Non-Mass Spectrometric Targeted Single-Cell Metabolomics.

Metabolic assays serve as pivotal tools in biomedical research, offering keen insights into cellular physiological and pathological states. While mass spectrometry (MS)-based metabolomics remains the gold standard for comprehensive, multiplexed analyses of cellular metabolites, innovative technologies are now emerging for the targeted, quantitative scrutiny of metabolites and metabolic pathways at the single-cell level. In this review, we elucidate an array of these advanced methodologies, spanning synthetic and surface chemistry techniques, imaging-based methods, and electrochemical approaches. We summarize the rationale, design principles, and practical applications for each method, and underscore the synergistic benefits of integrating single-cell metabolomics (scMet) with other single-cell omics technologies. Concluding, we identify prevailing challenges in the targeted scMet arena and offer a forward-looking commentary on future avenues and opportunities in this rapidly evolving field.

Single-Cell Profiling of Fatty Acid Uptake Using Surface-Immobilized Dendrimers.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

A Chemical Approach for Profiling Intracellular AKT Signaling Dynamics from Single Cells.

We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.

Galvanic Redox Potentiometry for Self-Driven in Vivo Measurement of Neurochemical Dynamics at Open-Circuit Potential.

Understanding the real-time correlation between chemical patterns and neural processes is critical for deciphering brain function. Voltammetry has enabled this task but with a number of challenges for current-based electrolysis in vivo. Herein, we report galvanic redox potentiometry (GRP) potentially as a universal strategy for in vivo monitoring of neurochemicals, with ascorbic acid (AA) as a typical example. The GRP sensor is constructed on a self-driven galvanic cell configuration, where AA is spontaneously oxidized at the indicating single-walled carbon nanotube-modified carbon fiber electrode (SWNT-CFE), while oxygen reduced at the laccase-modified reference CFE (Lac-CFE). At thermodynamic equilibrium, open-circuit potential (OCP) can be a linear indicator of the concentration of AA. The resulting sensor shows a high selectivity to AA dynamics in the presence of coexisting electroactive neurochemicals, which is mainly determined by the driving force for the cell reaction, as suggested by principal investigation. Sensing sensitivity of this OCP-based GRP method is not affected by nonspecific protein adsorption and electrode fouling. Moreover, a micropipette compartment of the reference electrode is designed to suppress mass crossover and prevent disturbance to oxygen reduction through confinement effect. The in vivo application of the GRP sensor is illustrated by measuring the basal level of cortical AA in live rat brain (230 ± 40 µM) and its dynamics during ischemia/reperfusion. The GRP concept is demonstrated as a prominent method for in vivo, real-time, quantitative analysis of brain neurochemistry.

Surface Immobilization of Redox-Labile Fluorescent Probes: Enabling Single-Cell Co-Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities.

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single-cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.

Monitoring of Heparin Activity in Live Rats Using Metal-Organic Framework Nanosheets as Peroxidase Mimics.

Metal-organic framework (MOF) nanosheets are a class of two-dimensional (2D) porous and crystalline materials that hold promise for catalysis and biodetection. Although 2D MOF nanosheets have been utilized for in vitro assays, ways of engineering them into diagnostic tools for live animals are much less explored. In this work, a series of MOF nanosheets are successfully engineered into a highly sensitive and selective diagnostic platform for in vivo monitoring of heparin (Hep) activity. The iron-porphyrin derivative is selected as a ligand to synthesize a series of archetypical MOF nanosheets with intrinsic heme-like catalytic sites, mimicking peroxidase. Hep-specific AG73 peptides as recognition motifs are physically adsorbed onto MOF nanosheets, blocking active sites from nonspecific substrate-catalyst interaction. Because of the highly specific interaction between Hep and AG73, the activity of AG73-MOF nanosheets is restored upon the binding of Hep, but not Hep analogues and other endogenous biomolecules. Furthermore, by taking advantages of biocompatibility and diagnostic property enabled by AG73-MOF nanosheets, the elimination process of Hep in live rats is quantitatively monitored by coupling with microdialysis technology. This work expands the biomedical applications of 2D MOF nanomaterials and provides access to a promising in vivo diagnostic platform.

