检测大肠杆菌O157:H7的电化学阻抗谱生物传感器的研究

我们提出了用掺锡的三氧化二铟(ITO)作为工作电极,通过硅烷化固定化技术,将抗大肠杆菌O157:H7单克隆抗体固定在ITO电极表面,利用电化学阻抗谱技术来构建一种新型的免疫传感器.该新型的免疫传感器的检测限为 4×103CFU/mL,检测线性范围为4×103-4×106CFU/mL.实验研究表明,该传感器具有灵敏度较高,检测时间短,操作简单等优点,在临床医学和环境监测中具有应用价值.     

Piezo-resistive method for tuberculosis detection using microcantilever biosensor

Microcantilever device is utilized in several biosensors for detection of bio-molecule of interest. Biosensors task is to identify presence of targeted molecule and supply result into a measurable signal. Comparing with conventional biological equipments, micro scale biosensors are very fast, reliable and price effective. Microelectromechanical systems (MEMS)/nanoelectromechanical systems are used for designing of such biosensors, which might be utilized in several biological applications. Sensing mechanism of biosensor is varied with the application. This paper is focused on detection of bio-molecule using microcantilever beam. Biosensor has shown here uses piezo-resistive method for detection of targeted bio-molecule. This Bio-MEMS device is designed and simulated using coventorware software. This biosensor wants to identify a presence of T.B. in a very suspected patient. For detection of primary stage of tuberculosis (ESAT-6), specific antibodies need to immobilize on top of microcantilever. Once patient sample (consist of ESAT-6) is placed on functionalized cantilever surface, biochemical reaction happen between tuberculosis antigens ESAT-6 and antibodies. Adsorption of antigens will increase mass working on cantilever and results in bending of microcantilever. This deflection shows the presence of tuberculosis within the patient sample.     

Parametric study on fluid structure interaction of a 3D suspended polymeric microfluidics (SPMF3)

Microsystem Technologies - Embedding microfluidic channel inside a microcantilever has drastically improved the sensitivity of microcantilever biosensors. The sensing principles in suspended...     

Urea biosensors

A biosensor is an analytical tool that comprises two essential components—an immobilized biocomponent, in intimate contact with a transducer that converts a biological signal into a measurable electrical signal. This review summarizes the studies carried on the development of biosensors for the analysis of urea in different fields of application, the various techniques of immobilization of urease enzyme, the stability and response time characteristics and the transducers used for biosensor development such as pH electrodes, ammonia gas sensing electrodes, ammonium ion-selective electrodes, optical, conductometric and amperometric transducers. Underlying the importance of this study is the fact that urea is toxic above certain concentrations and its continuous real time monitoring in clinical, environmental and food related environments is of utmost interest. The conventional analytical techniques used, although precise, are time consuming and mostly laboratory bound whereas biosensors have the advantages of ease of use, portability and the ability to furnish real time signals.     

A reusable capacitive immunosensor with a novel immobilization procedure based on 1,6-hexanedithiol and nano-Au self-assembled layers

A novel immobilization procedure of antibody proteins for capacitive immunosensing, based on thiolor sulfur compound (1,6-hexanedithiol, HDT) and colloid Au layers is proposed. The insulating organic monolayer film was first formed by the spontaneous assembly of HDT from solution onto gold. When these thiol-rich surfaces are exposed to Au colloid, the sulfurs form strong bonds to gold nanoparticles, anchoring the clusters to the electrode substrate. After the assembly of gold nanoparticles layer, the original formed organic thiols surface was restored, and a new nano-Au surface was obtained. Thus, the antibody could be immobilized through electrostatic adsorption between nano-Au and the antibody proteins. After use, the formed immunocomplex layer can be rinsed out, via a saline solution with extreme pH. Therefore, the immunosensor can be regenerated repeatedly, highlighting a clear advantage of this new approach with respect to classical immunoassays employing covalent immobilization.     

