基于适配体的生物膜干涉检测重组人促红细胞生成素α新方法

采用生物膜干涉(BLI)表面敏感光谱技术,建立了一种基于链DNA适配体39 nt的传感分析方法,实现了重组人促红细胞生成素α(EPO-α)的灵敏检测。将生物素化的39 nt固定于链霉亲和素传感芯片上,经2200 r/min高速振荡实现微升体积分析物的快速扩散。对适配体39 nt的取向、在BLI传感芯片上的固定浓度和非特异性吸附等影响因素进行了考察,发现50 nmol/L 3’末端生物素化39 nt具有最佳响应信号;加入Tween 20和牛血清白蛋白(BSA)可有效克服非特异吸附。基于麦胚凝集素构建了夹心法信号放大体系,在Tris-TB生理缓冲溶液中,检测EPO-α的线性范围为10～200 nmol/L,检出限为5 nmol/L。应用于人血浆等复杂基质中的EPO-α检测,回收率为86.7%～104.2%, RSD<11%,结果令人满意。此BLI传感体系为免标记蛋白相互作用和复杂生物样品检测等实际应用提供了有益参考。     

光电化学传感器的构建及应用

光电化学分析是基于光电化学过程和化学/生物识别过程建立起来的一种新的分析方法。该方法以光作为激发信号,以光电流作为检测信号,具有灵敏度高、响应快速、设备简单和易微型化等优点,在生物和环境等分析领域受到了广泛关注。电极表面修饰的光电层在吸收光子后被激发,所产生的载流子发生电荷分离和电子迁移,进而产生光电流。通过在光电层上进一步修饰传感识别单元,利用直接氧化还原、分子识别与结合、酶催化等方法所导致的光电流的变化与待测分子之间的数量关系,可实现对目标物的定量分析。因此,光电化学传感器在功能结构上包括光电转换单元和传感识别单元两部分,光电层的材料选择和传感识别策略是光电化学传感器构建的两大关键点。本文在对光电化学传感器基本原理及应用领域总结的基础上,对光电化学传感器的材料选择和传感模式进行了分析和综述。     

蛋白质微阵列SPR实时相位检测

将表面等离子体共振(surface plasmon resonance, SPR)与空间相位调制检测结合, 用一束准直的平行光照射传感芯片, 在反射光路中引入渥拉斯顿棱镜, 使光束中的p光和s光产生偏振光干涉形成条纹, 生化反应的有关信息从干涉条纹的相位变化中得出. 选择金膜厚度为30 nm和40 nm的两种芯片, 分别用浓度为50%和20%的酒精-水溶液进行实验, 得到SPR阵列图谱；选择金膜厚度为30 nm, 兔IgG作为阳性对照, 牛血清白蛋白(BSA)作为阴性对照, 与0.2 mL的羊抗兔IgG、1.8 mL水的混合溶液反应, 得到阵列反应图谱. 实验结果表明, 这种方法具有抗干扰能力强, 灵敏度高, 无需标记和可实时检测等优点, 能满足蛋白质芯片检测的要求.     

基于水溶性共轭聚合物的蛋白质检测*

近年来,以共轭聚合物作为生物传感元件,在生物大分子（如核酸、蛋白质）特异性识别、检测方面的研究越来越受到人们的关注。共轭聚合物具有强的光捕获能力,具有倍增光学响应性,可用来放大荧光传感信号,大大提高检测的灵敏度,为生物传感器的发展提供了新的传感模式。基于共轭聚合物的新型生物传感器在医疗诊断、环境检测以及国家安全防御等方面具有广泛的应用前景。本文简要介绍了共轭聚合物的荧光信号放大机制以及在蛋白质、酶、抗原－抗体检测方面的应用。最后对共轭聚合物在蛋白质检测方面的未来发展趋势进行了展望。     