Functional Nucleic Acid Probe for Parallel Monitoring K(+) and Protoporphyrin IX in Living Organisms.

Parallel monitoring of K(+) and protoporphyrin IX (PPIX) is of vital importance because they are not only involved in a variety of biological processes but also closely linked to each other in numerous cellular pathways. However, there are currently no existing methods that can meet the requirements for parallel and in vivo detection of K(+) and PPIX in living organisms. Herein, we demonstrated a functional nucleic acid (FNA)-based technique for parallel monitoring of K(+) and PPIX in living animals. Specifically, the selected G-rich FNA probe was selectively induced to form a parallel G-quadruplex by K(+). The parallel G-quadruplex then remarkably enhanced the fluorescence of PPIX. Thus, by modulating the fluorescence "turn on" with the G-quadruplex and K(+)/PPIX, both K(+) and PPIX could be detected. After validating the developed method for selective and sensitive detection of K(+) and PPIX in vitro, their dynamic changes in living organisms (i.e., living brains and tumors) following various physiological and pathological processes were simultaneously monitored. The current study not only provides a general method for the detection of metal ions and bioactive molecules but also presents a way to investigate their synergistic functions in the regulation of various biological processes. It may also be helpful for improving the imaging and therapeutic efficacy of PPIX and 5-ALA.

Integrated Nanozymes with Nanoscale Proximity for in Vivo Neurochemical Monitoring in Living Brains.

Nanozymes, the nanostructures with enzymatic activities, have attracted considerable attention because, in comparison with natural enzymes, they offer the possibility of lowered cost, improved stability, and excellent recyclability. However, the specificity and catalytic activity of current nanozymes are still far lower than that of their natural counterparts, which in turn has limited their use such as in bioanalysis. To address these challenges, herein we report the design and development of integrated nanozymes (INAzymes) by simultaneously embedding two cascade catalysts (i.e., a molecular catalyst hemin and a natural enzyme glucose oxidase, GOx) inside zeolitic imidazolate framework (ZIF-8) nanostructures. Such integrated design endowed the INAzymes with major advantage in improved catalytic efficiency as the first enzymatic reaction occurred in close (nanoscale) proximity to the second enzyme, so products of the first reaction can be used immediately as substrates for the second reaction, thus overcoming the problems of diffusion-limited kinetics and product instability. The considerable high catalytic activity and stability enabled the INAzymes to efficiently draw a colorimetric detection of glucose with good sensitivity and selectivity. When facilitated with in vivo microdialysis, the INAzyme was successfully used for facile colorimetric visualization of cerebral glucose in the brain of living rats. Moreover, when further combined with microfluidic technology, an integrative INAzyme-based online in vivo analytical platform was constructed. The promising application of the platform was successfully illustrated by continuously monitoring the dynamic changes of striatum glucose in living rats' brain following ischemia/reperfusion. This study developed a useful approach to not only functional nanomaterial design but also advanced platforms developments for diverse targets monitoring.

Ratiometric electrochemical sensor for effective and reliable detection of ascorbic acid in living brains.

The in vivo detection of ascorbic acid (AA), one of the physiologically important cerebral neurochemicals, is critical to probe and understand brain functions. Electrochemical sensors are convenient for AA detection. However, conventional electrochemical sensors usually suffer from several challenges, such as sluggish electron transfer kinetics for AA oxidation and poor reproducibility. To address these challenges, here we report ratiometric electrochemical sensors for effective and reliable detection of AA in living brains. The sensors were constructed by immobilizing preassembled thionine/Ketjen black (KB) nanocomposites onto glassy carbon (GC) electrodes or carbon fiber microelectrodes (CFMEs). The KB in the rationally functionalized nanocomposites efficiently facilitated AA oxidation at a relatively negative potential (∼-0.14 V) without particular physical or chemical pretreatment, forming the basis of selective measurement of AA. With a well-defined and reversible pair of redox wave at -0.22 V, the assembled thionine acted as an internal reference to substantially alleviate the lab-to-lab, person-to-person, and electrode-to-electrode variations. The in vitro experiments demonstrated that the sensors exhibited extremely high reproducibility and stability toward selective measurement of AA. More, with operational simplicity and robustness in analytical performance, the designed sensors were successfully applied to in vivo effectively, selectively, and reliably monitor the dynamic change of cerebral AA associated with pathological processes (i.e., salicylate-induced tinnitus as the model) in living rats' brains. This study not only offers a new strategy for construction of ratiometric electrochemical sensors but also opens a new way for selective and reliable detection of neurochemicals for probing brain functions.

