Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.     

Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy

Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was approximately 0.5 microM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.     

Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events

Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.     

Performance analysis of different pixel-wise processing methods for depth imaging with single photon detection data

We establish a long-range single photon counting three-dimensional (3D) imaging system based on cage optical structure. Five different pixel-wise processing methods for time-of-flight (TOF) photon counting data are compared with data collected by our 3D imaging system for ranges 40–700?m and a suitable representation model for photon counting data is proposed for pixel-wise processing. Experimental results show that these methods exploit the instrumental response function (IRF), yielding a high-quality 3D image. When the signal photon counts are greater than 13 per pixel, the resulting mean absolute error (MAE) values of the IRF-based methods are better than results from the non-IRF-based methods. Regarding IRF-based methods, the union of subspace (UOS) model-based approach and cross correlation are more suitable than the Markov chain Monte Carlo (MCMC) method in the condition of a small number of return signal photons. These results offer valuable information to promote the implementation of photon counting 3D imaging in real applications.     

Electrokinetic measurements of membrane capacitance and conductance for pancreatic beta-cells

Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary -cells and INS-1 insulinoma cells), determined using the methods of dielectrophoresis and electrorotation. The membrane capacitance value of 12.57 (+/-1.46) mFm(-2), obtained for -cells, and the values from 9.96 (+/-1.89) mFm(-2) to 10.65 (+/-2.1) mFm(-2), obtained for INS-1 cells, fall within the range expected for mammalian cells. The electrorotation results for the INS-1 cells lead to a value of 36 (+/-22) Sm(-2) for the membrane conductance associated with ion channels, if values in the range 2-3 nS are assumed for the membrane surface conductance. This membrane conductance value falls within the range reported for INS cells obtained using the whole-cell patch-clamp technique. However, the total 'effective' membrane conductance value of 601 (+/-182) Sm(-2) obtained for the INS-1 cells by dielectrophoresis is significantly larger (by a factor of around three) than the values obtained by electrorotation. This could result from an increased membrane surface conductance, or increased passive conduction of ions through membrane pores, induced by the larger electric field stresses experienced by cells in the dielectrophoresis experiments.     

浮游植物叶绿素a测定方法的比较分析

本文研究了乙酸纤维滤膜、混合纤维滤膜和玻璃纤维滤膜对浮游植物叶绿素a测定结果的影响,比较了丙酮浸提法、丙酮研磨法、乙醇法和热乙醇法四种方法的稳定性和可替代性。研究表明,三种滤膜对丙酮浸提法和研磨法的测定结果无显著影响,可选择任一种滤膜。丙酮浸提法和研磨法测定结果无显著差异,可相互替换,丙酮研磨法平均损耗率为32.91%,误差较大。乙醇法和热乙醇法可相互替换,但热乙醇法的平行性较差,加热过程难控制。丙酮法测定结果普遍偏高,其中丙酮浸提法的结果明显高于乙醇法,鉴于数据的可比性和简便操作,可采用丙酮浸提法,而乙醇法值得在日后水质监测中推广。     

Comparison of different automatic methods for estimating snow water equivalent

Manual measurements of snow water equivalent (SWE) require a considerable effort, which is why several automatic methods for single-point SWE estimates have been developed. This study provides a first comprehensive review and comparison of seven different automatic ways to determine SWE and/or the daily new snow water equivalent (HNW). We therefore deployed the methods tested over 4 subsequent winter seasons at a test site in the Swiss Alps. As reference we manually measured SWE on a biweekly schedule and HNW on a daily schedule over the same four winter periods. We tested 4 functional categories of methods: (1) direct recording of SWE (SNOWPILLOW, SNOWPOWER), (2) direct recording of HNW (PARSIVEL, GAUGE), (3) complex numerical models driven by meteorological data (SNOWPACK, COSMO-7), and (4) a simple stochastic model based on snow depth (HS) data (SIMPLE). As at our site melting/sublimation was insignificant for the mass balance during the snow accumulation period, differential SWE data could be converted into HNW, while HNW data could be cumulated to SWE. In general, our assessment showed that most of the methods performed reasonably well with respect to SWE estimations, but featured clear deficiencies with respect to HNW estimations. SNOWPILLOW, SNOWPACK, and SIMPLE showed a reasonable performance with respect to both, SWE and HNW estimates, and may hence be attractive for general-purpose applications. However, method-specific limitations have to be considered. This study is intended to facilitate finding the optimum instrumentation for specific applications, different purposes, and given boundary conditions.     

