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Packed capillary columns have become the standard front-end separation device for mass spectrometry-based proteomics. The development of simple, fast, and robust capillary column technology, especially that with mass-fabrication capacity, can greatly improve analytical throughput and reproducibility in omics research. In this technical note, we report a centrifugal packing technology, which has the capability to mass fabricate high quality capillary columns with a 2886 columns/day fabrication throughput. The centrifugally packed columns presented significantly improved efficiency (reduced plate height

Specific locations of carbon-carbon double bonds (C═C) in lipids often play an essential role in biological processes, and there has been a booming development in C═C composition analysis by mass spectrometry. However, a universal derivatization and fragmentation pattern for the annotation of C═C positions in lipids is still challenging and attractive. To expand this field in lipidomics, a flexible and convenient

Herein, we report the use of a polarity-sensitive, solvatochromic fluorophore Nile red to label and probe individual hydrogen nanobubbles on the surface of an indium-tin oxide (ITO) electrode. Nanobubbles are generated from the reduction of water on ITO and fluorescently imaged from the transient adsorption and desorption process of single Nile red molecules at the nanobubble surface. The ability to label and fluorescently image individual nanobubbles with Nile red suggests that the gas/solution interface is hydrophobic in nature. Compared to the short labeling events using rhodamine fluorophores, Nile red-labeled events appear to be longer in duration, suggesting that Nile red has a higher affinity to the bubble surface. The stronger fluorophore-bubble interaction also leads to certain nanobubbles being co-labeled by multiple Nile red molecules, resulting in the observation of super-bright and long-lasting labeling events. Based on these interesting observations, we hypothesize that Nile red molecules may start clustering and form some kind of molecular aggregates when they are co-adsorbed on the same nanobubble surface. The ability to observe super-bright and long-lasting multifluorophore labeling events also allows us to verify the high stability and long lifetime of electrochemically generated surface nanobubbles.

Nanoflow liquid chromatography-mass spectrometry (NanoLC-MS) has become the method of choice for the analysis of complex biological systems, especially when the available sample amount is limited. The preparation of high-performance capillary columns for nanoLC use is still a technical challenge. Here, we report a segmented microfluidic method for the preparation of packed capillary columns, where liquid segments were used as soft, dynamic, and well-dispersed slurry reservoirs for carrying and delivering micrometer packing particles. Based on this microfluidic packing technology, the column bed was assembled layer-by-layer at a 50 μm resolution, and ultralong capillary columns of 3, 5, and 10 m were fabricated in such a manner. The microfluidically packed columns demonstrated excellent separation efficiencies of 116 000 plates/m. The higher efficiencies obtained at higher slurry concentrations also indicate that a high-quality packed bed can be obtained without sacrificing the packing speed. Kinetic performance limit analysis shows that the microfluidic packed columns have higher peak capacity production efficiency in the high-resolution region, presenting an improved separation impedance of 2800, which is significantly better than columns packed with the conventional slurry packing method. In comparison with a commercial nanoLC column, a 5 m long microfluidic packed column was evaluated for proteomic analysis using a standard HeLa protein digest and presented 261% improvement in peptide identification capability, resulting in significantly enhanced protein identification confidence.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 10

Surfactants, mimics of contamination, play an important role in nanobubble nucleation, stability, and growth at the electrode surface. Herein, we utilize single-molecule fluorescence microscopy as a sensitive imaging tool to monitor nanobubble dynamics in the presence of a surfactant. Our results show that the presence of anionic and nonionic surfactants increase the rate of nanobubble nucleation at all potentials in a voltage scan. The fluorescence and electrochemical responses indicate the successful lowering of the critical gas concentration needed for nanobubble nucleation across all voltages. Furthermore, we demonstrate that the accumulation of surfactants at the gas-liquid interface changes the interaction of fluorophores with the nanobubble surface. Specifically, differences in fluorophore intensity and residence lifetime at the nanobubble surface suggest that the labeling of nanobubbles is affected by the nature of the nanobubble (size, shape, etc.) and the structure of the gas-liquid interface (surfactant charge, hydrophobicity, etc.).