G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain.

Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.

Hybridization of bioelectrochemically functional infinite coordination polymer nanoparticles with carbon nanotubes for highly sensitive and selective in vivo electrochemical monitoring.

This study demonstrates the formation of a three-dimensional conducting framework through hybridization of bioelectrochemically active infinite coordination polymer (ICP) nanoparticles with single-walled carbon nanotubes (SWNTs) for highly sensitive and selective in vivo electrochemical monitoring with combination with in vivo microdialysis. The bioelectrochemically active ICP nanoparticles are synthesized through the self-assembly process of NAD(+) and Tb(3+), in which all biosensing elements including an electrocatalyst (i.e., methylene green, MG), cofactor (i.e., ß-nicotinamide adenine dinucleotide, NAD(+)), and enzyme (i.e., glucose dehydrogenase, GDH) are adaptively encapsulated. The ICP/SWNT-based biosensors are simply prepared by drop-coating the as-formed ICP/SWNT nanocomposite onto a glassy carbon substrate. Electrochemical studies demonstrate that the simply prepared ICP/SWNT-based biosensors exhibit excellent biosensing properties with a higher sensitivity and stability than the ICP-based biosensors prepared only with ICP nanoparticles (i.e., without hybridization of SWNTs). By using a GDH-based electrochemical biosensor as an example, we demonstrate a technically simple yet effective online electroanalytical platform for continuously monitoring glucose in the brain of guinea pigs with the ICP/SWNT-based biosensor as an online detector in a continuous-flow system combined with in vivo microdialysis. Under the experimental conditions employed here, the dynamic linear range for glucose with the ICP/SWNT-biosensor is from 50 to 1000 µM. Moreover, in vivo selectivity investigations with the biosensors prepared by the GDH-free ICPs reveal that ICP/SWNT-based biosensors are very selective for the measurement of glucose in the cerebral system. The basal level of glucose in the microdialysates from the striatum of guinea pigs is determined to be 0.31 ± 0.03 mM (n = 3). The study offers a simple route to the preparation of electrochemical biosensors, which is envisaged to be particularly useful for probing the chemical events involved in some physiological and pathological processes.

Biofuel cell-based self-powered biogenerators for online continuous monitoring of neurochemicals in rat brain.

This study demonstrates a new electrochemical method for continuous neurochemical sensing with a biofuel cell-based self-powered biogenerator as the detector for the analysis of microdialysate continuously sampled from rat brain, with glucose as an example analyte. To assemble a glucose/O(2) biofuel cell that can be used as a self-powered biogenerator for glucose sensing, glucose dehydrogenase (GDH) was used as the bioanodic catalyst for the oxidation of glucose with methylene green (MG) adsorbed onto single-walled carbon nanotubes (SWNTs) as the electrocatalyst for the oxidation of dihydronicotinamide adenine dinucleotide (NADH). Laccase crosslinked onto SWNTs was used as the biocathodic catalyst for the O(2) reduction. To enable the bioanode and biocathode to work efficiently in their individually favorable solutions and to eliminate the interference between the glucose bioanode and O(2) biocathode, the biofuel cell-based biogenerator was built in a co-laminar microfluidic chip so that the bioanodic and biocathodic streams could be independently optimized to provide conditions favorable for each of the bioelectrodes. By using a home-made portable voltmeter to output the voltage generated on an external resistor, the biogenerator was used for glucose sensing based on a galvanic cell mechanism. In vitro experiments demonstrate that, under the optimized conditions, the voltage generated on an external resistor shows a linear relationship with the logarithmic glucose concentration within a concentration range of 0.2 mM to 1.0 mM. Moreover, the biogenerator exhibits a high stability and a good selectivity for glucose sensing. The validity of the biofuel cell-based self-powered biogenerator for continuous neurochemical sensing was illustrated by online continuous monitoring of striatum glucose in rat brain through the combination of in vivo microdialysis. This study offers a new and technically simple platform for continuously monitoring physiologically important species in cerebral systems.