Electric field-induced concentration and capture of DNA onto microtips

Simple, high-yield concentration of DNA is important for high-throughput genetic analysis and disease diagnosis. Glass-based microfilters are popular but the process requires centrifugation steps with cumbersome chemical processes. As an alternative, a concentration method using an electric field has been explored previously, but with limited efficiency. In this paper, electric field-induced concentration and capture of DNA are studied by using high-aspect-ratio microtips coated with a gold layer. The microtips are immersed longitudinally into a solution of 100???L containing ??-phage DNA. After DNA concentration using an electric field, the microtips are withdrawn from the solution. Under AC- and biased AC fields, DNA is concentrated by electrophoresis (EP), dielectrophoresis (DEP), and electroosmotic flow (EOF). To reduce capillary effects in the withdrawal process, the microtips are coated with positively charged poly-l-lysine (PLL). The pattern of captured DNA is analyzed by fluorescence microscopy. DEP attracts DNA molecules at the edges of microtips, where the highest gradient of electric field exists. EP attracts DNA onto the surface of microtips following the vectors of an electric field. EOF generates vortexes that deliver DNA onto microtips. Using this method, 85% of DNA is captured on the PLL-coated microtips after three sequential captures. The concentration mechanism can potentially facilitate rapid and simple preparation of DNA for downstream analysis.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

一种新的多层碳纳米管复合膜修饰的葡萄糖生物传感器制备

将碳纳米管(CNT)分散在壳聚糖(CHIT)溶液中固定到玻碳电极表面,用戊二醛交联甲苯胺蓝(TB)得到复合膜(TB-CNT-CHIT)修饰电极.由于碳纳米管具有良好的电子传递性能,与碱性生物染料甲苯胺蓝之间表现出协同作用,使甲苯胺蓝的电化学活性得到了较大的提高.此TB-CNT-CHIT复合膜修饰的玻碳电极在较低电位下对过氧化氢具有良好的电催化性能,与TB-CHIT膜比较,测定H2O2的灵敏度增大了近20倍.将复合组份多层修饰到电极上,通过戊二醛固定葡萄糖氧化酶,制备了一种新的葡萄生物传感器,该传感器在-0.2 V下对葡萄糖响应的线性范围为0.05～10 mM,检测下限为10μM.传感器的灵敏度较高,响应快,性能稳定.     

基于微/纳米加工技术的微型电化学免疫传感器研究

生物传感器的研制越来越趋向于微型化、集成化、智能化以及无创伤的方向发展.研制基于微/纳米加工技术的电化学免疫传感器顺应了这一趋势,利用微电子机械系统(MEMS)技术在硅基芯片上制备微型三电极系统和SU-8微型池,并采用自组装单层膜和纳米金修饰微型电极表面用于抗体的固定化,研制出新型的电化学免疫传感器.研究表明,这种微型电化学免疫传感器易于实现批量生产,便于集成,检测过程只需要少量的样品,大大降低有毒试剂的消耗,减少环境污染,同时具有分析成本低,响应时间快,检测下限低和适用于现场快速检测等优点.     

A micro amperometric immunosensor for detection of human immunoglobulin

1 Introduction In recent years, electrochemical immunosensors have gained considerable interests as bioanalytical devices[1―3]. They have many attractive features, such as convenience for manipulate, being easy to achieve high sensitivity and excellent d…     

电化学生物传感器的研究进展

电化学生物传感器将生物活性识别材料与电化学检测器件有机结合起来,广泛应用于临床医学、药物和食品分析与环境监测等领域。与其他电化学传感器相比,电化学生物传感器具有特异性好、检测灵敏度高和制作简便等优点。文章重点介绍了电化学生物传感器的基本原理、分类、研究进展及其在生物医学领域中的应用,并对电化学生物传感器的发展前景进行展望。     

Application of a functionalized parylene film as a linker layer of SPR biosensor

The surface plasmon resonance (SPR) biosensors have been used to detect various target analytes by using highly specific antigen-antibody interactions. In this work, a parylene film modified to have primary amine groups was applied as a linker layer of the SPR biosensor, and the primary amine groups were used for the covalent immobilization of proteins to the SPR biosensor. The feasibility of the parylene film as a linker layer was presented by estimating the influence of the parylene film on the SPR measurement parameters, such as the sensitivity and the detection range. Then, a model protein called horseradish peroxidase (HRP) was used to demonstrate the improved immobilization efficiency as well as the sensitivity of the SPR biosensor with the parylene-A film. Additionally, a reconstruction method of the immunoaffinity layer was presented by using oxygen plasma.     

