the simultaneous generation of distinct ICR and RPS signals. The ICR signals could be attributed to the balance between the effective orifice and surface charge density of the inner wall, while the RPS signals were induced by the complex of miRNA-10b and TDN passing through the nanopore. Such an operation contributed to a wide detection range of 1 fM-1 nM with a good linearity. The feasibility of this method is also validated in single-cell and real plasma detection.

PNP Nanofluidic Transistor with Actively Tunable Current Response and Ionic Signal Amplification.

Inspired by electronic transistors, electric field gating has been adopted to manipulate ionic currents of smart nanofluidic devices. Here, we report a PNP nanofluidic bipolar junction transistor (nBJT) consisting of one polyaniline (PANI) layer sandwiched between two polyethylene terephthalate (PET) nanoporous membranes. The PNP nBJT exhibits three different responses of currents (quasi-linear, rectification, and sigmoid) due to the counterbalance between surface charge distribution and base voltage applied in the nanofluidic channels; thus, they can be switched by base voltage. Four operating modes (cutoff, active, saturation, and breakdown mode) occur in the collector response currents. Under optimal conditions, the PNP nBJT exhibits an average current gain of up to 95 in 100 mM KCl solution at a low base voltage of 0.2 V. The present nBJT is promising for fabrication of nanofluidic devices with logical-control functions for analysis of single molecules.

Light-Enhanced Osmotic Energy Harvester Using Photoactive Porphyrin Metal-Organic Framework Membranes.

High ion selectivity and permeability, as two contradictory aspects for the membrane design, highly hamper the development of osmotic energy harvesting technologies. Metal-organic frameworks (MOFs) with ultra-small and high-density pores and functional surface groups show great promise in tackling these problems. Here, we propose a facile and mild cathodic deposition method to directly prepare crack-free porphyrin MOF membranes on a porous anodic aluminum oxide for osmotic energy harvesting. The abundant carboxyl groups of the functionalized porphyrin ligands together with the nanoporous structure endows the MOF membrane with high cation selectivity and ion permeability, thus a large output power density of 6.26 W m-2 is achieved. The photoactive porphyrin ligands further lead to an improvement of the power density to 7.74 W m-2 upon light irradiation. This work provides a promising strategy for the design of high-performance osmotic energy harvesting systems.

Reversible Electrochemical Tuning of Ion Sieving in Coordination Polymers.

Membrane-based ion separation is environmentally friendly, energy-efficient, and easy to integrate, being widely used in water desalination and purification systems. With the existing separation technologies, it is yet difficult to achieve real time, in situ, and reversible control of the separation process. Here, we design and fabricate a Prussian blue (PB) coordination polymer based membrane with uniform and electrochemically size-tunable subnanopores. The ion separation can be significantly and reversibly modulated through the electrochemical conversion between PB and Prussian white (PW). The permeation rates of small hydrated metal ions (Cs+ and K+) obviously increase upon switching from PB to PW, while the permeation rates of large hydrated metal ions (Li+, Na+, Mg2+, and La3+) remain constant. The membrane selectivity of small hydrated ions to large hydrated ions can be increased by more than 2 times during the electrochemical switch, which could be assigned to the slightly larger crystal size (e.g., pore window size) of PW than PB. The present approach provides a new strategy for constructing tunable seawater desalination and ion extraction systems.

High-performance bioanalysis based on ion concentration polarization of micro-/nanofluidic devices.

Micro-/nanofluidics has received considerable attention over the past two decades, which allows efficient biomolecule trapping and preconcentration due to ion concentration polarization (ICP) within nanostructures. The rich scientific content related to ICP has been widely exploited in different applications including protein concentration, biomolecules sensing and detection, cell analysis, and water purification. Compared to pure microfluidic devices, micro-/nanofluidic devices show a highly efficient sample enrichment capacity and nonlinear electrokinetic flow feature. These two unique characterizations make the micro-/nanofluidic systems promising in high-performance bioanalysis. This review provides a comprehensive description of the ICP phenomenon and its applications in bioanalysis. Perspectives are also provided for future developments and directions of this research field.

Electrogenerated Chemiluminescence Imaging of Electrocatalysis at a Single Au-Pt Janus Nanoparticle.

