16通道神经信息双模检测分析仪的研制与应用

研制了一种可检测分析神经电生理信号和递质化学信号的16通道双模检测分析仪.仪器山硬件和软件系统组成,其电流和电压分辨率分别为10 pA和0.6 μV,具备实时峰电位分离,数字IIR滤波,计时电流法,循环伏安法等常用电生理和电化学检测分析方法.为验证仪器的性能分别进行了电生理信号和电化学信号的检测实验.在SD大鼠脑部电生...     

基于双模检测仪器的大鼠神经放电和神经化学信号同步检测研究

研制了一种可同步检测神经电生理和化学信号的双模检测仪,并应用此仪器开展了双模同步检测实验。此仪器具有0.3μV电压分辨率的64通道神经电生理检测功能、pA级电流精度的4通道电化学检测功能,具备动作电位分离分类、计时电流法、循环伏安法等常用的检测功能,并且能实现对双模神经信号的同步观测和分析。在采用此仪器开展的单模实验中,完整地检测到了64通道的模拟神经信号,结合微电极阵列获得了信噪比( S/N)为6的神经动作电位发放;采用循环伏安法,获得了铁氰化钾在溶液浓度0.1~10 mmol/L范围内与电流响应的线性相关系数为0.9889;同时,采用计时电流法获得了抗坏血酸在溶液浓度10~800μmol/L 范围内与电流响应的线性相关系数为0.9841。结合大鼠脑缺血模型开展了双模检测实验,在该模型中成功地同步检测到了神经动作电位发放和抗坏血酸浓度变化引起的电流变化,并发现大鼠大脑初级感觉皮层抗坏血酸浓度与动作电位发放呈一定负相关关系。     

脑死亡过程中谷氨酸与场电位的同步检测微电极阵列研究

高浓度胞外K+会引起神经元的去极化、谷氨酸释放、甚至细胞死亡。为研究高浓度K+对在体神经元的影响,采用微机电系统( MEMS)方法制作了一种植入式微电极阵列( MEA),其上包含形状、位置固定的电化学(50伊150μm)和电生理(直径为15μm)检测位点,可同时进行脑内神经递质谷氨酸、局部场电位信号( LFP)双模检测。将这种MEA植入到大鼠纹状体后,给大鼠皮层施加高浓度K+刺激,结果表明,高钾刺激增加了纹状体内谷氨酸浓度,同时抑制了神经电生理活动。这是首次采用双模MEA研究神经元在体死亡过程,结果验证了双模微电极阵列在体检测的可行性,可用于研究脑内神经电化学、电生理的时空关系。     

纳米通道单分子检测低噪音电流放大器系统的研究

研制了一种新型微电流放大器系统,用于检测琢-Hemolysin生物蛋白纳米通道在单分子检测实验中所产生的微弱电流信号(<100 pA)。在1 mol/L KCl、10 mmol/L Tris-HCl,1 mmol/L EDTA的缓冲液(pH 8.0)中测定了DNA-PEG-DNA交联物与纳米通道的穿越和碰撞信号。实验中使用3 kHz贝塞尔滤波器和100 kHz模数转换器来对电流进行采样。结果表明,此放大器系统能够有效降低电流记录过程中的噪音,有利于分辨待测物分子与纳米通道作用所产生的较小阻断的电流信号(<10 pA)。     

纳米TiN修饰平面微电极阵列在神经信息双模检测中的应用

采用氮化钛(TiN)修饰平面微电极阵列(pMEA),对其性能进行改进,开展了离体神经电生理和神经递质电化学的检测研究。采用磁控溅射法在实验室自制微电极阵列上修饰具有纳米结构的TiN材料,修饰后的微电极阻抗降低近一个数量级,背景噪声基线降至±6μV,信噪比是修饰前的1.7倍。在SD大鼠离体脑片神经电生理信号检测中,信噪比可达10∶1,能分离提取±12μV的微弱信号。神经递质多巴胺电化学信号检测下限达50 nmol/L(信噪比2∶1),浓度在0.05~100μmol/L内与电流响应的线性相关系数为0.998。实验结果表明,在微电极表面修饰纳米TiN,降低了微电极阻抗,提高了信噪比,实现了对神经信息微弱信号的检测。     

NEWS——高灵敏检测腺苷的可再生电化学传感系统、绿色高效的三联吡啶钌电化学发光体系

湖南大学化学化工学院沈国励研究组研制出一种高灵敏检测腺苷的可再生电化学传感系统(Analytieal Chemistry,2007,79(7):2933-2939)。该传感系统具有如下优势:酪胺电聚合膜提供良好的再生性和稳定性;纳米金层提供相对大的比表面积;电活性物质二茂铁的实用性;目标物的识别反应引起检测探针脱落而有效削弱了残余峰电流。     

