利用图像SPR分析仪检测多组分蛋白芯片

介绍了一种新型高通量表面等离子体谐振(SPR)生化分析仪。基于图像分析技术和自动进样技术,该仪器可对微阵列SPR敏感芯片进行动态检测,实现高通量、多参数、多组分快速检测和定量分析。对兔IgG和人IgG分别与羊抗兔IgG和羊抗人IgG的免疫结合反应进行了实验检测。结果表明:该分析系统具有灵敏度高、免标记等优点,且阵列芯片中所设置的参比单元可以消除溶液本体折射率和温度变化的影响,提高了测量精度和准确性;另外,芯片可以再生重复使用,降低了测试成本。     

Phase-sensitive spatially-modulated surface plasmon resonance polarimetry for detection of biomolecular interactions

We here describe a scheme of spatially modulated surface plasmon resonance (SPR) polarimetry that enables to combine ultra-high phase sensitivity with good signal-to-noise background. The proposed approach uses spatial modulation of s-polarized component by birefringent elements and the extraction of phase-polarization information by Fourier-transform methods. This scheme was tested for monitoring the interactions between an antibody and its biological partner. Our experimental data, collected by amplitude-sensitive and phase-sensitive polarimetry demonstrate that the latter scheme provides at least one order of magnitude improvement in terms of detection limit.     

SPR生物传感器检测过敏致死动物血清类胰蛋白酶

应用表面等离子共振(SPR)技术检测过敏性休克死亡豚鼠血清类胰蛋白酶,试图为过敏性休克死亡提供客观诊断依据。首先,将16只健康豚鼠随机均分为实验组和对照组,每组再分为死亡即时组和冷冻3 d组。以人混合血清诱发动物过敏性休克致死。采用分子自组装方法将抗类胰蛋白酶单克隆抗体(Anti-TPSAB1)固定在SPR生物传感器芯片表面,检测豚鼠血清中类胰蛋白酶。实验结果表明:实验组类胰蛋白酶SPR响应值比对照组显著升高,在冷冻3 d的条件下对豚鼠血清类胰蛋白酶的表达无明显影响。因此,基于SPR生物传感器在动物血清类胰蛋白酶方面具有很好的应用前景,可作为诊断过敏性休克死亡的一项依据。     

SPR生物传感器在食品安全领域的应用研究

使用集成化手持式SpreetaTM SPR传感器快速检测大肠杆菌E.Coli 0157∶H7,利用大肠杆菌抗体的免疫吸附反应,采用亲和素—生物素系统放大检测的响应信号,并引入复合抗体作为第二抗体,整个检测过程在1 h内完成,实现病原微生物的快速检测。     

Ultrasensitive immunosensing of tuberculosis CFP-10 based on SPR spectroscopy

An antigen (Ag), CFP-10, found in tissue fluids of tuberculosis (TB) patients may be an ultimate candidate for use as a sensitive TB marker with a sensing method for early simplified diagnosis of TB. In this study, chemical and optical optimizations were carried out using novel immuno-materials for establishment of a self-assembled surface plasmon resonance (SPR) optical immunosensor system for detection of CFP-10, which is valuable for pre-clinical work, prior to conduct of massive clinical observations. For creation of a simple sensing interface, a monoclonal antibody (anti-CFP-10) was immobilized directly on a gold surface, followed by blocking with cystamine. Orientation and accessibility of anti-CFP-10 were assessed by the selective binding of CFP-10. Recent results indicate that the reusability of the sensor chip adopting the cystamine method was found to be preferable to other immobilization methods. A linear relationship was well correlated between SPR angle shift and CFP concentrations in the range from 100 ng mL⁻¹ to 1 μg mL⁻¹. Modification of the SPR chip with antibody provides a simple experimental platform for investigation of isolated proteins under experimental conditions resembling those of their native environment.     

Utilization of albumin-based sensor chips for the detection of metal content and characterization of metal–protein interaction by surface plasmon resonance

We report here the use of albumin-based biosensor chips for the determination of metal content and characterization of metal–protein interaction by surface plasmon resonance. Bovine serum albumin was immobilized onto a carboxymethylated dextran matrix and used for metal detection. The temperature for the analysis was defined and the highest interaction was observed at 25 °C. The albumin sensor chip binds cadmium, zinc or nickel in a concentration-dependent manner, but not magnesium, manganese and calcium. The optimal buffer condition used for the analysis contains 0.01 M HEPES, pH 7.4, 1 mM NaCl and 0.005% Tween-20. Using this condition, a linear calibration curve within the range of 10⁻⁸ to 10⁻⁴ M can be established for the metals. However, a dramatic increase in binding capacity was observed when metal concentration was higher than 10⁻⁴ M and reached a plateau at 10⁻² M. The detection limit for Cd can reach as low as 1 ppb. When measuring a solution containing two species of metal ions with the albumin chip, an additive effect was observed for Ni and Zn. However, 20–30% reduction in resonance response was found upon mixing Cd with Zn or Ni. These observations are consistent with the binding characteristics of albumin. The feasibility of measuring serum metal content by the albumin chip was examined. A linear calibration curve can be established if the samples are boiled and passed through a gel filtration column. The binding affinity of metal with albumin can also be achieved by using the sensor chip. The binding affinity follows the order of Ni > Zn > Cd. These results indicate that the albumin-based sensor chip is useful not only in the quantification of metal content, but also in the characterization of the biochemical properties of albumin.     