Here, we present a study of how liposomes are loaded and release their contents during their electrochemical detection. We loaded 200 nm liposomes with a redox mediator, ferrocyanide, and used amperometry to detect their collision on a carbon-fiber microelectrode (CFE). We found that we could control the favorability of their electroporation process and the amount of ferrocyanide released by modifying the osmolarity of the buffer in which the liposomes were suspended. Interestingly, we observed that the quantity of the released ferrocyanide varied significantly with buffer osmolarity in a nonmonotonic fashion. Using stimulated Raman scattering (SRS), we confirmed that this behavior was partly explained by fluctuations in the intravesicular redox concentration in response to osmotic pressure. To our surprise, the redox concentration obtained from SRS was much greater than that obtained from amperometry, implying that liposomes may release only a fraction of their contents during electroporation. Consistent with this hypothesis, we observed barrages of electrochemical signals that far exceeded the frequency predicted by Poisson statistics, suggesting that single liposomes can collide with the CFE and electroporate multiple times. With this study, we have resolved some outstanding questions surrounding electrochemical detection of liposomes while extending observations from giant unilamellar vesicles to 200 nm liposomes with high temporal resolution and sensitivity.

Here we report the development and characterization of a high throughput sensing device for single liposome detection. The device incorporates a quartz nanopipette positioned near a carbon-fiber microelectrode (CFE). Liposomes (∼200 nm diameter) loaded with Fe(CN)64- are driven out of the nanopipette orifice where they are sensed as a transient decrease in the measured ionic current (resistive-pulse analysis). Simultaneously, a redox signal is collected at the CFE due to the release of internalized redox molecules from translocating liposomes to the CFE surface. Interestingly, we observed that the redox signals arise coincidently with resistive-pulses, suggesting that leakage of liposome contents occurs during translocation. Further investigation suggested that liposome disruption occurs at the nanopore orifice and is not dependent on the nanopore electric field. The probability of this disruption appears to rely on the velocity of fluid flow in the nanopore as well as the nanopore geometry. The high-throughput nature of our technique may prove useful for rapid analysis of liposomal drug formulations or rapid, robust, direct measurement of neurotransmitter concentration in isolated vesicles from neurons and neuroendocrine cells.

Aggregation induced-emission (AIE) and antenna effect (AE) are two important luminescence behaviors. Connecting them into polymers is a promising, but very challenging work, which can supply opport...

In this report, we describe the fabrication, characterization, and use of a massive array of closed bipolar ultramicroelectrodes (UMEs) in electrochemical imaging applications. The bipolar UME array is 1 cm

When coupled online with mass spectrometry (MS), widely applied water-in-oil droplet-based microfluidics for single cell analysis met problems. For example, the oil phase rumpled the stability, efficiency, and accuracy of MS, the conventional interface between MS and the microfluidic chip suffered the low sample introduction efficiency, and the transportation rates sometimes unmatched the readout dwell times for transient signal acquisition. Considering cells are already "droplets" with hydrophilic surface and elastic hydrophobic membrane, we developed an oil-free passive microfluidic system (OFPMS) that consists of alternating straight-curved-straight microchannels and a direct infusion (dI) micronebulizer for inductively coupled plasma quadrupole-based mass spectrometry (ICP-qMS) of lined-up single-cell. OFPMS guarantees exact single cell isolation one by one just using a thermo-decomposable NH4HCO3 buffer, eliminating the use of any oil and incompatible polymer carriers. It is more flexible and facile to adapt to the dwell time of ICP-qMS owing to the adjustable throughput of 400 to 25000 cells/min and the controllable interval time of at least 20 ms between the lined-up adjacent single cells. Quantitative single-cell transportation and high detection efficiency of more than 70% was realized using OFPMS-dI-ICP-qMS exemplified here. Thus, cell-to-cell heterogeneity can be simply uncovered via the determination of metals in the individual cells.

In this work, we describe the preparation and use of a thin metal film modified Indium Tin Oxide (ITO) electrode as a highly conductive, transparent, and electrocatalytically active electrode material for studying nanobubbles generated at the electrode/solution interface. Hydrogen and oxygen nanobubbles were generated from water electrolysis on the surface of a Au/Pd alloy modified ITO electrode. The formation of single H2 and O2 nanobubbles was imaged in real time during a potential scan using single-molecule fluorescence microscopy. Our results show that while O2 nanobubbles can be detected at an early stage in the oxygen evolution reaction (OER), the formation of H2 nanobubbles requires a significant overpotential. Our study shows that thin-film-coated ITO electrodes are simple to make and can be useful electrode substrates for (single molecule) spectroelectrochemistry research.