基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

运用纳米金复合探针结合蛋白芯片,建立了一种检测心肌损伤标志物的新方法.构建了2种纳米金探针:标记有检测抗体和DNA探针1的检测探针和标记有DNA探针2(与DNA探针1的碱基互补配对)的信号探针.当目的抗原存在时,检测探针经检测抗体和抗原,与芯片上捕获抗体结合固定在芯片上,信号探针通过碱基互补配对与检测探针结合使信号放大,最后利用纳米金成核原理染色,通过显微镜观察结果并定量分析.该体系在40 min内可检测多种标志物,其中肌钙蛋白Ⅰ(c TnⅠ)的检出限为10 pg/m L,与临床电化学发光法(ECLIA)灵敏度相当;肌红蛋白(MYO)与新型脂肪酸结合蛋白(HFABP)的检出限分别为640和10 pg/m L,与ECLIA及酶联免疫吸附法(ELISA)相比,灵敏度显著提高.     

检测急性早幼粒细胞白血病PML/RARα融合基因的电化学传感器研制

针对急性早幼粒细胞白血病(APL)中PML/RARα融合基因的碱基序列,设计了锁核酸(LNA)修饰的发夹结构捕获探针,结合信号探针构建新型的"三明治"电化学传感模式.信号探针末端修饰的生物素可与酶上的亲和素结合,通过检测酶催化H2O2氧化底物3,3＇,5,5＇-四甲基联苯胺(TMB)产生的电化学信号,实现对靶序列的检测...     

SERS纳米探针对电刺激过程中细胞内产生活性氧的检测

电刺激是用于细胞内紊乱电活动引起疾病的一类重要治疗方式. 在电刺激过程中是否会诱导细胞内活性氧(ROS)水平的改变, 以及常规抗氧化抑制药物与电刺激治疗同时运用带来的影响, 目前尚未有相关研究. 本文设计了一种具有较好生物相容性的金/银核壳纳米棒表面增强拉曼(SERS)活性探针, 用于电刺激过程中细胞内产生ROS的检测. 将该探针与细胞共孵育, 使其内化入细胞, 对细胞进行不同时间的电刺激, 利用拉曼光谱对SERS探针的信号进行检测. 实验结果表明, 随着电刺激时间的延长, SERS信号减弱, 说明细胞内产生ROS的量明显增加. 该传感机制是利用ROS能刻蚀金/银核壳纳米棒的银壳, 从而使其变薄引起SERS信号减弱. 抗坏血酸(AA)和谷胱甘肽(GSH)两种抗氧化抑制剂类药物与电刺激同时运用时, 可观察到它们会对电刺激过程产生的ROS有清除作用. 该研究发展了一类用于细胞内ROS检测的光谱方法, 也为异常的氧化应激和肿瘤治疗过程中的组合用药提供了建议.     

基于纳米金探针和基因芯片的DNA检测新方法

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法. 荧光纳米金探针表面标记有两种DNA探针: 一种为带有Cy5荧光分子的信号探针BP1, 起信号放大作用; 另一种为与靶DNA一部分互补的检测探针P532, 两种探针比例为5∶1. 当靶DNA存在时, 芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA, 将靶DNA固定于芯片上; 荧光纳米金探针通过检测探针与靶DNA及芯片结合, 在芯片上形成“三明治”复合结构, 最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量. 新方法检测灵敏度高, 可以检测浓度为1 pmol/L的靶DNA, 操作简单, 检测时间短. 通过改进纳米金探针的标记和优化杂交条件, 可进一步提高核酸检测的灵敏度, 这将在核酸检测方面具有重要的应用价值.     

An Arrayed Micro‐glutamate Sensor Probe Integrated with On‐probe Ag/AgCl Reference and Counter Electrodes