Rational design and one-step formation of multifunctional gel transducer for simple fabrication of integrated electrochemical biosensors.

This study demonstrates a new strategy to simplify the biosensor fabrication and thus minimize the biosensor-to-biosensor deviation through rational design and one-step formation of a multifunctional gel electronic transducer integrating all elements necessitated for efficiently transducing the biorecognition events to signal readout, by using glucose dehydrogenase (GDH) based electrochemical biosensor as an example. To meet the requirements for preparing integrated biosensors and retaining electronic and ionic conductivities for electronically transducing process, ionic liquids (ILs) with enzyme cofactor (i.e., oxidized form of nicotinamide adenine dinucleotide) as the anion were synthesized and used to form a bucky gel with single-walled carbon nanotubes, in which methylene green electrocatalyst was stably encapsulated for the oxidation of nicotinamide adenine dinucleotide. With such kind of rationally designed and one-step-formed multifunctional gel as the electronic transducer, the GDH-based electrochemical biosensors were simply fabricated by polishing the electrodes onto the gel followed by enzyme immobilization. This capability greatly simplifies the biosensor fabrication, prolongs the stability of the biosensors, and, more remarkably, minimizes the biosensor-to-biosensor deviation. The relative standard deviations obtained both with one electrode for the repeated measurements of glucose and with the different electrodes prepared with the same method for the concurrent measurements of glucose with the same concentration were 3.30% (n = 7) and 4.70% (n = 6), respectively. These excellent properties of the multifunctional gel-based biosensors substantially enable them to well-satisfy the pressing need of rapid measurements, for example, environmental monitoring, food analysis, and clinical diagnoses.

Graphene as a spacer to layer-by-layer assemble electrochemically functionalized nanostructures for molecular bioelectronic devices.

This study demonstrates the capability of graphene as a spacer to form electrochemically functionalized multilayered nanostructures onto electrodes in a controllable manner through layer-by-layer (LBL) chemistry. Methylene green (MG) and positively charged methylimidazolium-functionalized multiwalled carbon nanotubes (MWNTs) were used as examples of electroactive species and electrochemically useful components for the assembly, respectively. By using graphene as the spacer, the multilayered nanostructures of graphene/MG and graphene/MWNT could be readily formed onto electrodes with the LBL method on the basis of the electrostatic and/or π-π interaction(s) between graphene and the electrochemically useful components. Scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), and cyclic voltammetry (CV) were used to characterize the assembly processes, and the results revealed that nanostructure assembly was uniform and effective with graphene as the spacer. Electrochemical studies demonstrate that the assembled nanostructures possess excellent electrochemical properties and electrocatalytic activity toward the oxidation of NADH and could thus be used as electronic transducers for bioelectronic devices. This potential was further demonstrated by using an alcohol dehydrogenase-based electrochemical biosensor and glucose dehydrogenase-based glucose/O(2) biofuel cell as typical examples. This study offers a simple route to the controllable formation of graphene-based electrochemically functionalized nanostructures that can be used for the development of molecular bioelectronic devices such as biosensors and biofuel cells.

Single-cell profiling of D-2-hydroxyglutarate using surface-immobilized resazurin analogs.

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.

Multi-omic single-cell snapshots reveal multiple independent trajectories to drug tolerance in a melanoma cell line.

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAFV600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population.

Surface-Enhanced Raman Scattering Active Gold Nanoparticles with Enzyme-Mimicking Activities for Measuring Glucose and Lactate in Living Tissues.