Knowledge of the chemical identity of metabolite molecules is critical for the understanding of the complex biological systems to which they belong. Since metabolite identities and their concentrations are often directly linked to the phenotype, such information can be used to map biochemical pathways and understand their role in health and disease. A very large number of metabolites however are still unknown; i.e., their spectroscopic signatures do not match those in existing databases, suggesting unknown molecule identification is both imperative and challenging. Although metabolites are structurally highly diverse, the majority shares a rather limited number of structural motifs, which are defined by sets of 1H and 13C chemical shifts of the same spin system. This allows one to characterize unknown metabolites by a divide-and-conquer strategy that identifies their structural motifs first. Here, we present the structural motif-based approach "SUMMIT Motif" for the de novo identification of unknown molecular structures in complex mixtures, without the need for extensive purification, using NMR in tandem with two newly curated NMR molecular structural motif metabolomics databases (MSMMDBs). For the identification of structural motif(s), first, the 1H and 13C chemical shifts of all the individual spin systems are extracted from 2D and 3D NMR spectra of the complex mixture. Next, the molecular structural motifs are identified by querying these chemical shifts against the new MSMMDBs. One database, COLMAR MSMMDB, was derived from experimental NMR chemical shifts of known metabolites taken from the COLMAR metabolomics database, while the other MSMMDB, pNMR MSMMDB, is based on predicted chemical shifts of metabolites of several existing large metabolomics databases. For molecules consisting of multiple spin systems, spin systems are connected via long-range scalar J-couplings. When this motif-based identification method was applied to the hydrophilic extract of mouse bile fluid, two unknown metabolites could be successfully identified. This approach is both accurate and efficient for the identification of unknown metabolites and hence enables the discovery of new biochemical processes and potential biomarkers.

When coupled online with mass spectrometry (MS), widely applied water-in-oil droplet-based microfluidics for single cell analysis met problems. For example, the oil phase rumpled the stability, efficiency, and accuracy of MS, the conventional interface between MS and the microfluidic chip suffered the low sample introduction efficiency, and the transportation rates sometimes unmatched the readout dwell times for transient signal acquisition. Considering cells are already "droplets" with hydrophilic surface and elastic hydrophobic membrane, we developed an oil-free passive microfluidic system (OFPMS) that consists of alternating straight-curved-straight microchannels and a direct infusion (

In this work, we describe the preparation and use of a thin metal film modified Indium Tin Oxide (ITO) electrode as a highly conductive, transparent, and electrocatalytically active electrode material for studying nanobubbles generated at the electrode/solution interface. Hydrogen and oxygen nanobubbles were generated from water electrolysis on the surface of a Au/Pd alloy modified ITO electrode. The formation of single H

Exocytosis is an ultrafast cellular process which facilitates neuron-neuron communication in the brain. Microelectrode electrochemistry has been an essential tool for measuring fast exocytosis events with high temporal resolution and high sensitivity. Due to carbon fiber's irreproducible and inhomogeneous surface conditions, however, it is often desirable to develop simple and reproducible modification schemes to enhance a microelectrode's analytical performance for single-cell analysis. Here we present carbon-fiber microelectrodes (CFEs) modified with a thin film of electrodeposited gold for the detection of exocytosis from rat pheochromocytoma cells (PC12), a model cell line for neurosecretion. These new probes are made by a novel voltage-pulsing deposition procedure and demonstrate improved electron-transfer characteristics for catecholamine oxidation, and their fabrication is tractable for many different probe designs. When we applied the probes to the detection of catecholamine release, we found that they outperformed unmodified CFEs. Further, the improved performance was conserved at cells incubated with L-DOPA (l-3,4-dihydroxyphenylalanine), a precursor to dopamine that increases the quantal size of the release events. Future use of this method may allow nanoelectrodes to be modified for highly sensitive detection of exocytosis from chemical synapses.

In this Technical Note, we describe a method to fabricate nanopore-supported Pt nanoparticle electrodes and their use in bipolar electrochemistry. A Pt nanoparticle is deposited on the orifice of a solid-state nanopore inside a focused-ion beam (FIB) system. Complete blockage of the nanopore with Pt metal forms a closed bipolar nanoparticle electrode whose size and shape can be tunable in one simple step. Nanoparticle electrodes and their arrays can be prepared on different substrates such as the tip of a glass pipet, a double-barrel pipet, and a freestanding silicon nitride membrane. Steady-state voltammetry can be performed on such nanoparticle electrodes via bipolar electrochemistry. Moreover, an array of Pt nanoparticles can be used for fluorescence-enabled electrochemical microscopy. Future use of highly advanced FIB systems may allow nanoparticles of10 nm to be fabricated which may enable coupled electrochemical reactions of single redox molecules. Pipette-supported single particle electrodes may also find useful applications in high resolution imaging with nanoscale scanning electrochemical microscopy (SECM) and neurochemical analysis inside single cells.