Complete all‐in‐one multi‐arrayed glutamate (Glut) sensors have been constructed on a silicon‐based micromachined probe composed of micro‐platinum (Pt) working electrodes, a micro‐silver/silver chloride (Ag/AgCl) reference electrode (RE), and a micro‐Pt counter electrode (CE). The OCP shift of the electrodeposited Ag/AgCl on‐probe micro‐reference electrode compared with a Ag/AgCl wire is <0.1 mV/h. The composition ratio of Ag, Cl, and Pt on the electrodeposited on‐probe micro‐reference electrode is observed to be 1.00 : 0.48 : 0.02 analyzed by EDS. The miniaturized amperometric Glut biosensors were constructed on working electrode sites (electrode area: ∼8.5×10⁻⁵ cm²) of the microprobe modified with glutamate oxidase (GlutOx) enzyme layers for the selective, fast, and continuous detection of L‐glutamate. The sensor selectivity towards common electroactive interferents has been improved significantly by coating the electrode surface with perm‐selective polymer layers, overoxidized polypyrrole (PPY) and Nafion®. The sensitivity, detection range, and response time of the proposed all‐in‐one Glut biosensors are 204.7±5.8 nA μM⁻¹ cm⁻² (N=5), 4.99–109 μM, and 2.7±0.3 sec, respectively and no interferent signals of AA and DA were observed. The sensor can be reused over 19 times of continuous repetitive operation (total measurement time: ∼4 hours) and the sensor sensitivity can retain up to four weeks of storage.     

A Neural Network Model in the Calibration of Glucose Sensor Based on the Immobilization of Glucose Oxidase into Polypyrrole Matrix

The immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power.     

Real Time In Vivo Measurement of Ascorbate in the Brain Using Carbon Nanotube‐Modified Microelectrodes

A carbon fiber microelectrode modified with a composite film of carbon nanotubes and Nafion was developed for in vivo ascorbate (AA) measurements in brain tissue. The modified‐microelectrodes exhibit an electrocatalytic activity for AA oxidation by shifting the peak potential negatively to ?0.040 V, showing a sensitivity of 37 pA/µM, a detection limit of 2.5 µM, a response time of 0.3 s and don’t respond to several electroactive compounds found in the brain extracellular space. In the rat hippocampus, the basal concentration of AA was 290 µM, and glutamate‐evoked changes in AA were biphasic comprising fast and slow components.     

基于导数直方图和神经网络的色谱基线提取算法

根据基线信号的变化特征,提出了一种新的色谱基线提取算法－基于一阶导数直方图和神经网络的色谱基线提取算法。鉴于信号总是存在着噪声,含噪声的基线信号一阶导数值也较大,并且分布无规律,而采用了多分辨率小波变换滤波技术。通过选择阈值门限,本算法可精确提取出信号的基线,尤其适用于非基线对称类的色谱信号的基线（趋势函数）提取。     

Analysis of glutamate in striatal microdialysates using capillary electrophoresis and laser-induced fluorescence detection

High-performance liquid chromatography (HPLC) with electrochemical detection has been used routinely to analyse the neurochemical constituents of brain microdialysates. However, conventional HPLC analysis requires large injection volumes and hence lengthy dialysis sampling times. Capillary electrophoresis (CE) is a rapid high-resolution separation technique with the ability to routinely handle very small sample volumes. If CE is coupled to a high-sensitivity detection system, such as laser-induced fluorescence (LIF), it becomes a powerful and rapid separation technique for the analysis of small-volume microdialysis samples.

These preliminary studies report reduced separation times for the excitatory amino acid glutamate, prederivatised with naphthalene 2,3-dialdehyde, and demonstrate its detection within small-volume brain microdialysis samples. The limit of detection for this system was 10⁻⁸ M.

Characterisation of striatal microdialysis samples comprised infusions of Ca²⁺-free artificial cerebrospinal fluid (aCSF) and Tetrodotoxin (TTx) (10 mM) to demonstrate that the detected transmitter is of neuronal origin and released in a calcium-dependent manner.

Removal of calcium from aCSF resulted in a decrease in glutamate in dialysis samples. Glutamate release significantly decreased (p<0.05) to ca. 40% of preinfusion control levels after 60 min and this level was maintained throughout the sampling period. These data suggest that glutamate release is, to some degree, a calcium-dependent process. TTx infusion (10 μM) produced a significant (p<0.05) reduction in glutamate release to ca. 10% of preinfusion levels. It would therefore appear that glutamate release is dependent on neuronal activity. In summary, we have demonstrated the establishment of CE-LIF and microdialysis for the measurement of glutamate.     

Laser-induced breakdown spectroscopy: Time-resolved spectrochemical applications

We have added time resolution to laser-induced breakdown spectroscopy in two forms, by gating an optical multichannel analyzer (OMA) and by time-resolving the output of a photomultiplier with a boxcar amplifier. Spectra were obtained for temporal segments of 25 to 100 ns, from 25 ns to 50 µs after initiation of the breakdown. OMA spectra of oxygen illustrate the power of this technique for survey purposes. The photomultiplier-boxcar arrangement was used to detect phosphorus atoms from diisopropylmethyl phosphonate in air, and also to detect chlorine in air, both in real time. In the former experiments we detected 690 ppm (w/w) of phosphorus and project a limit of detection with our current apparatus of 15 ppm (w/w). For chlorine, we observed signal from 120 ppm (w/w) and project a limit of detection of 60 ppm (w/w).     

