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1.
It is a great challenge to develop electrochemical sensors with superior sensitivity that concurrently possess high biocompatibility for monitoring at the single cell level. Herein we report a novel and reusable biomimetic micro-electrochemical sensor array with nitric oxide (NO) sensing-interface based on metalloporphyrin and 3-aminophenylboronic acid (APBA) co-functionalized reduced graphene oxide (rGO). The assembling of high specificity catalytic but semi-conductive metalloporphyrin with high electric conductive rGO confers the sensor with sub-nanomolar sensitivity. Further coupling with the small cell-adhesive molecule APBA obviously enhances the cytocompatibility of the microsensor without diminishing the sensitivity, while the reversible reactivity between APBA and cell membrane carbohydrates allows practical reusability. The microsensor was successfully used to sensitively monitor, in real-time, the release of NO molecules from human endothelial cells being cultured directly on the sensor. This demonstrates its potential application in the detection of NO with very low bioactive concentrations for the better understanding of its physiological function and for medical tracking of patient states.  相似文献   

2.
《Electroanalysis》2004,16(8):640-643
Nitric oxide (NO) is an important molecule in many different physiological phenomena. Investigation of nitric oxide production in vivo requires a specialized sensor capable of real‐time concentration measurement, with a high spatial resolution of NO gas production. In this study, a flexible microsensor is developed specifically for measurement of production of nitric oxide. The new sensor consists of a Pt/Ir working electrode coupled with an integrated Ag/AgCl reference electrode. The sensor is coated with a series of NO‐selective membrane polymers to prevent potential amperometric response due to interfering species. Presented experimental data demonstrates the ability for NO detection between 100 and 400 nM concentrations with a linear response (R2=0.9997). The detection limit of the sensor is 2.14 nM (S/N=3). Various selectivity experiments are indicative of a resistance to interfering species such as dopamine, norepinephrine, L ‐arginine.  相似文献   

3.
Neurogranin (Ng) is a neuron-specific protein kinase C (PKC) substrate, which contains four cysteine (Cys) residues. Recently, it has been shown that Ng is a redox-sensitive protein and is a likely target of nitric oxide (NO) and other oxidants [F.-S. Sheu, C.W. Mahoney, K. Seki, K.-P. Huang, Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin, J. Biol. Chem. 271 (1996) 22407-22413: J. Li, J.H. Pak, F.L. Huang, K.-P. Huang, N-methyl-D-aspartate induces neurogranin/RC3 oxidation in rat brain slices, J. Biol. Chem. 274 (1999) 1294-1300]. In this study, we directly examine the redox reactions between dissolved NO and Cys as well as between NO and bacterial expressed Ng in its reduced form, at concentrations approximate to the physiological levels in phosphate buffer solution (PBS) under aerobic conditions. The reaction kinetics are measured directly by our newly developed electrochemical sensor. Our sensor is based on the chemical modification of electrode with immobilized nanoparticles of transition metal palladium (Pd) which serves as catalytic centers for the electrochemical oxidation of thiol and NO selectively and quantitatively at different potentials. It detects Cys and Ng in a linear range from nano to micromolar concentration at + 450 mV, vs. a saturated calomel reference electrode (SCE), while the detection of NO at the sensor can be optimally achieved at + 700 mV (vs. SCE) with a linear current-to-concentration range of nM to microM. It thus provides a selective control to monitor two reactants independently. With this sensor as a detector, we found that (1) the oxidation of either Cys or Ng by NO is a fast reaction which reaches a near completion within 1-2 min at its physiological concentration; and (2) after the completion of reaction, NO is mostly, if not all, regenerated, an observation consistent with the reaction mechanism involving the formation of S-nitrosothiol as an intermediate. The reaction kinetics of both NO to Cys and NO to Ng implies that NO can achieve local action on cellular proteins in addition to its effect on targets located in neighboring cells via concentration-gradient-dependent diffusion.  相似文献   

4.
A new method employing a combination of microdialysis sampling and chemiluminescence (CL) detection was developed to monitor nitric oxide (NO) in vivo. A special probe was designed with an interference-free membrane to achieve very high selectivity for NO. High sensitivity was achieved by optimizing the working system and improving the NO sampling time. This system was used in vivo to monitor blood and brain tissue in rats and rabbits. The system is sensitive enough to detect variations of NO formation under different physiological states. The linear valid range of NO determination is 5 nM to 1 μM, with a 3σ detection limit of 1 nM; real NO concentrations in test animals used in this work were found to be in the range of 1-5 nM or even less. Finally, the effects of body temperature, NO donors, Viagra, NO activators, NO cofactors and NO interference (such as O2) were investigated carefully in different physiological situations.  相似文献   

