Fluorescent fibrous mats assembled with self-propagating probes for visual sensing of hydrogen peroxide and choline.

Challenges remain in the facile, rapid and sensitive detection of substances at ultralow levels. In the current study, visual sensors of hydrogen peroxide (H2O2) and choline are developed via the integration of an ultrafine fibrous substrate and self-propagating and aggregation-induced emission (AIE) probes. Self-immolative probes (SIPs) composed of phenylboronic acid triggers and choline units are grafted on electrospun polyethylene terephthalate (PET) fibers, followed by electrostatic adsorption of tetraphenylethene derivatives (TPE-SO3) to obtain fluorescent PET-Ch/TPE fibers. Choline oxidase (ChOX) is immobilized on polystyrene-co-maleic anhydride (PSMA) fibers to obtain PSMA-ChOX, followed by assembly into PET-Ch/TPE@PSMA-ChOX composite mats. The presence of H2O2 initiates the cleavage of phenylboronic acid triggers in SIPs to release choline and choline/TPE complexes from PET-Ch/TPE fibers. The released choline is oxidized by PSMA-ChOX fibers to generate H2O2 that then activates a cascade of self-propagating reactions until the release of all choline/TPE complexes, leading to the alleviation of AIE effect and gradual fluorescence fading of fibrous mats. Thus, the hydrogen peroxide and choline concentrations can be read out from the fluorescence fading time of fibrous mats with a detection limit of 0.5 µM H2O2 within 30 min, providing potential self-test devices for a real-time, naked-eye and sensitive detection of bioactive substances.

Microbiosensor fabrication by polydimethylsiloxane stamping for combined sensing of glucose and choline.

High performance microprobes for combined sensing of glucose and choline were fabricated using microcontact printing (µCP) to transfer choline oxidase (ChOx) and glucose oxidase (GOx) onto targeted sites on microelectrode arrays (MEAs). Most electroenzymatic sensing sites on MEAs for neuroscience applications are created by manual enzyme deposition, which becomes problematic when the array feature size is less than or equal to ∼100 µm. The µCP process used here relies on use of soft lithography to create features on a polydimethylsiloxane (PDMS) microstamp that correspond to the dimensions and array locations of targeted, microscale sites on a MEA. Precise alignment of the stamp with the MEA is also required to transfer enzyme only onto the specified microelectrode(s). The dual sensor fabrication process began with polyphenylenediamine (PPD) electrodeposition on all Pt microelectrodes to block common interferents (e.g., ascorbic acid and dopamine) found in brain extracellular fluid. Next, a chitosan film was electrodeposited to serve as an adhesive layer. The two enzymes, ChOx and GOx, were transferred onto different microelectrodes of 2 × 2 arrays using two different PDMS stamps and a microscope for stamp alignment. Using constant potential amperometry, the combined sensing microprobe was confirmed to have high sensitivity for choline and glucose (286 and 117 µA mM cm-2, respectively) accompanied by low detection limits (1 and 3 µM, respectively) and rapid response times (≤2 s). This work demonstrates the use of µCP for facile creation of multianalyte sensing microprobes by targeted deposition of enzymes onto preselected sites of a microelectrode array.

Monitoring Enzymatic Reactions in Real Time Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry.

We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of ∼5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused-silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected.

A novel biosensor array with a wheel-like pattern for glucose, lactate and choline based on electrochemiluminescence imaging.

Electrochemiluminescence (ECL) imaging provides a superior approach to achieve array detection because of its ability for ultrasensitive multiplex analysis. In this paper, we reported a novel ECL imaging biosensor array modified with an enzyme/carbon nanotubes/chitosan composite film for the determination of glucose, choline and lactate. The biosensor array was constructed by integrating a patterned indium tin oxide (ITO) glass plate with six perforated poly(dimethylsiloxane) (PDMS) covers. ECL is generated by the electrochemical reaction between luminol and hydrogen peroxide that is produced by the enzyme catalysed oxidation of different substrates with molecular oxygen, and ECL images were captured by a charge-coupled device (CCD) camera. The separated electrochemical micro-cells enabled simultaneous assay of six samples at different concentrations. From the established calibration curves, the detection limits were 14 µM for glucose, 40 µM for lactate and 97 µM for choline, respectively. Moreover, multicomponent assays and cross reactivity were also studied, both of which were satisfied for the analysis. This biosensing platform based on ECL imaging shows many distinct advantages, including miniaturization, low cost, and multi-functionalization. We believe that this novel ECL imaging biosensor platform will have potential applications in clinical diagnostics, medicine and food inspection.