Noble metal nanoparticles are promising catalysts in electrochemical reactions, while understanding the relationship between the structure and reactivity of the particles is important to achieve higher efficiency of electrocatalysis, and promote the development of single-molecule electrochemistry. Electrogenerated chemiluminescence (ECL) was employed to image the catalytic oxidation of luminophore at single Au, Pt, and Au-Pt Janus nanoparticles. Compared to the monometal nanoparticles, the Janus particle structure exhibited enhanced ECL intensity and stability, indicating better catalytic efficiency. On the basis of the experimental results and digital simulation, it was concluded that a concentration difference arose at the asymmetric bimetallic interface according to different heterogeneous electron-transfer rate constants at Au and Pt. The fluid slip around the Janus particle enhanced local redox reactions and protected the particle surface from passivation.

Illustrating the Mass-Transport Effect on Enzyme Cascade Reaction Kinetics by Use of a Rotating Ring-Disk Electrode.

Electrochemical biosensors based on enzymatic reaction have been applied to a wide range of fields. As the trend continues to grow, these biosensors are approaching the limit imposed by physics and chemistry. To further improve the performance of biosensors, the interplay of mass transport and enzymatic reaction kinetics, especially in enzyme cascade systems, should be considered in the design of biosensors. Herein, we propose a simple approach to studying the influence of mass transport and enzyme molecule motion on the kinetics of enzyme cascade reactions. ß-Galactosidase (ß-Gal) and glucose oxidase (GOx) of the enzyme cascade reaction are precisely immobilized onto the disk and ring electrodes, respectively, of a rotating ring-disk electrode (RRDE) via covalent attachment. At a low rotating speed (<600 rpm), convective transport promotes the enzyme cascade reaction. When the rotating speed is higher than 600 rpm, the cascade reaction becomes kinetically controlled. Further increase of the rotating speed results in a slow decline in reaction rate, possibly due to the production inhibition effect. In addition, the effect of conformation change of the enzyme at higher centrifugal forces on enzyme activity should be considered. This study would shine light on the effect of convective force on regulation of kinetics of enzyme cascade reaction, offering an ideal platform for studying other enzyme cascade reactions and providing fundamentals to design high-performance biosensors, biofuel cells, and bioelectronics.

Effect of Nanoemitters on Suppressing the Formation of Metal Adduct Ions in Electrospray Ionization Mass Spectrometry.

In the work, we showed that the use of nanoemitters (tip dimension <1 µm, typically ∼100 nm) could dramatically reduce the nonspecific metal adduction to peptide or protein ions as well as improve the matrix tolerance of electrospray ionization mass spectrometry (ESI-MS). The proton-enriched smaller initial droplets are supposed to have played a significant role in suppressing the formation of metal adduct ions in nanoemitters. The proton-enrichment effect in the nanoemitters is related to both the exclusion-enrichment effect (EEE) and the ion concentration polarization effect (ICP effect), which permit the molecular ions to be regulated to protonated ones. Smaller initial charged droplets generated from nanoemitters need less fission steps to release the gas-phase ions; thus, the enrichment effect of salt was not as significant as that of microemitters (tip dimension >1 µm), resulting in the disappearing of salt cluster peaks in high mass-to-charge (m/z) region. The use of nanoemitters demonstrates a novel method for tuning the distribution of the metal-adducted ions to be in a controlled manner. This method is also characterized by ease of use and high efficiency in eliminating the formation of adduct ions, and no pretreatment such as desalting is needed even in the presence of salt at millimole concentration.

Contribution of convection and diffusion to the cascade reaction kinetics of ß-galactosidase/glucose oxidase confined in a microchannel.