5.
A novel electrochemical device for the direct detection of S-nitrosothiol species (RSNO) is proposed by modifying an amperometric nitric oxide (NO) gas sensor with thin hydrogel layer containing an immobilized organoselenium catalyst. The diselenide, 3,3'-dipropionicdiselenide, is covalently coupled to primary amine groups in polyethylenimine (PEI), which is further cross-linked to form a hydrogel layer on a dialysis membrane support. Such a polymer film containing the organoselenium moiety is capable of decomposing S-nitrosothiols to generate NO(g) at the distal tip of the NO sensor. Under optimized conditions, various RSNOs (e.g., nitrosocysteine (CysNO), nitrosoglutathione (GSNO), etc.) are reversibly detected at 相似文献   

6.
Ye X  Rubakhin SS  Sweedler JV 《The Analyst》2008,133(4):423-433
Nitric oxide (NO) is endogenously generated by nitric oxide synthase (NOS) enzymes and is involved in a surprisingly wide range of biological functions. As efforts are made to elucidate the regulatory mechanisms of NOS expression and function, there is increasing interest in following NOS activity directly by monitoring NO production. Additionally, spatial and temporal measurements of NO are important for understanding its function and metabolism. In this work, developments in technology enabling NO detection in biological systems are reviewed. Measuring NO at single cell levels is important as NOS is heterogeneously distributed; however, such measurements are difficult as physiological NO levels are in the low nanomolar to low micromolar range. Here, three categories of analytical techniques enabling NO detection at single cell levels are highlighted: fluorescence microscopy, capillary electrophoresis with laser induced fluorescence detection, and electrochemistry. For each, the basic principles, performance, applications, figures of merits and limitations are presented in terms of single cell NO detection.  相似文献   

7.
In this work we report on the design, microfabrication and analytical performances of a new electrochemical sensor array (ESA) which allows for the first time the simultaneous amperometric detection of nitric oxide (NO) and peroxynitrite (ONOO(-)), two biologically relevant molecules. The on-chip device includes individually addressable sets of gold ultramicroelectrodes (UMEs) of 50 μm diameter, Ag/AgCl reference electrode and gold counter electrode. The electrodes are separated into two groups; each has one reference electrode, one counter electrode and 110 UMEs specifically tailored to detect a specific analyte. The ESA is incorporated on a custom interface with a cell culture well and spring contact pins that can be easily interconnected to an external multichannel potentiostat. Each UME of the network dedicated to the detection of NO is electrochemically modified by electrodepositing thin layers of poly(eugenol) and poly(phenol). The detection of NO is performed amperometrically at 0.8 V vs. Ag/AgCl in phosphate buffer solution (PBS, pH = 7.4) and other buffers adapted to biological cell culture, using a NO-donor. The network of UMEs dedicated to the detection of ONOO(-) is used without further chemical modification of the surface and the uncoated gold electrodes operate at -0.1 V vs. Ag/AgCl to detect the reduction of ONOOH in PBS. The selectivity issue of both sensors against major biologically relevant interfering analytes is examined. Simultaneous detection of NO and ONOO(-) in PBS is also achieved.  相似文献   

8.
A Hadamard transform-capillary electrophoresis-UV (HT-CE-UV) detection technique is described for the analysis of biological samples. Pseudorandom injections of sample and buffer according to a simplex matrix obtained from the corresponding Hadamard matrix is performed with conventional capillaries. Alternating injections are achieved with a novel capillary "T" connector created by drilling conventional capillary dimensions through a 1-cm diameter polycarbonate disc. This connector design coupled with a switching system allows for rapid, electrokinetic injections of solution into alternating sample and buffer capillary arms for UV detection. The standard mixtures of nitric oxide (NO) metabolites, nitrite and nitrate, dissolved in physiological saline solution are injected into the separation capillary according to an 83-element injection sequence to obtain a signal-to-noise ratio (S/N) enhancement of ca. 4.5 over a single injection. Nitrite, being the less concentrated metabolite in NO detection and thereby more difficult to detect, was calibrated with the HT-CE-UV method and a limit of detection (LOD) of 0.56 microM was obtained. Rat blood plasma was analyzed with this detection system and demonstrated to be comparable with NO metabolite concentrations of previously published results. This HT-CE-UV method is described where a unique reservoir tube design that contains 8-microL standard nitrite sample volumes is placed over the end of the capillary arm to explore low volume limits for biological samples.  相似文献   