An amperometric biosensor for choline determination prepared from choline oxidase immobilized in polypyrrole-polyvinylsulfonate film.

In this paper, a novel amperometric choline biosensor with immobilization of choline oxidase on electrochemically polymerized polypyrrole-polyvinylsulphonate (PPy-PVS) film has been accomplished via the entrapment technique. The effects of pH and temperature were investigated and optimum parameters were found to be 9.0 and 60 °C, respectively. There are two linear parts in the region between 1.0 × 10 (-7) - 1.0 × 10 (-6)M (R(2) = 0.997) and 1.0 × 10 (-5) - 1.0 × 10 (-3) M (R(2) = 0.986). The storage stability and operation stability of the enzyme electrode were also studied.

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix.

Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H(2)SO(4) medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M H(2)O(2) concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H(2)O(2)-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M.

Detection of choline in biological fluids from patients on haemodialysis by an amperometric biosensor based on a novel anti-interference bilayer.

A new and highly selective amperometric biosensor able to analyse choline in clinical samples from patients suffering from renal diseases and receiving repetitive haemodialysis treatment is described. The proposed biosensor is based on choline oxidase immobilized by co-crosslinking onto a novel anti-fouling and anti-interferent membrane. Between the several polymeric films electrosynthesized on a Pt electrode whose permselective behaviours were here investigated, those based on overoxidized polypyrrole/poly(o-aminophenol) bilayer revealed the most effective in rejecting common interferents usually present in biological fluids. The so realized biosensor showed notably analytical performances, displaying linear choline responses up to 100 µM, a sensitivity of 156 nA mM-1 mm-2 and a limit of detection, calculated at a signal-to-noise ratio equal to 3, of 1 µM; further, the within-a-day coefficients of variation for replicate (n = 3) were 2.7% and 1.2% at 100 µM and 10 µM choline levels, respectively. The remarkable performances and anti-interference behaviour allowed us the use of the proposed biosensor for the selective and fouling-free detection of choline in dialysate coming from patients on haemodialysis and even in their unpretreated human sera. Preliminary results gave choline levels in good agreement with the expected values.

Fabrication of polyethyleneimine-functionalized reduced graphene oxide-hemin-bovine serum albumin (PEI-rGO-hemin-BSA) nanocomposites as peroxidase mimetics for the detection of multiple metabolites.

The ultrasensitive bioassays are increasingly demanded for disease diagnosis and environmental monitoring. The combined unique natures of the components in nanocomposites have led to their wide applications in bioanalysis. In the current study, a simple strategy for preparing polyethyleneimine-functionalized reduced graphene oxide-hemin-bovine serum albumin (PEI-rGO-Hemin-BSA) nanocomposites as peroxidase mimetics was demonstrated. The developed nanocomposites of PEI-rGO-Hemin-BSA showed an excellent peroxidase-like activity. Importantly, through the glutaradelhyde crosslinking, PEI-rGO-Hemin-BSA could be further simply combined with various oxidases such as glucose oxidase, cholesterol oxidase, lactate oxidase and choline oxidase for the detection and quantitative measurement of multiple metabolites including glucose, cholesterol, l-lactate, and choline. The developed detection strategy, which is sensitive, convenient, low-costed, and in tiny sample consumption, could be expected wide applications in the disease diagnosis and management of metabolite disorders.

A cationic conjugated polymer and graphene oxide: Application to amplified fluorescence detection of sinapine.

An amplified fluorescence strategy is described for the detection of sinapine (SP) by using a cationic conjugated polymer (PFP) and graphene oxide (GO). It is observed that the fluorescein (FAM)-labeled single-stranded DNA (FAM-DNA) is absorbed on the surface of GO if SP is absent. This causes that fluorescence resonance energy transfer (FRET) from PFP to FAM is inefficient when adding PFP into FAM-DNA/GO complex. If SP is added to FAM-DNA/GO complex, FAM-DNA is desorbed from GO surface due to the competitive binding of SP and FAM-DNA toward GO. In this case, FAM-DNA is close to PFP in the presence of PFP through strong electrostatic interaction, leading to the occurrence of efficient FRET. Based on the above phenomenon, we demonstrate a method to amplify fluorescence signal of traditional GO-based SP assay by introducing PFP. In comparison to the use of single GO, the combination of PFP with GO-based strategy displays high turn-on ratio and enhanced sensitivity with a limit of detection as low as 7.3 ng mL-1 for SP detection. Satisfactory results in practical samples are also obtained by the recovery experiments, demonstrating the potential application of cationic conjugated polymer in plant-derived small molecule.

