A nanofluidic spiking synapse.

Currently, the nanofluidic synapse can only perform basic neuromorphic pulse patterns. One immediate problem that needs to be addressed to further its capability of brain-like computing is the realization of a nanofluidic spiking device. Here, we report the use of a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate membrane to achieve bionic ionic current-induced spiking. In addition to the simulation of various electrical pulse patterns, our synapse could produce transmembrane ionic current-induced spiking, which is highly analogous to biological action potentials with similar phases and excitability. Moreover, the spiking properties could be modulated by ions and neurochemicals. We expect that this work could contribute to biomimetic spiking computing in solution.

Chemical Redox Cycling in an Organic Photoelectrochemical Transistor: Toward Dual Chemical and Electronic Amplification for Bioanalysis.

The organic photoelectrochemical transistor (OPECT) has been proven to be a promising platform to study the rich light-matter-bio interplay toward advanced biomolecular detection, yet current OPECT is highly restrained to its intrinsic electronic amplification. Herein, this work first combines chemical amplification with electronic amplification in OPECT for dual-amplified bioanalytics with high current gain, which is exemplified by human immunoglobulin G (HIgG)-dependent sandwich immunorecognition and subsequent alkaline phosphatase (ALP)-mediated chemical redox cycling (CRC) on a metal-organic framework (MOF)-derived BiVO4/WO3 gate. The target-dependent redox cycling of ascorbic acid (AA) acting as an effective electron donor could lead to an amplified modulation against the polymer channel, as indicated by the channel current. The as-developed bioanalysis could achieve sensitive HIgG detection with a good analytical performance. This work features the dual chemical and electronic amplification for OPECT bioanalysis and is expected to stimulate further interest in the design of CRC-assisted OPECT bioassays.

Electrochemical Molecule Trap-Based Sensing of Low-Abundance Enzymes in One Living Cell.

Measuring the activity of low-abundance enzymes, down to a few molecules in one living cell, is important but challenging to elucidate their biological function. Here, an electrochemical molecule trap is established at the tip of a nanopipette with an electrochemical detector, in which the diffusion of the molecules away from the electrochemical detector is prevented by electro-osmotic flow (EOF). Accordingly, a limited amount of enzymes is trapped to continuously catalyze the conversion of the substrate to generate a sufficient amount of the byproduct hydrogen peroxide for electrochemical measurements. The resistive pulse sensing of the enzymes in single liposomes validates the detection sensitivity down to 15 molecules. Using this ultrasensitive electrochemical strategy, the activity of 60 sphingomyelinase molecules inside single unstimulated living J774 cells is measured, which was hardly detected by previous methods. The established electrochemical molecule trap-based sensing approach opens the door toward single-molecule electrochemical detection in one living cell. This success will solve the long-standing problem regarding the study of the activity of low-abundance proteins in cells in their native physiological state and greatly enhance the understanding of the roles of proteins in cellular behavior.

Liposome-Assisted Enzymatic Modulation of Plasmonic Photoelectrochemistry for Immunoassay.

Recently emerged liposomal photoelectrochemical (PEC) bioanalysis has brought new opportunities for biosensor development. This work presents the new concept of liposome-assisted enzymatic modulation of plasmonic photoelectrochemistry for PEC bioanalysis, which was exemplified by an Au nanoclusters (NCs)-sensitized nanoporous TiO2 nanotubes (Au NC@TiO2 NT) photoelectrode and an alkaline phosphatase (ALP)-loaded liposomal immunoassay of heart-type fatty acid binding protein in a 96-well plate. After sandwich immunorecognition and subsequent lysis treatment, enzymatically generated ascorbic acid by the released ALP was directed to reduce Au3+ into Au nanoparticles using the Au NCs as seeds, leading to the in situ change of the photoelectrochemistry of the electrode and corresponding reduction of the photocurrent. The depressed signal could be correlated with the target concentration with good analytical performance in terms of sensitivity and selectivity. This work features the liposome-assisted enzymatic modulation of plasmonic photoelectrochemistry, which provides a new protocol for general PEC bioanalysis development.