荧光传感器阵列

传感器阵列是基于对动物嗅觉系统的认识发展起来的一种有力的分子识别手段,其由一系列传感单元组成,通过各传感单元对样品响应后产生的特征图谱实现对特定物质的识别检测,尤其对混合样品的鉴定具有突出优势。其中,荧光传感器阵列由于具有灵敏度高、无需参照体系、输出信号丰富、能够成像等优点,已成为近年来传感器阵列发展的重点。本综述根据荧光传感单元形式的不同,分别介绍了溶液型、颗粒型、薄膜型荧光传感器阵列的发展情况,并重点阐述了荧光传感器阵列的设计方法、传感机理及其在对金属离子、有机化合物和生物分子识别中的应用。     

壳聚糖-纳米金复合膜修饰电化学发光免疫传感器检测人免疫球蛋白G

利用壳聚糖与纳米金良好的生物相容性及蛋白固定能力,制备了兼具导电性和透光性的人免疫球蛋白G(IgG)修饰膜,用于修饰玻碳电极,研制了新型电化学发光免疫传感器,并通过扫描电镜(SEM)及交流阻抗技术(EIS)考查了传感器表面性质.基于竞争免疫分析模式,以Ru(bpy)32+标记的羊抗人IgG为发光示踪物,采用新型共反应剂二丁基乙醇胺(DBAE)对光信号进行放大,建立了人IgG的检测方法,线性范围20ngmL-1～1.0μgmL-1,检测限6.5ngmL-1.将该电化学发光传感器应用于人血中IgG的检测,结果令人满意.     

基于CRISPR/Cas的生物传感器在食源性病原菌检测中的应用进展

食源性病原菌严重威胁食品安全及消费者的健康,因此,对食源性病原菌快速、准确的检测方法对保障食品安全尤为重要。规则成簇的短回文重复序列（Clustered regularly interspaced short palindromic repeats,CRISPR）/CRISPR相关系统（CRISPR-associated systems, Cas）生物传感器是在引导核糖核酸（RNA）的作用下,使Cas蛋白与RNA形成RNA复合体,并对靶标基因进行特异性识别,从而将靶标信号转化为可检测的物理和化学信号。CRISPR/Cas生物传感器具有特异性好、可编程以及使用简便等优势,在病原菌检测领域具有广阔的应用前景。本文根据Cas蛋白的种类和作用机制不同,分别介绍了不同CRISPR/Cas系统的作用原理和特点,概述了基于不同CRISPR/Cas的生物传感的信号识别、信号放大、信号输出策略及其在食源性病原菌检测中的应用进展,讨论了基于CRISPR/Cas的多重生物传感器的构建原理及其在多病原菌同时检测中的应用,分析了基于CRISPR/Cas的生物传感器在病原菌快速检测中面临的挑战及未来的发展趋势。     

用于氨氮检测的薄层氨气微传感芯片及其系统研究

设计并制备了一种具有微池薄液层结构的氨气微传感芯片,并构建了以此微传感芯片为敏感单元的氨氮检测系统,探索了使用安培型氨气微传感器检测氨氮的方法。此微传感芯片采用MEMS工艺制备,通过电化学方法在微电极表面修饰了对氨具有良好电催化氧化性能的纳米铂,提高了传感器的灵敏度。在芯片的SU-8微池中滴入微量碳酸丙烯酯(PC),形成可使氨气迅速扩散到电极表面的薄层电解液,使传感器具有较快的响应速度。使用自行设计的氨氮检测系统对氨氮进行检测,考察了氨氮检测的浓度响应特性、时间响应特性、重复性及选择性。氨氮检测系统的线性范围为0.1～5.0 mg/L;检测下限为0.1 mg/L;响应时间小于1 min;重复性偏差为4.0%。     