Frequency-selective absorbance detection: Refractive index and turbidity compensation with dual-wavelength measurement

A frequency-selective absorbance detection approach and its applications are described. First, a digital signal processor-lock-in amplifier (DSP-LIA)-based absorbance detector was evaluated. Compared to a simple operational amplifier (TL082CP)-based detector, the DSP-LIA-based detector showed lower noise levels, but the relative advantage was reduced under very low photocurrent levels (down to few nA). A 7 cm pathlength flow cell with this commercial LIA-based detector exhibited excellent Beer's law linearity (r² = 0.9999) and a noise level of 7 micro absorbance units (μAU). The limit of detection (LOD, S/N = 3) for methyl orange (MO) was 7 nM with this detector. Finally, as a more affordable alternative to an LIA, a balanced demodulator integrated circuit chip was used to fabricate a dual wavelength-frequency-selective LED-based absorbance detector. This device successfully compensated refractive index (RI) effect and turbidity effect in test flow systems. The LOD for MO with this system was 8 nM.     

End-column electrochemical detection for inorganic and organic species in high-voltage capillary electrophoresis

The electronics and construction for an end-column ultramicroelectrode (3–10 μm) detection system that permits the use of medium-sized capillaries (25 μm I.D.) without appreciable effects from the potential field at the end of the capillary. Normal peak-to-peak noise over 10 s was 0.01–0.1 pA. The background noise observed for a 200 × μm carbon-fiber electrode placed either 180 μm within a 25-μm capillary or at a point 500 μm away from the capillary was essentially the same. A study of detector response as a function of the position of the electrode has shown that accurate location of the electrode is important for sensitive and reproducible detection. These studies also showed that differences between the density of the electrolyte existing the capillary and the electrolyte in the detection cell could cause anomalous electrode response depending on the location of the electrode relative to the end of the capillary. Application of a carbon fiber or an Hg film electrode gave detection limits (twice the peak-to-peak noise over 10 s) of 2 · 10⁻⁸ mol/l for Pb²⁺, 1 · 10^{− 5} mol/l for NO₂⁻ and 5 · 10⁻¹⁰ mol/l for catechol.     

A low-noise,wideband preamplifier for a fourier-transform ion cyclotron resonance mass spectrometer

FTMS performance parameters such as limits of detection, dynamic range, sensitivity, and even mass accuracy and resolution can be greatly improved by enhancing its detection circuit. An extended investigation of significant design considerations for optimal signal-to-noise ratio in an FTMS detection circuit are presented. A low noise amplifier for an FTMS is developed based on the discussed design rules. The amplifier has a gain of approximately 3500 and a bandwidth of 10 kHz to 1 MHz corresponding to m/z range of 100 Da to 10 kDa (at 7 Tesla). The performance of the amplifier was tested on a MALDI-FTMS, and has demonstrated a 25-fold reduction in noise in a mass spectrum of C(60) compared with that of a commercial amplifier.     

Micro fluorescent analysis system integrating GaN-light-emitting-diode on a silicon platform

A micro fluorescent analysis system is proposed using silicon micromachining. GaN blue light-emitting diode (LED) monolithically integrated on a silicon substrate is used as a light source for the fluorescent analysis system. The blue light suits the excitation of several dyes used commonly in fluorescent analysis. Silicon photodiode (Si-PD) that matches the visible and near infrared fluorescent wavelengths of dyes is integrated on a silicon substrate. Polydimethylsiloxane (PDMS) micro-channels are also stacked for flowing dye-sensitized liquid. Therefore, the proposed system is an integrated system that can be composed on a silicon platform, i.e. a bottom layer of Si-PD, a middle layer of GaN-LED on silicon substrate and a top layer of micro PDMS channel. An aperture is opened into the GaN-LED layer by deep reactive ion etching to create a ring-shaped GaN-LED and a through-hole for detection. The light from the ring-shaped GaN-LED in the middle layer excites the dye-sensitized liquid in the top micro-channel layer. The fluorescence emitted from dye is detected by the Si-PD on the bottom layer at an angle larger than 90 degrees from the direction of excitation. Therefore, the detection optics consist basically of a dark-field illumination optical system. In order to evaluate the performance of the integrated system, fluorescence of fluorescein isothiocyanate (FITC) solution flowing in the micro channel is measured. From the measurement, the noise, sensitivity and limit of detection in the fabricated system are evaluated for FITC dye to be 0.57 pA, 1.21 pA μM(-1) and 469 nM, respectively. From these results, a compact fluorescence analysis system is demonstrated.