基于一维CCD的免疫层析试纸条检测系统

本文报道的基于一维CCD的免疫层析试纸条检测系统是一种以上转换磷光材料（UCP）作为标记物的生物传感器。该系统通过检测生物反应后试纸条上UCP颗粒的含量，计算出被测样品中特定生物分子的浓度，可以实现对多种病原体的快速定性与定量检测。本检测系统对0—60ns／ml系列标准样品的检测结果具有很好的线性响应特性，且与扫描型检测系统的检测结果十分接近。

Multicapas de FeNi/Ti sobre sustratos rígidos y flexibles para sensores de magnetoimpedancia especializados

En este trabajo se han estudiado las propiedades magnéticas de las multicapas de FeNi/Ti depositadas en sustrato rígido y flexible. Asimismo, se ha analizado la magnetoimpedancia gigante (GMI) y se ha estuadiado un prototipo de biosensor GMI utilizando ferrogeles.

Interfacial immobilization of monoclonal antibody and detection of human prostate-specific antigen.

Antibody orientation and its antigen binding efficiency at interface are of particular interest in many immunoassays and biosensor applications. In this paper, spectroscopic ellipsometry (SE), neutron reflection (NR), and dual polarization interferometry (DPI) have been used to investigate interfacial assembly of the antibody [mouse monoclonal anti-human prostate-specific antigen (anti-hPSA)] at the silicon oxide/water interface and subsequent antigen binding. It was found that the mass density of antibody adsorbed at the interface increased with solution concentration and adsorption time while the antigen binding efficiency showed a steady decline with increasing antibody amount at the interface over the concentration range studied. The amount of antigen bound to the interfacial immobilized antibody reached a maximum when the surface-adsorbed amount of antibody was around 1.5 mg/m(2). This phenomenon is well interpreted by the interfacial structural packing or crowding. NR revealed that the Y-shaped antibody laid flat on the interface at low surface mass density with a thickness around 40 Å, equivalent to the short axial length of the antibody molecule. The loose packing of the antibody within this range resulted in better antigen binding efficiency, while the subsequent increase of surface-adsorbed amount led to the crowding or overlapping of antibody fragments, hence reducing the antigen binding due to the steric hindrance. In situ studies of antigen binding by both NR and DPI demonstrated that the antigen inserted into the antibody layer rather than forming an additional layer on the top. Stability assaying revealed that the antibody immobilized at the silica surface remained stable and active over the monitoring period of 4 months. These results are useful in forming a general understanding of antibody interfacial behavior and particularly relevant to the control of their activity and stability in biosensor development.

Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation.

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ(42) fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ(42). Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.

Dynamic modulation of detection window in conducting polymer based biosensors.

Here we demonstrate a novel application that employs the ion exchange properties of conducting polymers (CP) to modulate the detection window of a CP based biosensor under electrical stimuli. The detection window can be modulated by electrochemically controlling the degree of swelling of the CP associated with ion transport in and out of the polymer. We show that the modulation in the detection window of a caffeine imprinted polypyrrole biosensor, and by extension other CP based biosensors, can be achieved with this mechanism. Such dynamic modulation in the detection window has great potential for the development of smart biosensors, where the sensitivity of the sensor can be dynamically optimized for a specific test solution.

Measuring the kinetics of amyloid fibril elongation using quartz crystal microbalances.

Kinetic measurements of amyloid growth provide insight into the free energy landscape of this supramolecular process and are crucial in the search for potent inhibitors of the main disorders with which it is associated, including Alzheimer's and Parkinson's diseases and Type II diabetes. In recent years, a new class of surface-bound biosensor assays, e.g., those based on surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) have been established as extremely valuable tools for kinetic measurements of amyloid formation. Here we describe detailed protocols of how QCM techniques can be used to monitor the elongation of amyloid fibrils in real time and to study the influence of external factors on the kinetics of amyloid growth with unprecedented accuracy.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

Direct detection of molecular biorecognition by dipole sensing mechanism.