抗污界面构建及其电化学生物传感应用进展

电化学生物传感器具有灵敏度高、便携性好、响应快速和易于集成等优点,在临床检测方面有很大应用潜力,并在可穿戴健康监测领域得到了快速发展。但在实际临床生物样本检测中,非靶标生物物质会在电极表面产生非特异性吸附（即生物污染）,影响了电化学生物传感器的性能。因此,构建具有防污染能力的传感界面（抗污界面）,防止非靶标物质吸附到电极表面,对于扩大电化学生物传感器的实际应用范围,实现在复杂生物样本中的检测至关重要。本文概述了物理、化学和生物抗污电极界面的构建及其在临床相关生物标志物检测中的应用,为电化学生物传感器实际应用性能的提升提供技术参考,并通过对界面抗污原理和存在问题的探讨,对抗污界面发展前景和未来趋势予以展望。     

NaYF_4：Yb,Er上转换荧光纳米粒子的表面修饰及其在免疫分析中的应用

稀土掺杂上转换荧光纳米材料因其近红外区激发,可见光区发射的特殊发光性能,在生物标记方面具有独特优势,可大幅度降低荧光背景.β-NaYF4：Yb,Er是目前已知的发光效率最高的上转换荧光纳米材料之一,已在生命分析及生物成像分析领域展现出了广阔的应用前景.然而,由于现有β-NaYF4：Yb,Er制备工艺多是在高温条件下于高沸点有机溶剂中反应制得,所得产品在水溶液中的分散性差,限制了其在生命分析中的广泛应用.本文采用聚丙烯酸（PAA）配体交换反应,对表面包覆油酸基团的疏水β-NaYF4：Yb,Er纳米粒子进行了有效的表面修饰.表面修饰后,上转换荧光纳米粒子表面的PAA具有众多游离羧基,使其在水溶液中具有良好的分散性.同时,由于羧基的存在,使得带有氨基的生物分子能够通过化学交联反应结合到纳米粒子表面.本文以PAA表面修饰后的β-NaYF4：Yb,Er纳米粒子为荧光探针,以磁珠作为免疫反应的载体,成功构建了一种新型免疫传感器,对模型靶标分子羊抗人IgG进行了灵敏检测.磁珠表面固定兔抗羊IgG,PAA修饰的β-NaYF4：Yb,Er纳米粒子表面连接人IgG,当样品中存在羊抗人IgG时,便会在磁珠表面形成（兔抗羊IgG-羊抗人IgG-上转换荧光纳米粒子标记的人IgG）三明治式免疫复合体,通过磁分离除去未反应的组分,在980nm激光激发下测定免疫复合体的上转换荧光强度,即可实现靶标分子的高灵敏分析,可检测到低至0.1ng/mL的羊抗人IgG.同时,以磁珠为载体的免疫复合体也可通过激光扫描共聚焦荧光显微镜进行荧光成像分析,背景荧光信号低,成像质量高.实验结果表明,PAA修饰的β-NaYF4：Yb,Er上转换荧光纳米粒子是一种理想的生物标记材料,有望在生物传感及生物成像分析领域获得广泛应用.     

Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody–antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance-based expensive sensors.     

High‐throughput and sensitive particle counting by a novel microfluidic differential resistive pulse sensor with multidetecting channels and a common reference channel

High‐throughput particle counting by a differential resistive pulse sensing method in a microfluidic chip is presented in this paper. A sensitive differential microfluidic sensor with multiple detecting channels and one common reference channel was devised. To test the particle counting performance of this chip, an experimental system which consists of the microfluidic chip, electric resistors, an amplification circuit, a LabView based data acquisition device was developed. The influence of the common reference channel on the S/N of particle detection was investigated. The relationship between the hydraulic pressure drop applied across the detecting channel and the counting throughput was experimentally obtained. The experimental results show that the reference channel designed in this work can improve the S/N by ten times, thus enabling sensitive high‐throughput particle counting. Because of the greatly improved S/N, the sensing gate with a size of 25 × 50 × 10 μm (W × L × H) in our chips can detect and count particles larger than 1.5 μm in diameter. The counting throughput increases with the increase in the flowing velocity of the sample solution. An average throughput of 7140/min under a flow rate of 10 μL/min was achieved. Comparing with other methods, the structure of the chip and particle detecting mechanism reported in this paper is simple and sensitive, and does not have the crosstalking problem. Counting throughput can be adjusted simply by changing the number of the detecting channels.     