Identification of metabolites in complex mixtures represents a key step in metabolomics. A new strategy is introduced, which is implemented in a new public web server, COLMARm, that permits the coanalysis of up to three two-dimensional (2D) NMR spectra, namely, 13C–1H HSQC (heteronuclear single quantum coherence spectroscopy), 1H–1H TOCSY (total correlation spectroscopy), and 13C–1H HSQC-TOCSY, for the comprehensive, accurate, and efficient performance of this task. The highly versatile and interactive nature of COLMARm permits its application to a wide range of metabolomics samples independent of the magnetic field. Database query is performed using the HSQC spectrum, and the top metabolite hits are then validated against the TOCSY-type experiment(s) by superimposing the expected cross-peaks on the mixture spectrum. In this way the user can directly accept or reject candidate metabolites by taking advantage of the complementary spectral information offered by these experiments and their different sensiti...

We report direct observation of electrochemical and thermal dealloying processes of individual metal alloy nanoparticles (NPs). Electrochemical dealloying of single Ag-Hg alloy NPs was achieved in a basic solution (e.g., pH 13) by oxidizing Hg under controlled potentials. Ag can also be oxidized during single-particle collision. However, it requires elevated potentials. The strong basic environment promoted the formation of metal oxides during collision leading to a unique core-shell type nanostructure which was further confirmed by transmission electron microscopy (TEM). In thermal dealloying, Hg was evaporated due to the use of a high-energy electron beam and the process was imaged in situ inside a TEM. Both electrochemical and thermal dealloying processes resulted in the transformation of an amorphous NP to a more stable Ag-Hg alloy nanocrystal. This work demonstrates that NP collision can be a useful tool to study dealloying processes of various nanomaterials at a single-particle level.

The hydrogen evolution reaction (HER) was studied on single platinum nanoparticles (NPs) using a microjet NP collision system. By ejecting NPs onto a closely positioned ultramicroelectrode (UME), one can study single-particle collision electrochemistry in acid concentrations as high as 3 M or 750 mM on the electrode surface. This is nearly 2 orders of magnitude greater than previously reported in NP-collision studies. The use of high-bandwidth recording and higher acid concentration allows us to temporally resolve the ultrafast hydrogen adsorption and evolution processes in HER on single colliding Pt NPs. Moreover, the electroactive surface area (EASA) and roughness factor ( Rf) of single Pt NPs can be readily derived from the integrated hydrogen adsorption charge. The use of high acid concentrations is critical toward obtaining a full monolayer of adsorbed hydrogen before overlapping with the evolution process. This method allows one to study electrochemical and electrocatalytic behavior of metal NPs in a chemical environment that is close to that used in real applications, for example, fuel cells and electrolyzers.

NMR-based metabolomics is a powerful tool to comprehensively monitor chemical processes in biological systems. Key to its success is the accurate and complete metabolite identification and quantification. Due to the inherent complexity of most metabolic mixtures, NMR peak overlap can make data analysis of 1D or even 2D NMR spectra challenging, especially for the(1)H spectral region from 3.2–4.5 ppm that is dominated by carbohydrates and their derivatives. To address this problem, we present an effective method for carbohydrate signal removal in complex metabolomics samples by oxidation via the addition of sodium periodate (NaIO(4)). In an optional step, reaction products can be removed with hydrazide beads. The treated samples show substantially simplified 1D and 2D NMR spectra with their carbohydrate peaks removed, whereas non-carbohydrate peaks remain mostly unaffected. This allows the unrestricted detection of those metabolites that are otherwise obscured by carbohydrate signals. The method was first tested for metabolite model mixtures and then applied to urine and serum samples. It revealed a significant number of non-carbohydrates that were made unambiguously observable and identifiable by this method. The proposed protocol is simple and it is suitable for high-throughput sample treatment for the comprehensive metabolite identification in a broad range of samples.