Three-Dimensional CdS@Carbon Fiber Networks: Innovative Synthesis and Application as a General Platform for Photoelectrochemical Bioanalysis.

This Letter reports a novel synthetic methodology for the fabrication of three-dimensional (3D) nanostructured CdS@carbon fiber (CF) networks and the validation of its feasibility for applications as a general platform for photoelectrochemical (PEC) bioanalysis. Specifically, 3D architectures are currently attracting increasing attention in various fields due to their intriguing properties, while CdS has been most widely utilized for PEC bioanalysis applications because of its narrow band gap, proper conduction band, and stable photocurrent generation. Using CdS as a representative material, this work realized the innovative synthesis of 3D CdS@CF networks via a simple solvothermal process. Exemplified by the sandwich immunoassay of fatty-acid-binding protein (FABP), the as-fabricated 3D CdS@CF networks exhibited superior properties, and the assay demonstrated good performance in terms of sensitivity and selectivity. This work features a novel fabrication of 3D CdS@CF networks that can serve as a general platform for PEC bioanalysis. The methodology reported here is expected to inspire new interest for the fabrication of other 3D nanostructured Cd-chalcogenide (S, Se, Te)@CF networks for wide applications in biomolecular detection and beyond.

Engineering of Electrochromic Materials as Activatable Probes for Molecular Imaging and Photodynamic Therapy.

Electrochromic materials (EMs) are widely used color-switchable materials, but their applications as stimuli-responsive biomaterials to monitor and control biological processes remain unexplored. This study reports the engineering of an organic π-electron structure-based EM (dicationic 1,1,4,4-tetraarylbutadiene, 12+) as a unique hydrogen sulfide (H2S)-responsive chromophore amenable to build H2S-activatable fluorescent probes (12+-semiconducting polymer nanoparticles, 12+-SNPs) for in vivo H2S detection. We demonstrate that EM 12+, with a strong absorption (500-850 nm), efficiently quenches the fluorescence (580, 700, or 830 nm) of different fluorophores within 12+-SNPs, while the selective conversion into colorless diene 2 via H2S-mediated two-electron reduction significantly recovers fluorescence, allowing for non-invasive imaging of hepatic and tumor H2S in mice in real time. Strikingly, EM 12+ is further applied to design a near-infrared photosensitizer with tumor-targeting and H2S-activatable ability for effective photodynamic therapy (PDT) of H2S-related tumors in mice. This study demonstrates promise for applying EMs to build activatable probes for molecular imaging of H2S and selective PDT of tumors, which may lead to the development of new EMs capable of detecting and regulating essential biological processes in vivo.

Cholesterol Oxidase/Triton X-100 Parked Microelectrodes for the Detection of Cholesterol in Plasma Membrane at Single Cells.

The classic electrochemical analysis of plasma membrane cholesterol at single cells utilizes a cholesterol oxidase modified microelectrode that oxidizes local cholesterol efflux from the plasma membrane to generate hydrogen peroxide for the electrochemical quantification. In this letter, a mixture of cholesterol oxidase and Triton X-100 was filled in the microcapillary that could park at the Pt layer coated tip due to slow hydrodynamic flow. During the contact of the tip with the cellular membrane, Triton X-100 at the tip permeabilized the contacted membrane to release cholesterol for the reaction with cholesterol oxidase. As compared with the linkage of cholesterol oxidase at the electrode surface, the oxidase parked in aqueous solution at the tip had a higher turnover rate resulting in larger electrochemical signal for single cell analysis. More charge collected at acyl-coA:cholesterol acyltransferase (ACAT) inhibited cells supported that this novel detection strategy could monitor the flunctation of membrane cholesterol at single cells. The successful detection of plasma membrane cholesterol at single cells using the oxidase parked microelectrode will provide a special strategy for the fabrication of biosensor that permits the integration of more molecules without functional groups at the electrode to measure active and inactive molecules in the plasma membrane. Moreover, the larger electrochemical signals collected could further increase the spatial resolution for single cell electrochemical analysis.

Fluorescence Lifetime-Resolved Ion-Selective Nanospheres for Simultaneous Imaging of Calcium Ion in Mitochondria and Lysosomes.