Influence of surface stress on frequency of microcantilever-based biosensors

Microcantilever-based biosensors have been found increasing applications in physical, chemical, and biological fields in recent years. When biosensors are used in those fields, surface stress and mass variations due to bio-molecular binding can cause the microcantilever deform or the shift of frequency. These simple biosensors allow biologists to study surface biochemistry on a micro or nano scale and offer new opportunities in developing microscopic biomedical analysis with unique characteristics. To compare and illustrate the influence of the surface stress on the frequency and avoid unnecessary and complicated numerical solution of the resonance frequency, some dimensionless numbers are derived in this paper by making governing equations dimensionless. Meanwhile, in order to analyze the influence of the general surface stress on the frequency, a new model is put forward, and the frequency of the microcantilever is calculated by using the subspace iteration method and the Rayleigh method. The sensitivity of microcantilever is also discussed.The supports from the Key Project from the Chinese Academy of Sciences (No. KJCX2-SW-L2), projects from the National Natural Science Foundation of China (No. 10225209, No. 19928205, No. 50131160739 and No. 10072068), and the National 973 project (No. G1999033103) are gratefully acknowledged.     

Development of an amperometric sulfite biosensor based on sulfite oxidase with cytochrome c,as electron acceptor,and a screen-printed transducer

An amperometric biosensor for sulfite has been developed. The enzyme sulfite oxidase (SOD) and electron acceptor cytochrome c are mixed into the carbon ink that is deposited onto the working electrode of a screen-printed strip. A silver–silver chloride electrode is printed alongside the working electrode and serves as reference/counter electrode. The electrochemical behaviour of the biosensor surface in plain buffer has been investigated by cyclic voltammetry. In the voltage range −0.5 to +0.5 V, a well-defined anodic peak appeared at −0.15 V and a less well-defined anodic peak at about +0.2 V. In the presence of SO₃²⁻, the cyclic voltammogram obtained with the biosensor exhibited an increase in magnitude of the more positive peak; this was considered to result from the electrocatalytic oxidation of SO₃²⁻ involving SOD and the heme (Fe²⁺/Fe³⁺) centre of cytochrome c. Amperometry in stirred solution was used to construct a hydrodynamic voltammogram for SO₃²⁻ using the biosensor; this exhibited a single wave with a plateau beginning at +0.3 V. This wave corresponds to the electrocatalytic response observed by cyclic voltammetry. The pH and concentration of buffer components have been optimised for the determination of SO₃²⁻ by amperometry in stirred solution. Using these conditions, a detection limit of 4 ppm was obtained. The stability of the biosensors was examined after storage in 0.05 M phosphate buffer pH 7.4 at 4°C; it was found that the initial response was retained for at least 45 days. The proposed biosensors were evaluated on samples of unspiked and spiked estuarine, river and tap waters. The recovery and precision data indicated that the devices could be expected to give reliable data in these waters.     

A simple and direct biomolecule detection scheme based on a microwave resonator

Although several successful biosensors exist, they often require complex fabrication sequence or time-consuming sensing processes such as an off-site verification of a sensing result. At the same time, the biosensors generally focus on high sensitivity. This paper reports a cost-competitive biosensor that is capable of simple and direct detection of biomolecules without any off-site verification. The biosensor is realized with a microwave passive with a simple structure, a coplanar waveguide (CPW)-to-slotline ring resonator (CSRR) that resonant frequency is 3.375 GHz. The CSRR biosensor was then modified for higher sensitivity by increasing the effective sensing area. Two kinds of the CSRR biosensor were realized using micromachining technology. After simple fabrication, the biosensors were electrically characterized by measuring the resonant frequency shift as the biotin and streptavidin attached on the CSRR biosensor. The biotin and streptavidin induce a resonant frequency decrease of 65 and 10 MHz for the original CSRR biosensor, and 79 and 18 MHz for the modified CSRR biosensor, respectively. Based on the measurement of the resonant frequency shift, the relative permittivity of the biomolecules was calculated by numerical simulation, and was found to be 9800 for biotin and 500 for streptavidin.     