Electrosynthesized poly(pyrrole)/poly(2-naphthol) bilayer membrane as an effective anti-interference layer for simultaneous determination of acethylcholine and choline by a dual electrode amperometric biosensor.

Several neural diseases appear related to the neurotransmitter acethylcholine (ACh) and its metabolite choline (Ch) brain levels so that their simultaneous determination is essential. A cross-talk and interference free dual electrode amperometric biosensor for the simultaneous determination of both analytes has been developed. Acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized by glutaraldehyde co-crosslinking with bovine serum albumin. A very efficient rejection of electroactive interferents has been achieved by a novel electrosynthesized polymeric bilayer membrane composed by overoxidised poly(pyrrole) and poly(2-naphthol) films. Sensitivities towards several electroactive interferents ranged from ca. 0.04% (e.g. ascorbate) to ca. 0.3% (e.g. dopamine) of those relevant to ACh and Ch (11 and 15 microA/microM, respectively). Detection limits (at S/N=3) in flow injection analysis were ca. 100 nM for both ACh and Ch at the ChO-AChE electrode and ca. 40 nM for Ch at the ChO sensor. Biosensor performances appear more than adequate for brain tissue homogenates and cerebrospinal fluids analysis where average levels in the low micromolar range are typically found.

A facile and practical biosensor for choline based on manganese dioxide nanoparticles synthesized in-situ at the surface of electrode by one-step electrodeposition.

In this paper, a facile and sensitive biocompatible biosensor based on Nafion/choline oxidase/manganese dioxide composite film was developed for the determination of choline chloride. Manganese dioxide (MnO2) nanoparticles, possessing the advantages of large specific surface areas, good hydrophilicity, great permeability as well as excellent biocompatibility, were synthesized in-situ at the surface of the glassy carbon electrode (GCE) by one-step electrodeposition. And then, choline oxidase (ChOx) was immobilized on the MnO2 modified GCE with coating a Nafion film to hold the ChOx/MnO2 film on the electrode surface firmly. Upon optimized conditions, a linear range of 8.0-1.0 mM was obtained for the sensor in a cyclic voltammetry method, with a detection limit as low as 5.0 µM. Besides, the biosensor was successfully employed to detect choline in milk, milk powder and feedstuff samples, providing a promising alternative for the practical application.

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline.

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.

Diagnostic capability of salivary biomarkers in the assessment of head and neck cancer: A systematic review and meta-analysis.

The purpose of this systematic review and meta-analysis was to evaluate the diagnostic value of salivary biological markers in the diagnosis of head and neck carcinoma. Studies were gathered by searching Cochrane, EMBASE, LILACS, MEDLINE, and PubMed. The references were also crosschecked and a partial grey literature search was undertaken using Google Scholar. The methodology of selected studies was evaluated using the 14-item Quality Assessment Tool for Diagnostic Accuracy Studies. After a two-step selection process, 15 articles were identified and subjected to qualitative and quantitative analyses. The studies were homogeneous, and all had high methodological quality. Combined biomarkers demonstrated better accuracy with higher sensitivity and specificity than those tested individually. Furthermore, the salivary biomarkers reviewed predicted the early stages of head and neck carcinoma better than the advanced stages. A restricted set of five single biomarkers (interleukin-8, choline, pipecolinic acid, l-phenylalanine, and S-carboxymethyl-l-cysteine) as well as combined biomarkers demonstrated excellent diagnostic test accuracy. The present systematic review confirms the potential value of a selected set of salivary biomarkers as diagnostic tools for head and neck carcinoma.

Simultaneous determination of acetylcholine, choline and physostigmine in microdialysis samples from rat hippocampus by microbore liquid chromatography/electrochemistry on peroxidase redox polymer coated electrodes.