9.
A novel flow injection analysis (FIA) system suitable for measurement of S-nitrosothiols (RSNOs) in blood plasma is described. In the proposed (FIA) system, samples and standards containing RSNO species are injected into a buffer carrier stream that is mixed with the reagent stream containing 3,3′-dipropionicdiselenide (SeDPA) and glutathione (GSH). SeDPA has been shown previously to catalytically decompose RSNOs in the presence of a reducing agent, such as GSH, to produce nitric oxide (NO). The liberated NO is then detected downstream by an amperometric NO sensor. This sensor is prepared using an electropolymerized m-phenylenediamine (m-PD)/resorcinol and Nafion composite films at the surface of a platinum electrode. Using optimized flow rates and reagent concentrations, detection of various RSNOs at levels in the range of 0.25–20 μM is possible. For plasma samples, detection of background sensor interference levels within the samples must first be carried out using an identical FIA arrangement, but without the added SeDPA and GSH reagents. Subtraction of this background sensor current response allows good analytical recovery of RSNOs spiked into animal plasma samples, with recoveries in the range of 90.4–101.0%.  相似文献   

10.
We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, l-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) μM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.  相似文献   

11.
A new real-time method for measuring a trace concentration of nitric oxide (NO) in a complex matrix routinely used in pharmacological studies of its bioactivity is described. NO was quantified as a gas by chemiluminescence after extraction from a continuous liquid sample flow with a limit of detection of 0.042 nmol dm(-3) at a signal to noise ratio of 3. Theories to calculate the concentration of NO in the liquid sample flow from a direct measurement of NO in the extraction carrier gas are presented. The efficiency of extraction is determined by a stopflow experiment. An example is presented of the measurement of the steady-state concentrations of NO in Krebs-bicarbonate buffer at pH 7.4 and 37 degrees C when its liquid surface is sequentially exposed to gases containing various concentrations of NO in O2 plus CO2.  相似文献   

12.
Trevin S  Bedioui F  Devynck J 《Talanta》1996,43(3):303-311
We describe in this paper an electrochemical and spectrophotometric study of the behavior of an electropolymerized nickel porphyrin film as a sensor for the determination of nitric oxide (NO) in aqueous solution. Our results show that the anodic oxidation of NO at the modified electrode may not be the result of a catalytic effect induced by the porphyrinic complex. However, the current (measured by differential pulse amperometry) and calculated NO concentration showed a linear relationship in the range 15 nM-6 muM in aerobic phosphate buffer solution (pH 7.4). These results provide a fruitful example of calibration of such electrochemical sensors for the selective detection of NO with a calculated detection limit, at a signal-to-noise ratio of three, equal to 1.5 nM.  相似文献   

13.
《Electroanalysis》2006,18(21):2043-2048
A new biosensor is described for the detection of S‐nitrosothiols (RSNOs) based on their decomposition by immobilized glutathione peroxidase (GPx), an enzyme containing selenocysteine residue that catalytically produces nitric oxide (NO) from RSNOs. The enzyme is entrapped at the distal tip of a planar amperometric NO sensor. The new biosensor shows good sensitivity, linearity, reversibility, and response times towards various RSNO species in PBS buffer, pH 7.4 . In most cases, the response time is less than 5 min, and the response is linear up to 6 μM of the tested RSNO species. The lowest detection limit is obtained for S‐nitrosocysteine (CysNO), at approx. 0.2 μM. The biosensor's sensitivity is not affected by the addition of EDTA as a chelating agent; an advantage over other potential catalytic enzymes that contain copper ion centers, such as CuZn‐superoxide dismutase and xanthine oxidase. However, lifetime of the new sensor is limited, with sensitivity decrease of 50% after two days of use. Nonetheless, the new amperometric GPx based RSNO sensor could prove useful for detecting relative RSNO levels in biological samples, including whole blood.  相似文献   