6-Chloro-N,N-Diethyl-1,3,5-Triazine-2,4-Diamine (Simazine) Electrochemical Sensing Chip Based on Biomimetic Recognition Utilizing a Molecularly Imprinted Polymer Layer on a Gold Chip

Abstract

Electrochemical sensing chip of 6-chloro-N,N-diethyl-1,3,5-triazine-2,4-diamine (Simazine) was developed on its reductive current. In order to give sensitivity and selectivity to the simazine sensing chip, a gold chip was modified with molecularly imprinted polymer (MIP) for simazine, which is worked as biomimetic recognition element and showed. Using simazine sensing chip, cathodic current of simazine was measured by cyclic voltammetry and the peak current depended on the concentration of simazine. The detection limit was 0.4 µM. Simazine sensing chip was 29 times more sensitive to simazine than bare gold electrode and showed selective response to simazine compared to atrazine and MCC, by the modification with Sim-MIP.     

Characterization of an ion-selective polypyrrole coating and application to the joint determination of potassium, sodium and ammonium by electrochemical impedance spectroscopy and partial least squares method

A stable immobilization of ionophores (INPHs) was achieved by selectively electropolymerizing overoxidized pyrrol on the working electrode surface. The optimal conditions found allowed the immobilization of a huge amount of INPH which remained its recognition properties. A single sensing chip, containing a generic INPH or an INPH mixture, was employed to the joint quantification of potassium, ammonium and sodium in fertilizer samples. Electrochemical impedance spectroscopy (EIS) and partial least square (PLS) regression were used to obtain and process the data, respectively. The sensing element (INPH) and the exposure time were optimized. The best results were obtained by using the dibenzo-18-crown-6 INPH after waiting for 60 s, the time required to stabilize the impedimetric measurement. Taking into account that a single chip was employed, acceptable relative errors were obtained in the determination of potassium and ammonium (17% and 9%, respectively) in real fertilizer samples, also containing sodium. Although sodium concentration could not be estimated with precision, it had to be introduced into the calibration set data in order to model its interference with the other two ions.     

An independently addressable microbiosensor array: what are the limits of sensing element density?

A microdisc sensor array, prepared by thin film technology, has been used as a model for miniaturized multi-functional biosensors. It consists of a series of wells, 20 microns in diameter, possessing a 1000 A Pt layer at the bottom that serves as the indicating electrode. The depth of the wells ranged from 2.3-24 microns, depending on the photoresist employed and the spinning speed used to coat the electrode interconnect grid. Ten such wells were arranged in a circular array within an area of radius 130 microns. The center to center distance between any two of the discs ranged from 30 to 155 microns. Each disc is connected by a conductive film line to corresponding pads on the side of the sensor chip. A cylinder placed on top of the chip array formed the electrochemical cell into which a common reference and counter electrode were placed. The reference electrode was operated at ground potential. Prior to the evaluation of enzyme sensors, an assessment of "chemical cross-talk", the perturbation of sensor response resulting from the overlap of proximal diffusion layers, was made using Fe(CN)6(4-). The preliminary conclusion is that the sensing elements probably must be separated by about 100 microns in order to avoid interference from adjacent sensors. A technique was developed for the precision delivery of enzyme and cross-linking agent to the 2.3 microns cavity, having a capacity of 4 pL. This procedure makes possible the preparation of sensor arrays capable of detecting different analytes by employing different enzymes. The sensors gave reasonably rapid (2-4 s) response with linearity (up to about 10 mM. However, the sensors in the center of the array clearly showed the effects of depletion of substrates by the surrounding sensors.     