Systematic investigation of biomolecular interactions using combined frequency and motional resistance measurements

The resonance frequency of acoustic biosensors is today used as a label-free technique for detecting mass changes on sensor surfaces. In combination with an appropriate continuous flow system it has earlier been used for affinity and kinetic rate determination. Here, we assess the potential of a modified acoustic biosensor, monitoring also the real-time dissipation through the resistance of the sensor, to obtain additional kinetic information related to the structure and conformation of the molecules on the surface. Actual interaction studies, including an attempt to determine avidity, are presented along with thorough verification of the experimental setup utilizing true viscous load exposure together with protein and DNA immobilizations.True viscous loads show a linear relationship between resistance and frequency as expected. However, in the interaction studies between antibodies and proteins, as well as in the immobilization of DNA and proteins, higher surface concentrations of interacting molecules led to a decrease (i.e. deviation from the linear trend) in the differential resistance to frequency ratio. This is interpreted as increased surface rigidity at higher surface concentrations of immobilized molecules. Consequently, studies that aim at obtaining biological binding information, such as avidity, from real-time resistance and dissipation data should be conducted at low surface concentrations. In addition, the differential resistance to frequency relationship was found to be highly dependent on the rigidity of the preceding layer(s) of immobilized molecules. This dependence can be utilized to obtain a higher signal-to-noise ratio for resistance measurement by using low surface densities of immobilized interaction partners.     

Electrochemical performance of a DNA-based sensor device for detecting toxic algae

This work illustrates the electrochemical performance of a DNA-based sensor device for detecting toxic algae. This biosensor uses an electrochemical detection of the species in a sandwich hybridisation. A thiol (biotin) labelled capture probe was immobilized onto gold (carbon) electrodes. Synthetic positive control DNA was applied to the sensor and allowed to hybridize to the capture probe. A signal probe with a horseradish peroxidase (HRP) label was then applied, followed by an antibody to the HRP and a substrate. The electrical signal obtained from the redox reaction was proportional to the amount of DNA applied to the biosensor, which in turn would be proportional to the number of cells harvested when applied to real samples. Optimization of the hybridization process was already achieved in a previous work. Elucidation of the different steps of the fabrication process from the electrochemical point of view, proof of concept with different algal species and evaluation of the influence of the transducer platform geometry and material in the biosensor analytical performance are the main achievements reported here.     

多孔阳极氧化铝的化学修饰及其应用于过氧化氢的测定

多孔阳极氧化铝经化学修饰后吸附细胞色素C,制备了过氧化氢生物传感器电极。多孔阳极氧化铝通过电化学和化学腐蚀阻挡层后,用两步无电沉积方法制备了纳米金修饰的多孔阳极氧化铝电极,再在含有L-半胱氨酸的细胞色素C的溶液中通过吸附制备细胞色素C电极。用循环伏安法和计时电流法测试细胞色素C电极的电化学性能及催化对过氧化氢的还原。结果表明,包覆的细胞色素C电极显示较好的稳定性,在扫描速度为80 mV/s时于-50 mV、-190 mV附近出现一对稳定的氧化还原峰。该电极对过氧化氢具有良好的电催化还原性能,在1.5×10-5 mol/L～4.8×10-4 mol/L浓度范围内,电流与浓度呈良好的线性关系。多孔阳极氧化铝经化学修饰后,可应用于生物传感器。     

纳米金与二茂铁在葡萄糖传感器中的相互作用

针对纳米金颗粒修饰的葡萄糖生物传感器对葡萄糖的响应电流随着工作电压的下降快速下降的问题,进一步利用电子媒介体二茂铁对其进行修饰,并选用丝网印刷电极研究了纳米金颗粒和二茂铁之间的相互作用。实验结果表明:二茂铁有效地降低了纳米金颗粒修饰的葡萄糖生物传感器响应电流的下降值,纳米金颗粒降低了电子媒介体二茂铁的氧化还原反应电位,并且,纳米金颗粒与电子媒介体二茂铁在葡萄糖生物传感器中表现协同增效效应。     

Signal enhancement of surface plasmon resonance-based immunoassays for the allergen detection

A surface plasmon resonance (SPR) biosensor was used to determine the recombinant group 1 house dust mite allergen (rDer f1) in both HBS-EP buffer and fetal bovine serum (FBS). The monoclonal antibody was immobilized onto the CM5 sensor chip surface using an amine coupling method. The procedures of antibody immobilization and the subsequent primary and enhanced immunoassay were monitored in real time. The sensitivity for rDer f1 detection was remarkably improved by using intact polyclonal antibody as signal amplifying agent. Using this signal enhanced SPR immunosensor, rDer f1 in HBS-EP buffer and FBS was detected at a concentration of 15.4 and 32.1 ng/ml, respectively. The result demonstrates that SPR biosensor is a simple and reliable method for allergen detection.