This work investigates the feasibility of transducing molecular-recognition events into a measurable potentiometric signal. It is shown for the first time that biorecognition of acetylcholine (ACh) can be translated to conformational changes in the enzyme, acetylcholine-esterase (AChE), which in turn induces a measurable change in surface potential. Our results show that a highly sensitive detector for ACh can be obtained by the dilute assembly of AChE on a floating gate derived field effect transistor (FG-FET). A wide concentration range response is observed for ACh (10(-2)-10(-9)M) and for the inhibitor carbamylcholine CCh (10(-6)-10(-11)M). These enhanced sensitivities are modeled theoretically and explained by the combined response of the device to local pH changes and molecular dipole variations due to the enzyme-substrate recognition event.

Towards the formation of neuro-transistor artificial chemical synapse

Highly sensitive biosensor for detection of acetylcholine (ACh) and competitive acetylcholinesterase (AChE) inhibitor, eserine, is investigated. Peculiar microelectronic configuration of an ion-sensitive field-effect transistor (ISFET) in addition to a right choice of the pH-transducing nanolayers allows recording a response of the enzyme-modified ISFET (EnFET) to a wide range of ACh concentrations. We demonstrate a remarkable improvement of at least three orders of magnitude in dose response to ACh. Described bioelectronic system reveals clear response, when the catalytic activity of the immobilized AChE is inhibited in a reversible manner by eserine, competitive inhibitor of AChE. ©2007 IEEE.

Amorphous silicon thin film transistor biosensing system

Electronic systems are a very good platform for sensing biological signals for fast point-of-care diagnostics or threat detection. One of the solutions is the lab-on-a-chip integrated circuit (IC), which is low cost and high reliability, offering the possibility for label-free detection. In recent years, similar integrated biosensors based on the conventional complementary metal oxide semiconductor (CMOS) technology have been reported. However, post-fabrication processes are essential for all classes of CMOS biochips, requiring biocompatible electrode deposition and circuit encapsulation. In this work, we present an amorphous silicon (a-Si) thin film transistor (TFT) array based sensing approach, which greatly simplifies the fabrication procedures and even decreases the cost of the biosensor. The device contains several identical sensor pixels with amplifiers to boost the sensitivity. Ring oscillator and logic circuits are also integrated to achieve different measurement methodologies, including electro-analytical methods such as amperometric and cyclic voltammetric modes. The system also supports different operational modes. For example, depending on the required detection arrangement, a sample droplet could be placed on the sensing pads or the device could be immersed into the sample solution for real time in-situ measurement. The entire system is designed and fabricated using a low temperature TFT process that is compatible to plastic substrates. No additional processing is required prior to biological measurement. A Cr/Au double layer is used for the biological-electronic interface. The success of the TFT-based system used in this work will open new avenues for flexible label-free or low-cost disposable biosensors. © 2013 Materials Research Society.

Low potential detection of glutamate based on the electrocatalytic oxidation of NADH at thionine/single-walled carbon nanotubes composite modified electrode

A glutamate biosensor based on the electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH), which was generated by the enzymatic reaction, was developed via employing a single-walled carbon nanotubes/thionine (Th-SWNTs) nanocomposite as a mediator and an enzyme immobilization matrix. The biosensor, which was fabricated by immobilizing glutamate dehydrogenase (GIDH) on the surface of Th-SWNTs, exhibited a rapid response (ca. 5 s), a low detection limit (0.1 mu M), a wide and useful linear range (0.5-400 mu M), high sensitivity (137.3 +/- 15.7) mu A mM(-1) cm(-2), higher biological affinity, as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid, and 4-acetamidophenol, did not cause any interference due to the use of a low operating potential (190 mV vs. NHE). The biosensor can be used to quantify the concentration of glutamate in the physiological level. The Th-SWNTs system represents a simple and effective approach to the integration of dehydrogenase and electrodes, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications including biosensors and biofuel cells.