透明导电玻璃(ITO)基材自加热传感静态芯片聚合酶链反应(PCR)

利用商品化ITO玻璃导电层的温阻效应, 无需任何微加工手段, 实现了自加热和传感的芯片温度自动程序控制, 最大程度地减小了传感滞后对温度控制稳定性的影响, 温度控制的稳定性达到了0.2 ℃, 升温速度最快可达20 ℃/s以上, 在冷却风扇辅助下降温速度最快达到了8 ℃/s. 芯片温控单元的引线从传统的两对(一对用于传感, 一对用于加热)减少为一对. 通过在该芯片上直接构建多个开放微池反应器的方法成功地实现了λDNA 157 bp片段的并行扩增. 将该芯片置于倒置荧光显微镜样品台上, 以蓝色(575 nm)发光二极管为光源, 以光电倍增管为检测手段检测了dsDNA和SYBR Green Ⅰ嵌合物的荧光强度随温度的实时变化曲线.     

Preparation of Large-area Controllable Patterned Silver Nanocrystals for High Sensitive and Stable Surface-enhanced Raman Spectroscopy

A novel and facile method for fabricating large-area patterned silver nanocrystals was introduced and the investigation on the high sensitive and stable surface-enhanced Raman spectroscopy(SERS) of the nanocrystals was carried out. Nanostructured silicon substrate was processed by laser interference and used as a template for growing silver nanocrystals via galvanic battery reaction method. The substrate with large area for violent chemical reaction was tailored into a nanocell array. The limited reaction area hindered the growth of silver nanocrystals and made their size uniform and controllable. The size and gaps of the nanocrystals could be controlled by template period and ratio, which were easily reproduced by laser interference. Taking 10^-8 to 10^-11 mol/L Rh6G for example, the optimized silver arrays exhibited great potential for ultrasensitive molecular sensing in terms of its high SERS enhancement ability, favorable stability, and excellent reproducibility.     

Specific detection of oxytetracycline using DNA aptamer-immobilized interdigitated array electrode chip

An electrochemical sensing system for oxytetracycline (OTC) detection was developed using ssDNA aptamer immobilized on gold interdigitated array (IDA) electrode chip. A highly specific ssDNA aptamer that bind to OTC with high affinity was employed to discriminate other tetracyclines (TCs), such as doxycycline (DOX) and tetracycline (TET). The immobilized thiol-modified aptamer on gold electrode chip served as a biorecognition element for the target molecules and the electrochemical signals generated from interactions between the aptamers and the target molecules was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV). The current decrease due to the interference of bound OTC, DOX or TET was analyzed with the electron flow produced by a redox reaction between ferro- and ferricyanide. The specificity of developed EC-biosensor for OTC was highly distinguishable from the structurally similar antibiotics (DOX and TET). The dynamic range was determined to be 1-100 nM of OTC concentration in semi-logarithmic coordinates.     

Counting bacteria on a microfluidic chip

This paper reports a lab-on-a-chip device that counts the number of bacteria flowing through a microchannel. The bacteria number counting is realized by a microfluidic differential Resistive Pulse Sensor (RPS). By using a single microfluidic channel with two detecting arm channels placed at the two ends of the sensing section, the microfluidic differential RPS can achieve a high signal-to-noise ratio. This method is applied to detect and count bacteria in aqueous solution. The detected RPS signals amplitude for Pseudomonas aeruginosa ranges from 0.05 V to 0.17 V and the signal-to-noise ratio is 5-17. The number rate of the bacteria flowing through the sensing gate per minute is a linear function of the sample concentration. Using this experimentally obtained correlation curve, the concentration of bacteria in the sample solution can be evaluated within several minutes by measuring the number rate of the bacteria flowing through the sensing gate of this microfluidic differential RPS chip. The method described in this paper is simple and automatic, and have wide applications in determining the bacteria and cell concentrations for microbiological and other biological applications.     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.