Comparison of different methods for shielding design in computed tomography

The purpose of this work is to compare different methods for shielding calculation in computed tomography (CT). The BIR-IPEM (British Institute of Radiology and Institute of Physics in Engineering in Medicine) and NCRP (National Council on Radiation Protection) method were used for shielding thickness calculation. Scattered dose levels and calculated barrier thickness were also compared with those obtained by scatter dose measurements in the vicinity of a dedicated CT unit. Minimal requirement for protective barriers based on BIR-IPEM method ranged between 1.1 and 1.4 mm of lead demonstrating underestimation of up to 20 % and overestimation of up to 30 % when compared with thicknesses based on measured dose levels. For NCRP method, calculated thicknesses were 33 % higher (27-42 %). BIR-IPEM methodology-based results were comparable with values based on scattered dose measurements, while results obtained using NCRP methodology demonstrated an overestimation of the minimal required barrier thickness.     

Palladium film decoupler for amperometric detection in electrophoresis chips

Demonstrated in this article is that a palladium metal film can be applied to decouple the electric circuitry of electrochemical detection from that of the electrophoretic separation in an electrophoresis chip. The Pd solid-state field decoupler, as well as the working electrodes, is thermally evaporated onto the plastic chip and oriented vertically across the separation channel. After the sample zones flow over the Pd decoupler, their electrochemical response is measured at working electrodes in the downstream pathway. Because the electrodes are on the separation channel, the electrode channel alignment is no longer a problem. For a separation channel of roughly 200 microm in width and 75 microm in depth in 10 mM phosphate (pH 5.1), the noise level at the working electrode is < 15 pA at an electric field of 570 V/cm.     

Comparison of different methods for presenting variable amplitude loading fatigue results

This paper discusses eight methods for presenting fatigue test results for variable amplitude loading and their comparison with constant amplitude loading. While the maximum amplitude method compares constant and variable amplitude loading results by the Woehler and Gassner curves, all other seven methods try to transform the variable amplitude results into the Woehler curve by applying different equations. The advantage of the maximum amplitude method is the direct comparison of the maximum amplitude of the spectrum with the yield strength and with the high‐cycle fatigue strength, which is an important step in structural design. Among the other methods, the best results were obtained by following: most damaging, half damage and mean damage amplitudes. However, the presentation of constant and variable amplitude results by these methods in one scatter band is possible only when the real damage sum is close to D = 1.0.     

Visible photolysis and amperometric detection of S-nitrosothiols

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S-N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500-550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA μM(-1)) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA μM(-1)). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.     

Study of different carbon materials for amperometric enzyme biosensor development

Diamond-like carbon (DLC) electrodes which constitute a new research area in electrochemistry and glassy carbon (GC) electrodes were used as transducers for fabrication of glucose oxidase (GOD) biosensors. The amperometric signal of the enzyme electrode was due to the electro-oxidation of H₂O₂ generated in the enzyme layer. This work has shown that the detection limit of glucose on GOD/GC electrode is 20 μM while it is 50 μM on GOD/DLC electrode. The sensitivity of GOD/GC electrode decreases 4% after 8 days and we have good repeatability of measurements for GOD/DLC electrode in the same day.     

Comparison of different test methods for determining the mar resistance of clearcoats

This paper presents a number of different methods for determining the mar resistance of automotive clearcoats. The various methods are compared on the basis of measurements on different clearcoat systems and the results discussed with other physical parameters (microhardness, crosslinking density). Micro-scratching tests (individual scratches) were also carried out in addition to more practical tests (simulated car washes).     

Dual conductivity/amperometric detection system for microchip capillary electrophoresis

The performance characteristics and advantages of a new dual electrochemical microchip detection system based on simultaneous conductivity and amperometric measurements are described. The system relies on the combination of a contactless conductivity detector with an end-column thick-film amperometric detector. Such coupling of the conductivity and amperometric detection modes in a single separation channel greatly enhances the sample characterization to offer simultaneous measurements of both ionic and electroactive species, improved reproducibility, and confirmation of peak identity. The simultaneous measurement of nitroaromatic and ionic explosives is used for demonstrating the ability to detect both electroactive and ionic species. Major improvements are also observed for analytes responding at both detectors. For example, the generation of dual response ratios can be used to improve the reproducibility and confirm the peak identity/integrity. Such dual response ratios reflect the distinct redox and conductivity properties of the individual analytes. The independence of the two detectors is reflected in the absence of "cross-talk" effects. The behavior of the dual detector is comparable with those of the individual detectors. Such a dual electrochemical detection system is easy to implement and requires inherently portable low-cost instrumentation.     