In this paper, novel calcium-selective nanospheres incorporating Pluronic F127 and (4-carboxybutyl) triphenylphosphonium bromide (TPP) as shell layers were designed to monitor the level of free calcium ion in mitochondria and lysosomes at living cells simultaneously. TPP as a target for mitochondria drove the nanospheres to bind intracellular mitochondria, while the lipophilic F127 layer resulted in the partial accumulation of nanospheres in lysosomes. This dual feature of the shell layer led to the colocation of nanospheres in both mitochondria and lysosomes. Chromoionophore III (ETH 5350) was chosen as the chromoionophore in the nanospheres that had different fluorescence lifetimes in either mitochondria or lysosomes, and therefore, the locations of the nanospheres at these two cellular compartments were identified. After the stimulation of cells using ionomycin, a burst of calcium concentration in mitochondria was observed that was associated with almost constant calcium concentration in lysosomes. The simultaneous recording of calcium ions in both of the compartments using fluorescence lifetime-solved nanospheres offered a special strategy to spatially monitor subcellular fluctuation of ions in living cells.

Cu Nanoclusters-Encapsulated Liposomes: Toward Sensitive Liposomal Photoelectrochemical Immunoassay.

Herein we report the strategy of liposome-mediated Cu2+-induced exciton trapping upon CdS quantum dots (QDs) for amplified photoelectrochemical (PEC) bioanalysis application. Specifically, the Cu nanoclusters (NCs)-encapsulated liposomes were first fabricated and then processed with antibodies bound to their external surfaces. After the sandwich immunocomplexing, the confined liposomal labels were subjected to sequential lysis treatments for the release of Cu NCs and numerous Cu2+ ions, which were then directed to interact with the CdS QDs electrode. The interaction of Cu2+ ions with CdS QDs could generate CuxS and form the trapping sites to block the photocurrent generation. Since the photocurrent inhibition is closely related with the Cu NCs-loaded liposomal labels, a novel and general "signal-off" PEC immunoassay could thus be tailored with high sensitivity. Meanwhile, a complementary "signal-on" fluorescent detection could be accomplished by measuring the fluorescence intensity originated from the Cu NCs. This work features the first use of Cu NCs in PEC bioanalysis and also the first NCs-loaded liposomal PEC bioanalysis. More importantly, by using other specific ions/reagents-semiconductors interactions, this protocol could serve as a common basis for the general development of a new class of liposome-mediated PEC bioanalysis.

Semiconducting Organic-Inorganic Nanodots Heterojunctions: Platforms for General Photoelectrochemical Bioanalysis Application.

In this study, semiconducting organic polymer dots (Pdots) and inorganic quantum dots (Qdots) were first utilized to construct the organic-inorganic nanodots heterojunction for the photoelectrochemical (PEC) bioanalysis application. Specifically, n-type CdS Qdots, p-type CdTe Qdots, and tetraphenylporphyrin (TPP)-doped poly[(9,9-dioctylfluorenyl-2,7-diyl)- co-(1,4-benzo-{2,1',3}-thiadazole)] (PFBT) Pdots were fabricated, and their energy levels, that is, their valence band (VB)/conduction band (CB) or lowest unoccupied molecular orbital (LUMO)/highest occupied molecular orbital (HOMO) values, were also determined. Then, these nanodots were integrated to construct four types of p-n and p-p organic-inorganic nanodots heterojunctions, that is, CdS Qdots/TPP-doped PFBT Pdots, TPP-doped PFBT Pdots/CdS Qdots, CdTe Qdots/TPP-doped PFBT Pdots, and TPP-doped PFBT Pdots/CdTe Qdots, on the transparent glass electrode. Upon light irradiation, four heterojunctions exhibited different PEC behaviors with some having prominent photocurrent enhancement. With the model molecule l-cysteine (l-cys) as target, the proposed PEC sensor exhibited good performances. In brief, this work presents the first semiconducting organic-inorganic nanodots heterojunction for PEC bioanalysis application, which could be easily used as a general platform for future PEC bioanalysis building. Besides, it is expected to inspire more interest in the design, development, and implementation of various organic-inorganic heterojunctions for advanced PEC bioanalysis in the future.