Here, we report a nanopipette-based electrochemical approach to prepare metal nanoelectrodes with excellent control over electrode size, shape, and thickness of the insulation wall. Nanoelectrodes are prepared by electrochemical plating in a laser-pulled quartz nanopipette tip immersed in a liquid gallium/indium alloy electrode, which not only protects the ultrasmall quartz tip but also starts electrodeposition from the tip orifice. This versatile approach enables reproducible fabrication of electrodes of several different metals, including gold, platinum, silver, and copper. Moreover, nanoelectrodes with varying sizes can be easily prepared by focused ion-beam milling. A unique aspect of this method is the control over the thickness of quartz insulation walls relative to the size of the electroactive surface enabling control of the RG (defined as the radius of the insulating sheath over the radius of the active metal electrode). As such, these nanoelectrodes may be especially attractive as useful nanoprobes in high-resolution imaging applications, such as scanning electrochemical microscopy.

Among human body fluids, serum plays a key role for diagnostic tests and, increasingly, for metabolomics analysis. However, the high protein content of serum poses significant challenges for nuclear magnetic resonance (NMR)-based metabolomics studies because it can strongly interfere with metabolite signal detection and quantitation. Although several methods for protein removal have been proposed, including ultrafiltration and organic-solvent-induced protein precipitation, there is currently no standard operating procedure for the elimination of protein from human serum samples. Here, we introduce novel procedures for the removal of protein from serum by the addition of nanoparticles. It is demonstrated how serum protein can be efficiently, cost-effectively, and environmentally friendly removed at physiological pH (pH 7.4) through attractive interactions with silica nanoparticles. It is further shown how serum can be processed with nanoparticles prior to ultrafiltration or organic-solvent-induced protein precipitation for optimal protein removal. After examination of all of the procedures, the combination of nanoparticle treatment and ultrafiltration is found to have a minimal effect on the metabolite content, leading to remarkably clean homo- and heteronuclear NMR spectra of the serum metabolome that compare favorably to other methods for protein removal.

Electrostatic interactions play an essential role in many analytical applications including molecular sensing and transport studies using nanopores and separation of charged species. Here, we report the voltammetric quantification of electrostatic ion enrichment in a 5-20 nm thin electrochemical cell. A simple lithographic micro/ nanofabrication process was used to create ultrathin-layer cells (UTLCs) with a critical dimension (i.e., cell thickness) as small as 5 nm. The voltammetric response of a UTLC was found to be largely dominated by the electrostatic interaction between charges on the cell walls and the redox species. We show that the ultrasmall cell dimension yielded a 100–300-fold enrichment for cationic redox species. An interesting surface adsorption effect was also demonstrated.

Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signal. This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+). Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 μm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

P450 3A4 (CYP3A4) is one of the most important isoforms in the human cytochrome P450 superfamily. It was used as an example in this proof-of-concept study in order to demonstrate an activity-based labeling and then click chemistry (CC) mediated element-tagging strategy for simultaneously specific quantification and activity measurement of an enzyme using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICPMS). A dual functional hexynylated 17α-ethynylestradiol activity-based probe was synthesized for specifically labeling CYP3A4 and then CC-mediated Eu-tagging with an azido-DOTA-Eu complex for CYP3A4 quantification and activity measurement in human liver microsome and serum samples using (153)Eu SUID ICPMS. The LOD (3σ) of CYP3A4 reached 20.3 fmol when monitoring (151/153)Eu ICPMS signals, in addition to the merits of specificity and simultaneous activity measurement achieved. We believe that this activity-based CC-mediated element-tagging strategy will liberate more potential advantages of ICPMS in bioanalysis.

We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.

A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

We report the design and synthesis of a trifunctional probe for seeing and counting cancer cells using both fluorescence imaging (FI) and inductively coupled plasma mass spectrometry (ICPMS) for the first time. It consisted of a guiding cyclic RGD peptide unit to catch cancer cells via targeting the α(v)β(3) integrin overexpressed on their surface, a 5-amino-fluorescein moiety for FI using confocal laser scanning microscopy (CLSM) as well as a 2-aminoethyl-monoamide-DOTA group for loading stable europium ion and subsequent ICPMS quantification of the cancer cells without the use of radioactive isotopes. In addition to FI, the LOD (3σ) of the α(v)β(3) integrin was down to 69.2-309.4 amol per cell depending on the type of the α(v)β(3)-positive cancer cells when using ICPMS and those of the cancer cell number reached 17-75. This probe developed enables us not only to see but also to count the α(v)β(3)-positive cancer cells ultrasensitively, paving a new way for early diagnosis of cancer.