The spatial positioning of enzymes and mass transport play crucial roles in the functionality and efficiency of enzyme cascade reactions. To fully understand the mass transport regulating kinetics of enzyme cascade reactions, we investigated the contribution of convective and diffusive transports to a cascade reaction of ß-galactosidase (ß-Gal)/glucose oxidase (GOx) confined in a microchannel. ß-Gal and GOx are assembled on two separated gold films patterned in a polydimethylsiloxane (PDMS) microchannel with a controllable distance from 50 to 100 µm. Experimental results demonstrated that the reaction yield increases with decreasing distance between two enzymes and increasing substrate flow rate. Together with the simulation results, we extracted individual reaction kinetics of the enzyme cascade reaction and found that the reaction rate catalyzed by ß-Gal occurred much faster than by GOx, and thus, the ß-Gal catalytic reaction showed diffusion controll, whereas the GOx catalytic reaction showed kinetic controll. Since the decrease in the enzymes distance shortens the transport length of intermediate glucose to GOx, the amount of glucose reaching GOx will be increased in the unit time, and in turn, the enzyme cascade reaction yield will be increased with decreasing the gap distance. This phenomenon is similar to the intermediates pool of tricarboxylic acid (TCA) cycle in the metabolic system. This study promotes the understanding of the metabolic/signal transduction processes and active transport in biological systems and promises to design high performance biosensors and biofuel cells systems.

Water transport within carbon nanotubes on a wave.

Water molecules possess discontinuous properties in confined surroundings as compared to the bulk, their transport velocity shows a step change with the increase in the radius of hydrophobic carbon nanotubes (CNTs). Here, we report that the chain of water molecules in CNTs behaves as a "spring" owing to hydrogen bonding. Thus, the transport of water molecules in confined systems proceeds as a wave motion with eigen frequencies in the terahertz region which is determined by the CNT size. Water velocities derived from molecular dynamics (MD) fit well with the ones from finite element methods (FEM) on consideration of both the no-slip and slip boundary conditions for CNT diameters less than 1 nm and more than 1 nm, respectively. The present work helps clarify the features of mass and momentum transfers in confined surroundings, and provides perspectives for mass transfer applications.

Propagation of concentration polarization affecting ions transport in branching nanochannel array.

A new ionic current rectification device responsive to a broad range of pH stimuli has been fabricated using porous anodic alumina membrane with tailor-made branching nanochannel array. The asymmetric geometry is achieved by changing oxidation voltage using a simple two-step anodization process. Due to the protonation/deprotonation of the intrinsic hydroxyl groups upon solution pH variation, the nanochannels array-based device is able to regulate ionic current rectification properties. The ion rectification ratio of the device is mainly determined by the branching size and surface charges, which is also confirmed by theoretical simulations. In addition, theoretical simulations show that the slight difference in ion rectification ratio for the nanochannel devices with different branching numbers is due to the propagation of concentration polarization. Three dimensional simulations clearly show the nonuniform concentration profiles in stem nanochannel near the junction interface due to the presence of branching nanochannels. The present ionic device is promising for biosensing, molecular transport and separation, and drug delivery in confined environments.

Morpholino-functionalized nanochannel array for label-free single nucleotide polymorphisms detection.

The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected.

Fast serial analysis of active cholesterol at the plasma membrane in single cells.

Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases.

Sensitive electrochemical flow injection analysis of H2O2 released from cells with a pass-through mode.

Conventional plate electrodes were commonly used in electrochemical flow injection analysis and only part of molecules diffused to the plane of electrodes could be detected, which would limit the performance of electrochemical detection. In this study, a low-cost native stainless steel wire mesh (SSWM) electrode was integrated into a 3D-printed device for electrochemical flow injection analysis with a pass-through mode, which is different compared with previous flow-through mode. This strategy was applied for sensitive analysis of hydrogen peroxide (H2O2) released from cells. Under the optimal conditions (the applied potentials, the flow rate and the sample volume), the device exhibits high sensitivity toward H2O2. Linear relationships could be achieved between electrochemical responses and the concentration of H2O2 ranging from 1 nM to 1 mM. The excellent analytical performance of the SSWM-based device could be attributed to the pass-through mode based on the mesh microstructure and intrinsic catalytic properties for H2O2 by stainless steel. This approach could be further successfully extended for screening of H2O2 released from HeLa cells with electrochemical responses linear to the number of cells in a range of 3 - 1.35 × 104 cells with an injection volume of 30 µL. This study revealed the potential of mesh electrodes in electrochemical flow injection analysis for cellular function and pathology and its possible extension in cell counting and on-line analysis.