3D Semiconducting Polymer/Graphene Networks: Toward Sensitive Photocathodic Enzymatic Bioanalysis.

This work reports the development of three-dimensional (3D) semiconducting polymer/graphene (SP/G) networks toward sensitive photocathodic enzymatic bioanalysis. Specifically, the porous 3D graphene was first synthesized via the hydrothermal and freeze-dry processes and then mixed with semiconducting polymer to obtain the designed hierarchical structure with unique porosity and large surface area. Afterward, the as-prepared hybrid was immobilized onto the indium tin oxide (ITO) for further characterizations. Exemplified by sarcosine oxidase (SOx) as a model biocatalyst, an innovative 3D SP/G-based photocathodic bioanalysis capable of sensitive and specific sarcosine detection was achieved. The suppression of cathodic photocurrent was observed in the as-developed photocathodic enzymatic biosystem due to the competition of oxygen consumption between the enzyme-biocatalyst process and O2-dependent photocathodic electrode. This work not only presented a unique protocol for 3D SP/G-based photocathodic enzymatic bioanalysis but also provided a new horizon for the design, development, and utilization of numerous 3D platforms in the broad field of general photoelectrochemical (PEC) bioanalysis.

Water as a Universal Infrared Probe for Bioanalysis in Aqueous Solution by Attenuated Total Reflection-Surface Enhanced Infrared Absorption Spectroscopy.

Monitoring the properties and reactions of biomolecules at their interface has attracted ever-growing interest. Here, we propose an approach of infrared analysis technique that utilizes water molecule as a universal probe for in situ and label free monitoring of interfacial bioevents in aqueous solution with high sensitivity. The strong infrared (IR) signal of O-H stretching vibrations from the repelled water is used to sensitively reveal the kinetics of interfacial bioevents at molecular level based on the steric displacement of water using an attenuated total reflection-surface enhanced infrared absorption spectroscopy. Using interfacial immuno-recognition and DNA hybridization as demonstrations, water IR probe offers 26 and 34 times higher sensitivity and even 200 and 86 times lower detection limit for immunosensing and DNA sensing, respectively, as compared to the traditional IR molecular fingerprints.

C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells.

Here, a g-C3N4 nanosheet modified microwell array providing enhanced electrochemiluminescence (ECL) and better visible sensitivity was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. The detection limit for ECL visualization of hydrogen peroxide at microwell array was improved to be 500 nM that guaranteed the detection of low concentration cholesterol at single cells in parallel. To achieve single cell cholesterol analysis, the individual cells cultured at the microwell array were exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol, and then treated with triton X-100, cholesterol esterase, and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the codetection of membrane and intracellular cholesterol at single cells. The inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.

Enediol-Ligands-Encapsulated Liposomes Enables Sensitive Immunoassay: A Proof-of-Concept for General Liposomes-Based Photoelectrochemical Bioanalysis.

Sensitive photoelectrochemical (PEC) bioanalysis usually relies on enzyme-assisted signal amplification. This work describes the first proof-of-concept study for liposome-based PEC bioanalysis. Specifically, unilamellar liposomes were prepared and then utilized to carry the enediol-ligands and antibodies within their internal cavities and upon their external surfaces, respectively. On the other hand, the 96-well plate was used for accommodating the sandwich immunocomplexing, and then the confined liposomes were directed to release the encapsulated enediol-ligands into an individual well. The subsequent in situ sensitization of the TiO2 nanoparticles (NPs) electrode was then used to transduce the recognition events. This facile strategy allows for sensitive immunoassay without the involvement of laborious electrode fabrication and enzymatic amplification. Importantly, the protocol can be extended as a general PEC method for numerous other targets of interest. We believe this work could offer a new perspective for the rational implementation of various liposome complexes for novel PEC bioanalysis.

Polymer Dots for Photoelectrochemical Bioanalysis.