Gold nanoparticles (AuNPs) with simultaneous plasmonic and biocatalytic properties provide a promising approach to developing versatile bioassays. However, the combination of AuNPs' intrinsic enzyme-mimicking properties with their surface-enhanced Raman scattering (SERS) activities has yet to be explored. Here we designed a peroxidase-mimicking nanozyme by in situ growing AuNPs into a highly porous and thermally stable metal-organic framework called MIL-101. The obtained AuNPs@MIL-101 nanozymes acted as peroxidase mimics to oxidize Raman-inactive reporter leucomalachite green into the active malachite green (MG) with hydrogen peroxide and simultaneously as the SERS substrates to enhance the Raman signals of the as-produced MG. We then assembled glucose oxidase (GOx) and lactate oxidase (LOx) onto AuNPs@MIL-101 to form AuNPs@MIL-101@GOx and AuNPs@MIL-101@LOx integrative nanozymes for in vitro detection of glucose and lactate via SERS. Moreover, the integrative nanozymes were further explored for monitoring the change of glucose and lactate in living brains, which are associated with ischemic stroke. The integrative nanozymes were then used to evaluate the therapeutic efficacy of potential drugs (such as astaxanthin for alleviating cerebral ischemic injuries) in living rats. They were also employed to determine glucose and lactate metabolism in tumors. This study not only demonstrated the great promise of combining AuNPs' multiple functionalities for versatile bioassays but also provided an interesting approach to designing nanozymes for biomedical and catalytic applications.

Modulating luminescence of Tb(3+) with biomolecules for sensing heparin and its contaminant OSCS.

The detection of heparin (Hep) and its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance in clinics but remains challenging. Here, we report a sensitive and selective time-resolved luminescence (TRL) biosensing system for Hep by modulating the photoluminescence of Tb(3+) with guanine-rich ssDNA and Hep-specific AG73 peptide (RKRLQVQLSIRT). With the developed system, Hep including both unfractionated Hep (UFH) and the low molecular weight Hep (LMWH) has been successfully detected with a satisfactory detection limit. Owing to the highly specific interaction between Hep and AG73 peptide, major interfering substances in Hep detection, such as Hep analogs of chondrotin sulfate (Chs) and hyaluronic acid (HA), did not interfere with Hep detection. The established TRL sensing system was then successfully used for monitoring Hep metabolism in living rats by microdialysis. Moreover, the proposed TRL sensing system was further applied to analyze OSCS contaminant in Hep with heparinases treatment by exploring the inhibition effects of OSCS on the activity of heparinases. As low as 0.002% of OSCS in Hep was identified.

Ionic liquid-assisted preparation of laccase-based biocathodes with improved biocompatibility.

Laccase enzyme has been widely used as the catalyst of the biocathodes in enzymatic biofuel cells (BFCs); the poor biocompatibility of this enzyme (e.g., poor catalytic activity in neutral media and low tolerance against chloride ion) and the lack of selectivity for oxygen reduction at the laccase-based biocathode against ascorbic acid, unfortunately, offer a great limitation to future biological applications of laccase-based BFCs. This study demonstrates a facial yet effective solution to these limitations with the assistance of hydrophobic room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (Bmim(+)PF(6)(-)). With the Bmim(+)PF(6)(-) overcoating, the laccase-based biocathodes possess a good bioelectrocatalytic activity toward O(2) reduction in neutral media and a high tolerance against Cl(-). Moreover, the Bmim(+)PF(6)(-) overcoating applied to the laccase-based biocathodes also well suppresses the oxidation of ascorbic acid (AA) at the biocathodes and thereby avoids the AA-induced decrease in the power output of the laccase-based BFCs. The mechanisms underlying the excellent properties of the Bmim(+)PF(6)(-) overcoating are proposed based on the intrinsic features of ionic liquid Bmim(+)PF(6)(-). To demonstrate the applications of the BFCs with the as-prepared biocathodes in biologically relevant systems, an AA/O(2) BFC is assembled with single-walled carbon nanotubes (SWNTs) as electrode materials both for accelerating AA oxidation at the bioanode and for promoting direct electron transfer of laccase at the biocathode. With the presence of 0.50 mM AA in 0.10 M quiescent phosphate buffer (pH 7.2), the assembled BFC has an open circuit voltage of 0.73 V and a maximum power output of 24 µW cm(-2) at 0.40 V under ambient air and room temperature. This study essentially offers a new strategy for the development of enzymatic BFCs with a high biocompatibility.