Comparison of different methods for the assessment of the environmental gamma dose

Four different techniques for the assessment of environmental gamma dose are discussed and compared: high pressure ionisation chamber (Reuter-Stokes type), TLD dosemeters (GR-200), in situ gamma ray spectrometry and dose evaluation from the activity concentration of radionuclides in the soil. Soil samples gathered from a field near Turin (North-West Italy) have been analysed with a p-type HPGe, and their radionuclide concentrations have been used to evaluate the dose at I m above the soil surface. At the same location the dose rate was evaluated with the in situ gamma ray spectrometry (with a portable n-type HPGe) and with the ionisation chamber and the TLD dosemeters. The contribution of cosmic rays was added to the dose rate calculated from the soil samples and the in situ gamma spectrometry. The results obtained with these four techniques agree well within 20%.     

不同分析标准测试土壤铅镉结果比较

土壤的金属测试重点在于前处理,不同的消解方式对于测试结果有着较大的影响,我国目前现行的土壤铅、镉分析标准,主要选用《GB/T 17141-1997土壤质量铅、镉的测定石墨炉原子吸收分光光度法》(以下简称石墨炉法),该方法通过加入四酸加热使土壤完全消解,与《HJ 803-2016土壤和沉积物12种金属元素的测定王水提取-电感耦合等离子体质谱法》(以下简称ICP-MS法)王水提取或国外常用的王水提取法(如美国EPA 6020)在前处理上有较大区别。本文通过对不同质地的实际土壤样品分别使用四酸完全消解法和王水提取法分析铅、镉含量,比较发现上述两种前处理方法的分析结果相近,相对偏差较小。     

Forbidden light detection from single molecules

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to more than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to approximately 10(-17) L. This allows for an exceptional discrimination of bulk-generated against surface-generated fluorescence, which may be of great value when surface-binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determining antigen concentrations down to 5 fmol.     

橡胶防老剂RD不同分析方法的比较

为比较橡胶防老剂RD(TMQ 2,2,4-三甲基-1,2-二氢化喹啉聚合体(C12H15N)n(n=2～4))高效液相色溶剂梯度洗脱的方法和GC-Ms高温裂解方法测定聚合物的含量,采用高效液相色谱法,以水-甲醇作为流动相,检测波长254nm,流速为1mL/min,C18 4.6mm×150mm/5μm色谱柱;GC-Ms法的热分解温度590℃,分解时间6s,热分解室温度为320℃,注射温度250℃,色谱柱DB-1 30mm×0.32mm×0.25μm,升温速度10℃/min,终温为120℃。载气N2流速为1mL/min,FID检测温度280℃,H2流速为30mL/min,空气流速为300mL/min;面积归一法计算含量。结果表明,高效液相色谱法比气相色谱法稳定、可靠,高效液相色谱法测定橡胶防老剂RD的含量优于气相色谱法。     

Comparison of different dispersion models for single layer optical thin film index determination

We here determine the optical properties of different single-layer thin films containing Ta₂O₅, Si, Indium Tin Oxide and Au in the ultraviolet-visible and near infrared ranges. More specifically, we deduce the complex refractive index and thickness from the reflectance and transmittance measured using a spectrophotometer at normal incidence. One major difficulty is to find an appropriate selection of dispersion laws for various types of material (dielectric, semiconductors, and metals). For this purpose, a number of models have been investigated from a theoretical point of view in consideration of the Kramers-Kronig relation. These include the Forouhi-Bloomer model, combined with the modified Drude, Tauc-Lorentz and multiple-oscillator Tauc-Lorentz models. A global optimization procedure had to be employed because of the large number of parameters (from 3 to 15) required to describe the optical dispersion laws. The calculated reflectance and transmittance are in good agreement with experimental data and the complex refractive index is consistent with our knowledge and that already reported.