电化学SPR生物传感器的研究及应用

围绕论文题目“电化学SPR生物传感器的研究及应用”，我们将SPR传感金膜同时用作电化学研究的界面，在自行组建的电化学SPR (EC-SPR)池中进行了相关的EC-SPR研究。 本论文研究工作的主要内容包括以下几个方面： 1． 发展了一种电化学薄化控制SPR金膜厚度，优化SPR信号的方法。这种方法主要是利用在较高电位下金与氯离子发生络合反应使SPR金膜表面的金部分溶解进入溶液从而达到薄化金基底的目的。通过调节溶液中氯离子的浓度和电化学扫描的次数，可以现场调控SPR基底的金膜厚度。我们用这种处理过的金膜进行了生物分子的吸附试验，结果证明了这种处理过的金膜适用于一般的SPR分析。 2． 采用湿化学镀膜法结合光刻法制备SPR金膜微阵列，拟将用于SPR成像分析。这种方法属于湿化学法制备SPR金膜微阵列，主要是在胶体金纳米粒子的自组装膜上刻蚀出金纳米粒子的微阵列，然后用湿化学法生长出合适的金微阵列。这种方法对制备条件要求比较简单，在制备纳米金微阵列的过程中腐蚀时间比较好控制，同时催化生长出新的金面。重复试验证实了这种方法能够制备出稳定的，尺寸可控的金微阵列，有望用于SPR成像系统研究生物分子相互作用。 3． 在SPR金膜表面利用电沉积法制备了超薄的壳聚糖薄膜，并将之应用于生物分子相互作用的研究。通过一步电沉积的方法制备了超薄的壳聚糖修饰的SPR金基片，并研究了几种常见蛋白与壳聚糖薄膜的非特异性作用，进一步用鼠IgG和抗鼠IgG作为一个典型的例子研究了壳聚糖修饰膜的生物相容性。试验表明壳聚糖修饰膜有好的生物相容性。 4． 首次提出利用生物催化沉积金属纳米粒子放大SPR信号测定小分子的方法。生物小分子抗坏血酸能够还原银离子，使其在金纳米表面沉积形成金属银原子。银原子的沉积将会极大地增强SPR信号，从而实现SPR光谱对小分子抗坏血酸浓度的放大测定。每次测定后，通过电化学剥脱Ag原子，SPR芯片的表面能够完全再生。同时，剥脱的银原子的量也能够被电化学测定，这也实现了抗坏血酸的间接电化学测定。 5． 结合电化学和SPR技术表征了DNA/Zr4+多层膜在金膜表面的生长过程，并研究了这种多层膜与细胞色素c的相互作用。SPR技术被用于测定 (DNA/ Zr4+)1双层中DNA单层的有效膜厚，及其表面覆盖率。利用红外反射光谱和X-射线光电子能谱表征这种多层膜的组成。通过EC-SPR方法，这种多层膜和细胞色素c的相互作用被进一步分析。结果表明这种多层膜不仅增强了细胞色素c的固定量，而且保持了细胞色素c的生物活性。 6． 利用EC-SPR技术测定了聚苯胺支撑的双层磷脂膜中的酶促反应。通过泡囊融合法在聚苯胺表面形成HRP掺杂的磷脂双层膜。这种磷脂双层膜能够很好的保存膜内的辣根过氧化酶(HRP)的活性，同时，这种膜允许质子的跨膜传输，能够提供聚苯胺和HRP在双氧水存在下反应所需的质子，实现酶促开关控制聚苯胺氧化还原态的变化，通过SPR检测这种聚苯胺膜的氧化还原态的变化，从而达到利用SPR测定酶底物小分子的目的。 7． 开展了适配子(aptamer)的EC-SPR研究。利用亚甲基兰为外在电化学探针分子，我们设计了一种简单的、可再生的电化学方法测定小分子腺苷。结果表明这种方法对腺苷的检测具有较高的灵敏性和选择性。这种设计思路有望进一步用于构建一个可再生的SPR传感器平台，用于研究适配子与蛋白质相互作用。