Different from the most extensively used inorganic quantum dots (Qdots) for the current state-of-the-art photoelectrochemical (PEC) bioanalysis, this work reports the first demonstration of polymer dots (Pdots) for novel PEC bioanalysis. The semiconducting Pdots were prepared via the reprecipitation method and then immobilized onto the transparent indium tin oxide glass electrode for PEC biodetection of the model molecule l-cysteine. The experimental results revealed that the as-fabricated Pdots exhibited excellent and interesting PEC activity and good analytical performance of rapid response, high stability, wide linear range, and excellent selectivity. In particular, the PEC sensor could easily discriminate l-cysteine from reduced l-glutathione (l-GSH). This work manifested the great promise of Pdots in the field of PEC bioanalysis, and it is believed that our work could inspire the development of numerous functional Pdots with unique properties for innovative PEC bioanalytical purposes in the future.

Endogenous MicroRNA-Triggered and Real-Time Monitored Drug Release via Cascaded Energy Transfer Payloads.

It is a great challenge to design a drug delivery system with a controlled manner, especially one triggered by an exclusive endogenous disease marker and with an easily tracked release process. Herein, we developed a drug delivery platform of carbon dots which were connected to a stem-loop molecular beacon loaded with doxorubicin and polyethylene glycol modified folic acid. Such a platform enables one to release drugs on demand under the stimuli of endogenous microRNA-21, and turn on the fluorescence of carbon dots and doxorubicin, which allows one to monitor the drug release process. The intracellular experiment indicated that folic acid could mediate endocytosis of the nanocarrier, and the overexpressed endogenous microRNA-21 served as a unique key to unlock the drug nanocarrier by competitive hybridization with the molecular beacon, which finally resulted in fluorescence recovery and realized a chemotherapeutic effect within human breast cancer cells. The nanocarrier may have potential application in personalized treatment of different cancer subtypes in which the corresponding miRNAs are overexpressed.

Two-photon excitation nanoparticles for photodynamic therapy.

Two-photon excitation (TPE) nanoparticle-based photosensitizers (PSs) that combine the advantages of TPE and nanotechnology have emerged as attractive therapeutic agents for near-infrared red (NIR) light excited photodynamic therapy (PDT) for cancer treatment. TPE PDT is characterized by nonlinear absorption of two relatively low-energy photons of NIR light with the resulting emission of high-energy visible light. This high-energy light can sensitize oxygen to produce cytotoxic reactive oxygen species (ROS) and singlet oxygen (1O2) which can kill cancer cells. The long-wavelength light used to excite TPE NPs allows for deeper tissue penetration to achieve efficient PDT of deep-seated tumors. Moreover, TPE nanoparticles normally have large two-photon absorption (TPA) cross-sections, which hold great potential as efficient two-photon donors in PDT. In this review, we will summarize the recent advances made in the development of TPE nanoparticles for cancer PDT. Five different TPE nanoparticles, including quantum dots (QDs), carbon nanomaterials, silica nanoparticles, gold nanomaterials, and polymer nanoparticles, are summarized in detail, and the existing challenges as well as the future perspectives are also discussed.

Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells.

In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1-2 µm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.

Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging.

Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity.

Probing Low-Copy-Number Proteins in a Single Living Cell.

Single-cell analysis techniques are essential for understanding the microheterogeneity and functions of cells. Low-copy-number proteins play important roles in cell functioning, but their measurement in single cells remains challenging. Herein, we report an approach, called plasmonic immunosandwich assay (PISA), for probing low-copy-number proteins in single cells. This approach combined in vivo immunoaffinity extraction and plasmon-enhanced Raman scattering (PERS). Target proteins were specifically extracted from the cells by microprobes modified with monoclonal antibody or molecularly-imprinted polymer (MIP), followed by labeling with Raman-active nanotags. The PERS detection, with Raman intensity enhanced by 9 orders of magnitude, provided ultrasensitive detection at the single-molecule level. Using this approach, we found that alkaline phosphatase and survivin were expressed in distinct levels in cancer and normal cells, and that extended culture passage resulted in reduced expression of survivin. We further developed acupuncture needle-based PISA for probing low-copy-number proteins in living bodies.