14.
A highly selective nitric oxide(NO) sensor is fabricated and applied to devise an enhanced flow injection analysis(FIA) system for S-nitrosothiols(RSNOs) measurement in biological samples.The NO sensor is prepared using a polytetrafluoroethylene(PTFE) gas-permeable membrane loaded with Teflon AF? solution,a copolymer of tetrafluoroethylene and 2,2-bis(trifluoroethylene)-4,5-difluoro -l,3-dioxole,to improve selectivity.This method is much simpler and possesses good performance over a wide range of RSNOs concentrations.Standard deviation for three parallel measurements of blood plasma is 4.0%.The use of the gas sensing configuration as the detector enhances selectivity of the FIA measurement vs.using less selective electrochemical detectors that do not use PTFE/Teflon type outer membranes.  相似文献   

15.
16.
This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N) 3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.  相似文献   

17.
A new approach for preparing potentially more blood-compatible nitric oxide (NO)-generating polymeric materials is described. The method involves creating polymeric films that have catalytic sites within (lipophilic copper(II) complex) that are capable of converting endogenous S-nitrosothiols present in blood (S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO), etc.) to NO. The catalytic NO generation reaction involves the initial reduction of Cu(II) to Cu(I) within the complex by appropriate reducing agents (e.g., thiolates or ascorbate), followed by the reduction of S-nitrosothiols to NO by the Cu(I) complex at the polymer/solution interface. The NO fluxes observed when PVC or polyurethane films containing the copper(II) complex are placed in solutions containing physiological levels of nitrosothiols (muM levels) reach ca. 8 x 10-10 mol cm-2 min-1, greater than that produced by normal endothelial cells that line all healthy blood vessels. It is thus anticipated that this spontaneous catalytic generation of NO from endogenous nitrosothiols will render such polymeric materials more thromboresistant when in contact with blood in vivo.  相似文献   

18.
An amperometric sol-gel derived sensor that both releases nitric oxide (NO) and measures physiologically relevant concentrations of oxygen (PO2) is described. The sensor consists of a platinum electrode coated with an aminosilane/ethyltrimethoxysilane hybrid xerogel film. Hydrophilic polyurethane (HPU) is doped into the hybrid film to reduce sensor hydration time and increase oxygen permeability. Diazeniumdiolate NO donors are formed within the polymer matrix by exposing the cured film to high pressures of NO. These coatings release up to 7.2 pmol s(-1) cm(-2) of NO over the first 12 h and maintain detectable levels of NO release through 48 h. Sensors modified with HPU-doped, NO-releasing xerogels exhibit a linear response to O2 within 30 min of polarization at -0.65 V vs. Ag/AgCl, and have a sensitivity of approximately 6 nA/mmHg O2. The xerogel coating is stable in buffer solution with minimal fragmentation over 48 h. In vitro biocompatibility studies indicate that these materials effectively reduce platelet adhesion.  相似文献   

19.
A planar ultramicroelectrode nitric oxide (NO) sensor was fabricated to measure the local NO surface concentrations from NO-releasing microarrays of varying geometries. The sensor consisted of platinized Pt (25 microm) working electrode and a silver paint reference electrode coated with a thin silicone rubber gas permeable membrane. An internal hydrogel layer separated the Pt working electrode and gas permeable membrane. The total diameter of the sensor was 相似文献   

20.
Nitric oxide (NO) is recognized as one of the major players in various biochemical processes, including blood pressure, neurotransmission and immune responses. However, experimental studies involving NO are often limited by difficulties associated with the use of NO gas, including its toxicity and precise control over NO concentration. Moreover, the reactions of NO with biological molecules, which frequently occur on time scales of microseconds or faster, are limited by the millisecond time scale of conventional stopped-flow techniques. Here we present a new approach for studying rapid biological reactions involving NO. The method is based on designed ruthenium and manganese nitrosyls, [Ru(PaPy3)(NO)](BF4)2 and [Mn(PaPy3)(NO)](ClO4) (PaPy3H = N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide), which upon photolysis produce NO on a fast time scale. The kinetics of the binding of the photogenerated NO to reduced cytochrome c oxidase (CcO) and myoglobin (Mb) was investigated using time-resolved optical absorption spectroscopy. The NO was found to bind to reduced CcO with an apparent lifetime of 77 micros using the [Mn(PaPy3)(NO)]+ complex; the corresponding rate is 10-20 times faster than can be detected by conventional stopped-flow methods. Second-order rate constants of approximately 1 x 10(8) M(-1) s(-1) and approximately 3 x 10(7) M(-1) s(-1) were determined for NO binding to reduced CcO and Mb, respectively. The generation of NO by photolysis of these complexes circumvents the rate limitation of stopped-flow techniques and offers a novel alternative to study other fast biological reactions involving NO.  相似文献   

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