新型安培生物传感器的研制

由于生物传感器在临床、环境、食品等领域具有广阔的应用前景，近年来发展迅速。本论文从合成新型溶胶-凝胶衍生的生物相容性固定化载体和在碳基底表面构筑有序的低聚苯胺分子线两方面对安培酶电极进行了研究。主要结果如下：1．利用溶胶-凝胶／水凝胶复合材料作为载体固定辣根过氧化物酶，构造了无试剂、高稳定的过氧化氢生物传感器。该传感器有望应用于实际在线检测过氧化氢。2．合成了新型功能化溶胶-凝胶／EastmanAQ聚合物复合材料，成功地固定了带正电荷的媒介体，构造了一种响应快速，灵敏度高的无试剂过氧化氢传感器。该功能化复合材料可应用于其它分析检测中。3．合成了钛溶胶-凝胶／聚乙烯醇接枝4-乙烯吡啶复合材料。以该材料为基础制备的酪氨酸生物传感器和葡萄糖生物传感器均表现出高灵敏度，好长期稳定性等优点。该复合材料可应用于其它生物分子的固定。4，合成了有机修饰的溶胶-凝胶／壳聚糖复合材料。由于该材料生物相容性好且价格低廉，因此以其为固定化载体制备的葡萄糖生物传感器有望实现商品化。5．利用电化学还原重氮盐法在碳电极表面构筑了有序的低聚苯胺分子线。利用循环伏安法、X-射线光电子能谱法、扫描隧道显微镜法进行了表征。该设计在传感器、诊断及分子电子领域具有潜在应用前景。

高稳定生物传感器的研究与开发

由于生物传感器在临床、环境、食品等领域具有广阔的应用前景，近年来发展迅速。本论文以“十五”国家科技攻关重大项目《科学仪器研制与开发》的子课题《有机-无机杂化材料膜BOD生物传感器及BOD快速测定仪的研制与开发》为契机，进行了高稳定的BOD生物传感器的研究与开发；将自组装技术和纳米技术相结合，探索了一种制备性能稳定的酶生物传感器的新方法。主要结果如下：目前水污染经常必测的水质监测指标之一的生化需氧量（BOD）存在着测量时间长、不能及时反映水质变化、不能有效地进行信息反馈等缺点，为了解决这些问题，展开了快速、稳定的BOD生物传感器方面的研究：通过对微生物进行筛选，选择性能稳定的、对有机物降解能力强的微生物制备微生物膜；通过对各种固定化材料的比较，筛选出以硅的溶胶掺杂聚乙烯醇接枝聚乙烯毗睫的有机-无机杂化材料固定微生物制备微生物膜，该新型有机-无机掺杂材料不仅有效地防止了薄膜的开裂，而且由于其含有大量的轻基而具有良好的生物相容性。选择适当的溶液作标准溶液，能够改进生物传感器法与传统的五天法结果的一致性，可以进一步拓宽BOD生物传感器的应用范围。所研制的BOD生物传感器能在10 min左右测定BoD含量，稳定性和重现性良好；用于实际样品的测走，所得结果与标准稀释法一致；该传感器保存12个月经活化后至少具有80％以上的活性，可连续使用90天以上。通过对流路系统、恒温系统等的研究，与江苏江分电分析仪器厂的研究人员一起制备了XSF-1型在线BOD监测仪样机三台，并顺利通过了中国科学院院级鉴定。首先将金电极或玻碳电极表面功能化，再依次组装金纳米粒子和酶，制得性能稳定的生物传感器。这样制备的HRP生物传感器，实现了HRP的直接电化学，对H_2O_2还原具有很高的催化活性，响应速度快（＜2.5s）、灵敏度高、重现性好，并且具有长期稳定性。由此提供了一种组装金纳米粒子固定生物大分子的方法，由于金纳米粒子与生物大分子之间有较强的相互作用，酶或蛋白质能被牢固地固定在电极表面，而且金纳米粒子能够促进电子转移，从而有助于实现酶或蛋白质的直接电化学；该方法可多层组装金纳米粒子，从而增加酶载量；此外，该传感器也可与产物含过氧化氢的氧化还原酶联用制备双酶或多酶生物传感器。