A novel photometric glucose biosensor based on decolorizing of silver nanoparticles

A novel glucose biosensor based on chromophore (silver nanoparticles) decolorizing for the photometric determination of glucose was developed. Silver nanoparticles are directly synthesized in the sol-gel matrix by a one-step method based on the reduction of the inorganic precursor AgNO₃ and were used for the preparation, characterization and calibration of a highly sensitive and cost-effective localized surface plasmon resonance-based glucose biosensor. In the presence of glucose oxidase (GOx) and due to the enzyme-substrate (glucose) reaction, H₂O₂ was produced and silver nanoparticles in the sol-gel glass have the ability for the decomposition of hydrogen peroxide. Due to the degradation of silver nanoparticles a remarkable change in the localized surface plasmon resonance absorbance strength could be observed which have been monitored as a suitable signal for determination of substrate concentration. Beer's law is obeyed in the range from 50 to 800 mg/L glucose and the limit of detection is 23 mg/L. The proposed optical biosensor has been successfully applied to the determination of glucose in various real samples.     

Thermodynamic analysis of the interaction between 3-aminophenylboronic acid and monosaccharides for development of biosensor

The special interaction between 3-aminophenylboronic acid and monosaccharides, particularly glucose, fructose and galactose, has been investigated and used for the development of electrochemical and surface plasmon resonance (SPR) based saccharide sensors. The binding constants and thermodynamic parameters of interaction such as free energy of binding (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the first time using isothermal titration calorimetry revealed the enthalpic and entropic contributions to the free energies of binding. Effects of pH and buffer on the binding constants (k) between 3-aminophenylboronic acid and monosaccharides were also examined. The binding constant of fructose was found to be six times higher than glucose and four times higher than galactose at pH 11, leading the specific detection of fructose. SPR and potentiometric calibration for fructose were linear in the concentration range of 10–120 mM and 8–84 mM, respectively. Limit of detection of the SPR and potentiometric method were found to be as 8.9 mM and 1.4 mM, respectively.     

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.     

一种小型化SPR生物传感器的设计与实现

介绍了一种采用不规则四边形棱镜设计的小型化表面等离子体共振(SPR)生物传感器。棱镜结构与已有的TI Spreeta传感器类似,但是在尺寸、光学性能等方面做了较大优化。新研制的SPR传感器在光学检测精度和系统集成性等方面也有了很大提高。在光路设计中,采用波长为630 nm的宽光束红光LED作为光源,5 000像素点线阵CCD作为光电检测器,光学检测效果要大大优于TI Spreeta波长为830 nm的近红外光源和128像素点的线阵硅光二极管。在光路优化的同时,系统集成了流动控制模块、信号采集处理模块,形成了一个可实现生物大分子相互作用分析的集成小型化SPR检测装置。利用空气、水及乙醇等进行的SPR实验表明:该装置能够对单一样本进行精确检测,共振角的检测精度高达0.01°,且检测结果线性度高,稳定性好,单一样本的检测偏差小于0.5%。     

SPR生物传感器温度特性的理论研究

表面等离子体共振(SPR)生物传感器在生物大分子检测时具有非常高的理论分析精度,但在实际应用过程中传感响应特性容易受到环境温度的影响.在小型化SPR传感器中由于缺乏温度控制模块,这种情况尤为明显.利用理论模型分析研究了环境温度变化对传感系统中光源、棱镜、金属薄膜和待测溶液等不同介质、光学检测器等的物理特性的影响以及由此...     

Ag-TiO2薄膜表面等离子体共振光谱特性研究

以拓宽Ag膜波长调制型表面等离子体共振(SPR)传感器的工作波长区间,将TiO2膜沉积于Ag膜表面制成Ag-TiO2复合薄膜.利用数值计算方法对不同厚度Ag膜和As-TiO2复合膜波长调制型SPR光谱特性进行仿真研究.仿真结果表明:共振吸收峰显著依赖于Ag膜厚度;当保持Ag-TiO2复合膜厚度60 nm时,复合薄膜SPR波长随TiO2厚度的增大而向长波长方向移动,发现红移;与60 nm厚Ag膜共振波长相比,12 nm厚TiO2与48 rnm厚Ag组成的复合膜共振波长红移超过200 nm.     

Enhanced surface plasmon resonance detection of DNA hybridization based on ZnO nanorod arrays

We demonstrated an enhanced surface plasmon resonance detection incorporating ZnO nanorod arrays (NRAs) built on a thin gold film. ZnO NRAs were fabricated by wet chemical growth method and used for the detection of DNA hybridization. Experimental results exhibited that ZnO NRAs provided a notable sensitivity improvement by more than 3 times, which is attributed to an increase in the surface reaction area. The measured sensitivity enhancement matched well with the numerical analyses based on the effective medium theory. Our approach is intended to show the feasibility and extend the applicability of the ZnO-based SPR biosensor to diverse biomolecular binding events.     