CS/PVP Hydrogel-Based Nanocapillary for Monitoring Bacterial Growth and Rapid Antibiotic Susceptibility Testing.

Infection with drug-resistant bacteria poses a significant threat to human health. Judicious use of antibiotics could reduce the likelihood of bacterial resistance, which can be evaluated through antibiotic susceptibility testing (AST). This paper focuses on the application of a needle-like nanocapillary tip filled with chitosan (CS)/polyethylene pyrrolidone (PVP) hydrogel based on its specific pH-sensitive properties. The gel-filled nanocapillary has the potential to be used for electrical pH detection with a sensitivity of 3.06 nA/pH and a linear range from 7.3 to 4.3. Such sensitivity for pH measurement could be extended for monitoring of bacterial (such as Escherichia coli and Streptococcus salivarius) growth because of the relationship between pH and bacterial growth. Bacterial growth curves obtained using the hydrogel-filled nanocapillary showed good agreement with the OD600 method. Moreover, this device could be applied for rapid AST for tetracycline and norfloxacin on E. coli with minimum inhibitory concentrations of 2 and 0.125 µg/mL, respectively. This study expands the application of the hydrogel-based nanocapillary for bacterial research by monitoring changes in pH values.

Dependence of the direct electron transfer activity and adsorption kinetics of cytochrome c on interfacial charge properties.

With the advantages of in situ analysis and high surface sensitivity, surface-enhanced infrared absorption spectroscopy in attenuated total reflection mode (ATR-SEIRAS) combined with electrochemical methods has been employed to examine the interfacial direct electron transfer activity and adsorption kinetics of cytochrome c (cyt c). This work presents data on cyt c adsorption onto negatively charged mercaptohexanoic acid (MHA) and positively charged 6-amino-1-hexanethiol (MHN) self-assembled monolayers (SAMs) on gold nanofilm surfaces. The adsorbed cyt c displays a higher apparent electron transfer rate constant (33.5 ± 2.4 s(-1)) and apparent binding rate constant (73.1 ± 5.2 M(-1) s(-1)) at the MHA SAMs surface than those on the MHN SAMs surface. The results demonstrate that the surface charge density determines the protein adsorption kinetics, while the surface charge character determines the conformation and orientation of proteins assembled which in turn affects the direct electron transfer activity.

Construction and Evaluation of Zeolitic Imidazolate Framework-Encapsulated Hemoglobin Microparticles as Oxygen Carriers.

Artificial oxygen carriers, such as favorably hemoglobin-based oxygen carriers, have received considerable attention due to some drawbacks of human donor blood. Among all oxygen carriers, the metal organic framework (MOF) exhibits excellent oxygen-carrying capacity due to its good encapsulation efficiency and competitive biocompatibility. Recently, zeolitic imidazolate frameworks (ZIFs) with unique structure have attracted much attention due to their outstanding solvothermal stability. Notably, ZIF-8, the prototypical ZIF, has been utilized to load hemoglobin (Hb) as a potential blood substitute. In this work, another ZIF material, which possesses a high oxygen binding/release capability, suitable safety profile, high stability, and efficiency as a potential oxygen carrier, was used to encapsulate Hb in an environment-friendly condition.

Rational design of mesoporous chiral MOFs as reactive pockets in nanochannels for enzyme-free identification of monosaccharide enantiomers.

Monosaccharides play significant roles in daily metabolism in living organisms. Although various devices have been constructed for monosaccharide identification, most rely on the specificity of the natural enzyme. Herein, inspired by natural ionic channels, an asymmetrical MOF-in-nanochannel architecture is developed to discriminate monosaccharide enantiomers based on cascade reactions by combining oxidase-mimicking and Fenton-like catalysis in homochiral mesoporous CuMOF pockets. The identification performance is remarkably enhanced by the increased oxidase-mimicking activity of Au nanoparticles under a local surface plasmon resonance (LSPR) excitation. The apparent steady-state kinetic parameters and nano-fluidic simulation indicate that the different affinities induced by Au-LSPR excitation and the confinement effect from MOF pockets precipitate the high chiral sensitivity. This study offers a promising strategy for designing an enantiomer discrimination device and helps to gain insight into the origin of stereoselectivity in a natural enzyme.