Here we report the voltammetric study of coupled electrochemical reactions on microelectrodes and nanoelectrodes in a closed bipolar cell. We use steady-state cyclic voltammetry to discuss the overall voltammetric response of closed bipolar electrodes (BPEs) and understand its dependence on the concentration of redox species and electrode size. Much of the previous work in bipolar electroanalytical chemistry has focused on the use of an "open" cell with the BPE located in an open microchannel. A closed BPE, on the other hand, has two poles placed in separate compartments and has remained relatively unexplored in this field. In this work, we demonstrated that carbon-fiber microelectrodes when backfilled with an electrolyte to establish conductivity are closed BPEs. The coupling between the oxidation reaction, e.g., dopamine oxidation, on the carbon disk/cylinder and the reduction of oxygen on the interior fiber is likely to be responsible for the conductivity. We also demonstrated the ability to quantitatively measure voltammetric properties of both the cathodic and anodic poles in a closed bipolar cell from a single cyclic voltammetry (CV) scan. It was found that "secondary" reactions such as oxygen reduction play an important role in this process. We also described the fabrication and use of Pt bipolar nanoelectrodes which may serve as a useful platform for future advances in nanoscale bipolar electrochemistry.

Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods has been quantitatively compared, and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.

Large perfusive silica beads (particle size 110 microm, through pore approximately 2 microm) held in place by the keystone effect were used as single-particle frits for the manufacture of particulate packed capillary columns. High-quality capillary electrochromatographic separations of a standard test mixture of alkylbenzenes were obtained over the full voltage range of 5-30 kV, with no requirement for pressurization. Excellent robustness was demonstrated by the reproducibility of migration times, peak efficiencies, and resolution during 100 consecutive runs at the highest voltage (30 kV) without thermostating and pressurization. Superior performance relative to traditional sinter-fritted columns is ascribed to the heat-free fritting process and short frit length of approximately 110 microm.

A simple benchtop method of fabricating glass-sealed nanometer-sized Au and Pt disk electrodes, glass nanopore electrodes, and glass nanopore membranes is reported. The synthesis of all three structures is initiated by sealing the tips of electrochemically sharpened Au and Pt microwires into glass membranes at the end of a soda lime or lead glass capillary. Pt and Au nanodisk electrodes are obtained by hand polishing using a high-input impedance metal oxide semiconductor field effect transistor (MOSFET)-based circuit to monitor the radius of the metal disk. Proper biasing of the MOSFET circuit, based on a numerical analysis of the polishing circuit impedance, allows for the reproducible fabrication of Pt disk electrodes of radii as small as 10 nm. Significantly smaller background currents in voltammetric measurements are obtained using lead glass capillaries, a consequence of the lower mobility of Pb(2+) (relative to Na(+)) in the glass matrix. Glass nanopore electrodes and glass nanopore membranes are fabricated, respectively, by removal of part or all of the metal sealed in the glass membranes. The nanostructures are characterized by atomic force microscopy, steady-state voltammetry, and ion conductivity measurements.

Metabolomics aims at a complete characterization of all metabolites in biological samples in terms of both their identities and concentrations. Because changes of metabolites and their concentrations are a direct reflection of cellular activity, it allows for a better understanding of cellular processes and function to be obtained. Although NMR spectroscopy is routinely applied to complex biological mixtures without purification, overlapping NMR peaks often pose a challenge for the comprehensive and accurate identification of the underlying metabolites. To address this problem, we present a novel nanoparticle-based strategy that differentiates between metabolites based on their electric charge. By adding electrically charged silica nanoparticles to the solution NMR sample, metabolites of opposite charge bind to the nanoparticles and their NMR signals are weakened or entirely suppressed due to peak broadening caused by the slow rotational tumbling of the nanometer-sized nanoparticles. Comparison of the edited with the original spectrum significantly facilitates analysis and reduces ambiguities in the identification of metabolites. This method makes NMR directly sensitive to the detection of molecular charges at constant pH, as demonstrated here both for model mixtures and human urine. The simplicity of the approach should make it useful for a wide range of metabolomics applications.