Surface plasmon resonance in chalcogenide glass-based optical system

The surface plasmon resonance phenomenon has been studied in a chalcogenide glass-based optical system. IR transmission properties of these materials combined to their high refractive indices lead to advantageous properties for sensing. In this study, numerical simulations have been carried out to investigate the potentialities of sulfide glass from the GeGaSbS system as a coupling prism material. Then, an angular modulation SPR biosensor has been set up in the Kretschmann–Raether arrangement. Experimental data are consistent with numerical calculation and the detection limit of the sensor is 3 × 10⁻⁵ RIU. These preliminary results are promising. Further investigations have to be carried out to confirm the great potentialities of those materials for SPR-based biosensor.     

An application of a plasmonic chip with enhanced fluorescence to a simple biosensor with extended dynamic range

Plasmonic chips coated with thin silver and SiO₂ layers were applied to a simple biosensor with wide dynamic range, which has some advantages in the points of sensitivity and surface selectivity in spite of a simple fluorescence detection system without complex optical stuffs. The enhanced fluorescence of fluorescent labeled-marker proteins, cy5-streptavidin (SA), excited by irradiation light from the rear panel of the chip was measured. To obtain the maximum fluorescence enhancement by excitation under an electric field enhanced by grating-coupled surface plasmon resonance from the rear irradiation, the optimal silver thickness was studied and found to be 30-35 nm. A directional fluorescence due to re-coupling between fluorescence and plasmon polaritons was also observed on the plasmonic chips and enhanced fluorescence 90 times compared with that on glass slides was superior to the others reported. The limit of detection (LOD) studied here was improved up to 50 pM after 10-min incubation and dynamic range was also extended by 2 order, compared with that on glass slides. The irradiation from the rear panel demonstrated advantage in the surface-selective detection of cy5-SA at 100 nM concentration. Our plasmonic chip has great potential in biosensing application due to its advantages of rapid detection, simple operation, high sensitivity (wide dynamic range), surface selectivity, and suppression of non-specific adsorption.     

A single chip multi-channel surface plasmon resonance imaging system

A single chip, multi-channel surface plasmon resonance (SPR) imaging system has been developed. The equipment has no moving parts and uses a single sensor chip on which multiple channels can be incorporated. A light emitting diode is used as a photon source while a CCD camera forms the detector. The optical configuration has been designed to achieve a uniform illumination of the sample over a fixed area with a range of incident angles. A calibration test using sucrose solutions shows that the sensitivity of the equipment is 4.3×10⁻⁴ refractive index units per pixel line-pair. The use of the system for simultaneous interrogation of different polyelectrolyte thin films, formed by the electrostatic layer-by-layer deposition method, is demonstrated. A reversible pH-dependent response for these organic layers is also reported.     

Manufacture of robust surface plasmon resonance fiber optic based dip-probes

Surface plasmon resonance (SPR) spectroscopy has been conducted on both prism and fiber optic (FO) based sensors for several years. This technique measures the refractive index (RI) of a solution or layer adsorbed to a thin (50 nm) Au layer on the sensor substrate. To date a succinct set of protocols have not been published regarding the optimization of fiber-based SPR dip-probe sensors. Such sensors would allow application of SPR to a wider variety of applications. This paper focuses on consideration of the choice of fiber, isolation of the mirror from the sensing area, and orientation of the probes in the metal layer sputter deposition chamber in the manufacture of SPR dip-probes for reproducibility and robustness. Optimization of the process yields sensors with a batch to batch reproducibility as low as 0.5 nm in the location of the SPR spectral minima. Further study of RI measurements by the same probe over 2 months show these SPR dip-probes have a long shelf-life. A selection of probes was exposed to various solutions to monitor their drift. The data shows the probes’ response indicated a lowering of the RI measured over a period of 3 or 7 days depending on the probe type. Evidence of surface porosity and damage upon exposure to hydrothermal water seems to indicate these sensors are prone to chemical attack. Further research is needed to characterize this attack and allow creation of more robust sensors.     

SPR传感器阵列中微流通池的流场仿真

在自制表面等离子体激元共振(SPR)传感器阵列检测装置的微流通池的研制中,为了获得更好的检测精度和重复性,需要流经各检测点的流量尽可能一致。设计了几种不同的微流通池结构,并利用Comsol Multiphysics软件对其内部的流场进行仿真分析。首先,分析了常用梭状流通池的内部流场分布,并研究了不同长度过渡段对流场分布的影响。在此基础上,讨论了不同流通池形状,特别是流通池外形轮廓,如椭圆状流通池和边界流线型更好的流通池对流场分布的影响。此后,还研究了过渡区中微结构阵列设计对检测区流场分布的影响。仿真研究结果表明:适当延长过渡段长度,有利于改善流体流动的稳定性和均匀性,但这种改善将随长度的进一步增加逐渐减弱。而不同的边界外形轮廓对检测区域流场的影响不大;当采用一定的微结构阵列扰流设计后,流场均匀性一定程度上得到改善。