Microbore column liquid chromatography with post-column immobilized enzyme reactor (IMER) and electrochemical detection on redox polymer coated electrodes was used for detection of acetylcholine (ACh) and choline (Ch) in microdialysis samples. The sensitivity of the coated electrodes, decreased gradually by about 10%/day, with highest reduction of 30% within the first 16 h of use. A number of choline derivatives were tested as possible internal standards, of those acetylethylhomocholine (AEHCh) and butyrylcholine (BCh) were found the most suitable candidates since they both provided high enzymatic conversion in the IMER. Physostigmine produced a negative peak, possibly reflecting oxidation of eseroline--a decarbamoylated product of reversible reaction of physostigmine with immobilized acetylcholine esterase. The probes, implanted in the ventral hippocampi of awake rats were perfused at a flow-rate of 1.25 microl/min with Ringer solution containing 10 microM physostigmine or with artificial cerebrospinal fluid only. The concentrations of ACh in 10-microl samples at basal conditions were between 0.9-2.5 nM, whereas in the presence of physostigmine the ACh levels raised to 41-48 nM. Physostigmine concentration was reduced to 8.8 microM, indicating its in vivo delivery of about 12%. The coefficients of variation were reduced from 7.4% for external standard method after every sixth sample to 5.8% and 5.9% for internal standardization with AEHCh and BCh, respectively. The latter method shortened the total analysis time by about 15%, thus being especially suitable for continuous long-lasting off-line or on-line monitoring. Additionally, other endogenous cholines such as butyrylcholine or synthetic choline derivatives could be detected by the present method.

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer.

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2':5',2"-terthiophene-3'-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H(2)O(2) in a choline solution at +0.6V. The other one modified with ChO/HRP utilized the reduction process of H(2)O(2) in a choline solution at -0.2V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0 x 10(-6) to 8.0 x 10(-5) M and the other based on ChO/CPME from 1.0 x 10(-6) to 5.0 x 10(-5) M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0 x 10(-7) and 4.0 x 10(-7) M, respectively. The response time of sensors was less than 5s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor.

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).

High-field localized 1H NMR spectroscopy in the anesthetized and in the awake monkey.

Localized cerebral in vivo 1H NMR spectroscopy (MRS) was performed in the anesthetized as well as the awake monkey using a novel vertical 7 T/60 cm MR system. The increased sensitivity and spectral dispersion gained at high field enabled the quantification of up to 16 metabolites in 0.1- to 1-ml volumes. Quantification was accomplished by using simulations of 18 metabolite spectra and a macromolecule (MM) background spectrum consisting of 12 components. Major cerebral metabolites (concentrations >3 mM) such as glutamate (Glu), N-acetylaspartate (NAA), creatine (Cr)/phosphocreatine (PCr) and myo-inositol (Ins) were identified with an error below 3%; most other metabolites were quantified with errors in the order of 10%. Metabolite ratios were 1.39:1 for total NAA, 1.38:1 for glutamate (Glu)/glutamine (Gln) and 0.09:1 for cholines (Cho) relative to total Cr. Taurine (Tau) was detectable at concentrations lower than 1 mM, while lactate (Lac) remained below the detection limit. The spectral dispersion was sufficient to separate metabolites of similar spectral patterns, such as Gln and Glu, N-acetylaspartylglutamate (NAAG) and NAA, and PCr-Cr. MRS in the awake monkey required the development and refinement of acquisition and correction strategies to minimize magnetic susceptibility artifacts induced by respiration and movement of the mouth or body. Periods with major motion artifacts were rejected, while a frequency/phase correction was performed on the remaining single spectra before averaging. In resting periods, both spectral amplitude and line width, that is, the voxel shim, were unaffected permitting reliable measurements. The corrected spectra obtained from the awake monkey afforded the reliable detection of 6-10 cerebral metabolites of 1-ml volumes.

Exocellular phospholipase C activity from a Propionibacterium acnes strain isolated from a periodontal pocket.

The culture supernatant of a strain of Propionibacterium acnes was investigated for its phospholipase (PL) activity. The microorganism was isolated from a periodontal pocket of a patient with periodontal disease. Supernatants from cultures of this microorganism were used as a source to obtain enzymes. Proteins from the supernatants were concentrated, and their enzymatic activity was partially purified through molecular sieving. The procedure yielded two peaks of activity. This activity was shown to hydrolyze phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) and was effective in a pH range of 5 to 9, with an optimal activity at pH 7.0. Divalent cations were not required for activity of the enzymes. Analysis of the products obtained from the hydrolysis of PC labeled in the choline, phosphoryl, or acyl moieties and PE containing labeled oleic acid indicated that the supernatants' activity was mostly phospholipase C (PL-C). Phospholipase C can act synergistically with other factors to produce tissue damage, and hence may contribute to the pathogenesis of periodontal disease.

The determination of choline in human semen by the enzymic method.

Studies are reported on the specific enzymic microdetermination of free choline in human semen. The concentration of choline in normal semen was found to be 0.9 - 1.4 mg/ml. The minimum detectable concentration of choline using the method described was 1.5 micrograms. Normal levels of choline in vaginal fluid, saliva, serum and urine could not be detected by this procedure.