Fluorescence-enabled electrochemical microscopy (FEEM) is demonstrated as a new technique to image transient concentration profiles of redox species generated on ultramicroelectrodes (UMEs). FEEM converts an electrical signal into an optical signal by electrically coupling a conventional redox reaction to a fluorogenic reporter reaction on a closed bipolar electrode. We describe the implementation of FEEM for diffusion layer imaging and use an array of thousands of parallel bipolar electrodes to image the diffusion layers of UMEs in two and three dimensions. This new technique provides a way to image an entire 2-dimensional lateral cross section of a dynamic diffusion layer in a single experiment. By taking several of these lateral cross sections at different axial positions in the diffusion layer, a 3-dimensional image of the diffusion layer can be built. We image the diffusion layer of a 10 μm diameter carbon fiber electrode over the course of a cyclic voltammetry experiment and compare the FEEM-generated images to concentration profiles generated from numerical simulation. We also image the diffusion layer of a two electrode array consisting of two 10 μm diameter carbon fibers over the course of a potential step experiment.

The steady-state voltammetric response of the truncated conical-shaped glass nanopore electrode is presented. Analytical theory, finite-element simulations, and experimental measurement of the diffusive flux of a redox molecule through the pore orifice demonstrate that the steady-state current decreases rapidly as the pore depth increases and then asymptotically approaches a constant value when the pore depth is approximately 50x larger than the pore orifice. The asymptotic limit of the steady-state current is only a function of the pore orifice radius and the cone angle of the pore and has a finite value for all cone angles greater than zero. Experimental confirmation of the predicted dependence on pore depth is obtained using nanopore electrodes with 100-1000 nm orifice radii, by measuring the steady-state voltammetric current corresponding to the oxidation of ferrocene in acetonitrile solutions containing an excess of supporting electrolyte.

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.

We investigate the principle of the open circuit potential (OCP) change upon a particle collision event based on mixed potential theory and confirmed by a mimic experiment in which we studied the changes in the OCP when two different electrodes (Pt and Au) are brought into contact in a solution that contains some irreversible redox couples. A micrometer-sized Au ultramicroelectrode, when connected in parallel to a Pt micro- or nanoelectrode, showed clearly measurable OCP changes whose magnitude matches well with that predicted by a simplified mixed potential theory for a pair of different electrode materials. On the basis of the study, each electrode establishes a different mixed potential involving two or more half reactions that have different heterogeneous electron transfer kinetics at different electrodes and the OCP changes are very sensitive to the relative ratio of the rate constant of the individual half reaction at different materials.

Continuous plasma separation will be greatly helpful for dynamic metabolite monitoring in kinetics research and drug development. In this work, we proposed a continuous on-chip plasma separation method based on the natural aggregating and sedimentation behavior of red blood cells at low shear rate. In this approach, a glass capillary was first used to realize quick and obvious delamination of blood cells from plasma. A novel "dual-elbow" connector was designed to change the direction of delamination. The blood was finally separated by laminar flow and bifurcation on the microchip. Results demonstrated that the present device can efficiently and stably separate plasma from blood in a continuous means, e.g., in a 4 h separation we did not observe clogging or a trend of clogging. In addition, the present approach can avoid the damage to cells which usually occurs in separation with high shear rate in a microchannel and possible contaminants to plasma. The proposed microchip device is robust, simple, and inexpensive for long time plasma separation with high plasma recovery and less sample consumption. The present work provides an effective tool for metabolite monitoring in pharmacokinetics research and drug development.

This report presents a fundamental study of nanoparticle transport phenomena in conical-shaped pores contained within glass membranes. The electrophoretic translocation of charged polystyrene (PS) nanoparticles (80- and 160-nm-radius) was investigated using the Coulter counter principle (or "resistive-pulse" method) in which the time-dependent nanopore current is recorded as the nanoparticle is driven across the membrane. Particle translocation through the conical-shaped nanopore results in a direction-dependent and asymmetric triangular-shaped resistive pulse. Because the sensing zone of conical-shaped nanopores is localized at the orifice, the translocation of nanoparticles through this zone is very rapid, resulting in pulse widths of ~200 μs for the nanopores used in this study. A linear dependence between translocation rate and nanoparticle concentration was observed from 10(7) to 10(11) particles/mL for both 80- and 160-nm-radius particles, and the magnitude of the resistive pulse scaled approximately in proportion to the particle volume. A finite-element simulation based on continuum theory to compute ion fluxes was combined with a dynamic electric force-based nanoparticle trajectory calculation to compute the position- and time-dependent nanoparticle velocity as the nanoparticle translocates through the conical-shaped nanopore. The computational results were used to compute the resistive pulse current-time response for conical-shaped pores, allowing comparison between experimental and simulated pulse heights and translocation times. The simulation and experimental results indicate that nanoparticle size can be differentiated based on pulse height, and to a lesser extent based on translocation time.

Here we report a new type of microelectrode sensor for single-cell exocytotic dopamine release. The new microsensor is built by forming a gold-nanoparticle (AuNP) network on a carbon fiber microelectrode. First a gold surface is obtained on a carbon fiber microdisk electrode by partially etching away the carbon followed by electrochemical deposition of gold into the pore. The gold surface is chemically functionalized with a sol-gel silicate network derived from (3-mercaptopropyl)trimethoxysilane (MPTS). A AuNP network is formed by immobilizing Au nanoparticles onto the thiol groups in the sol-gel silicate network. The AuNP-network microelectrode has been characterized by scanning electron microscopy (SEM) and steady-state voltammetry. The AuNP-network microelectrode has been used for amperometric detection of exocytotic dopamine secretion from individual pheochromocytoma (PC12) cells. The results show significant differences in the kinetic peak parameters including shorter rise time, decay time, and half-width as compared to a bare carbon fiber electrode equivalent. These results indicate AuNP-network microelectrodes possess an excellent sensing activity for single-cell exocytotic catecholamine release, specifically dopamine. Moreover, key advantageous properties inherent to bare carbon fiber microelectrodes (i.e., rigidity, flexibility, and small size) are maintained in addition to an observed prolonged shelf life stability and resistance to cellular debris fouling and dopamine polymerization.

In this technical note, we report a process in scaling down the fabrication of Au disk nanoelectrodes as small as approximately 4 nm in radii. We have developed a bottom-up approach toward the fabrication of individual disk-shape Au nanoelectrodes. This new approach is based upon electrochemical deposition of Au in a silica nanopore electrode and involves the following four steps. First, a laser-assisted pulling process is employed to fabricate a disk-shape Pt nanoelectrode. Second, a Pt nanopore electrode is obtained by electrochemically etching the Pt from the disk nanoelectrode. Third, a Au metal nanowire is electrochemically deposited using the Pt nanopore electrode as a template. In the last step, the Au electrode is slightly polished to expose a disk-shape Au nanoelectrode, whose size is determined by the size of the initial Pt nanoelectrode. Steady-state voltammetry in the presence of ferrocene has been used to characterize these Au nanoelectrodes. The Au nanoelectrodes are also characterized using cyclic voltammetry in a H2SO4 solution. The results show characteristic peaks corresponding to the formation of Au surface oxides and their subsequent reduction. The Au nanoelectrodes are modified with 6-(ferrocenyl)hexanethiol molecules, and cyclic voltammetry is used to characterize the ferrocene molecules attached at the Au. As an application, we have constructed Au single-nanoparticle electrodes (SNPEs) using the Au disk nanoelectrodes fabricated by electrochemical deposition. Our initial results of such SNPEs show excellent electrochemical response from single Au nanoparticles.

By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick's first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick's law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters Deltai (change in current) and nanotube length. Catechol was used to determine the Deltai attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data.

The preparation and characterization of Pt nanoelectrodes in the range of 1 to 3 nm in radii are reported. A Pt microwire is sealed into a bilayer quartz capillary and pulled into an ultrasharp Pt nanowire sealed in a silica tip using a laser-assisted pulling process. The ultrasharp tip is then sealed into a piece of glass tubing, which is manually polished to expose the Pt. Transmission electron microscopy and steady-state voltammetry are utilized to characterize the nanoelectrodes. The results show that the minimum size of the Pt nanoelectrode is determined by the size of the Pt microwire and parameters used in the pulling process. The heterogeneous electron transfer rate constant for the oxidation of ferrocene, ferrocenemethanol, and potassium hexachloroiridate (III) are determined from steady-state voltammetry using the method of Mirkin and Bard and are found to be k(o) = 7.6 +/- 3.4 cm/s and alpha = 0.85 +/- 0.06 for ferrocene, k(o) = 7.4 +/- 6.9 cm/s and alpha = 0.78 +/- 0.16 for ferrocenemethanol, and k(o) = 6.0 +/- 4.2 cm/s and alpha = 0.72 +/- 0.15 for IrCl(6)(3-).