表面等离子体(SPR)检测电路设计

讨论了表面等离子体(SPR)光电检测电路的设计方案，采用电荷耦合器件(CCD)检测SPR角谱，设计了一种单片机CCD驱动电路。     

Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors

The reaction between antibody immobilized to surfaces, with and without a dextran matrix, and antigen in solution was studied using surface plasmon resonance detection. The use of a reference surface made it possible to identify conditions where a response related to changes in matrix conformation could be ignored. It was possible therefore to improve data quality by separating signals related to binding events from signals due to differences in refractive index between sample and running buffer. When antigen was injected over antibody immobilized to surfaces with and without dextran matrix the binding curves were virtually superimposable. Consequently, no binding artifacts associated with the dextran matrix were observed. Sets of binding curves obtained with different antigen concentrations were analyzed using numerical integration of differential rate equations and global fitting. When data was inconsistent with a one to one reaction it was possible to obtain good fits to an entire data set assuming several other reaction schemes including parallel, competitive and two-state reactions. Thus data analysis alone was not sufficient to discriminate between different reaction schemes. In contrast several reaction schemes could be ruled out with simple experiments; the duration of antigen injection, and reanalysis of antigen recovered in fractions from the antibody surface. In view of these findings experimental design appears to be the key to successful interaction analysis.     

局域表面等离子共振传感器概述

近年来,基于贵金属局域表面等离子共振(localized surface plasmon resonance,LSPR)而开发的生物传感器被陆续应用在环境检测和基础生物医学研究中。和传统的表面等离子共振(surface plasmon resonance,SPR)传感器相比,LSPR基于先进的纳米材料合成技术而省略了复杂的光学器件,具有成本低,精度高,便于小型化等诸多优点。在实际应用特别是临床检测中,由于样品的复杂性和LSPR技术本身的低特异性,LSPR技术目前尚处于实验室开发阶段。随着表面化学研究的进步和新型受体的出现,LSPR的特异性也不断提高,使实时样品检测成为可能。本文主要介绍LSPR技术与传统的SPR的异同及其在临床生物医学研究中的应用。     

一种表面等离子共振芯片的制备及其应用

目的 研制一种新型的还原态牛血清白蛋白（rBSA）芯片,使其适用于Biacore系列仪器并具有与CM5芯片相同的功能。方法 利用二硫键还原剂将天然态牛血清白蛋白（nBSA）的所有巯基还原出来,形成rBSA,然后通过物理吸附将rBSA固定于裸金芯片表面,经交联和羧基化处理后得到rBSA芯片,接着利用原子力显微镜（AFM）表征其表面修饰效果,并与CM5芯片比较在磺胺甲唾唑（SMX）检测中的应用效果。结果 通过AFM扫描,发现rBSA芯片表面基质均一;其检测SMX的抑制标准曲线与CM5芯片相比较,曲线形状十分相似,各抑制浓度的抗体结合信号均具有很好的稳定性;此外,rBSA芯片还具有再生更容易、分析时间更短的优势。结论 rBSA芯片修饰成功,制备的芯片具有优良的性能,能够满足实验的需要。     

用表面等离子体谐振(SPR)技术测试抗原抗体结合反应

利用自行初步设计的SPR传感仪，对抗原抗体结合反应进行测试，分析研究结合反应中两者的适宜比例。实验表明，所得曲线与理论基本相符。     

Systematic screening of viral entry inhibitors using surface plasmon resonance

Viral binding and entry into host cells for various viruses have been studied extensively, yielding a detailed understanding of the overall viral entry process. As cell entry is an essential and requisite process by which a virus initiates infection, it is an attractive target for therapeutic intervention. The advantages of targeting viral entry are an extracellular target site, relatively easy access for biological interventions, and lower toxicity. Several cell‐based strategies and biophysical techniques have been used to screen compounds that block viral entry. These studies led to the discovery of inhibitors against HIV, HCV, influenza, Ebola, and RSV. In recent years, several compounds screened by fragment‐based drug discovery have been approved as drugs or are in the final stages of clinical trials. Among fragment screening technologies, surface plasmon resonance has been widely used because it provides accurate information on binding kinetics, allows real‐time monitoring of ligand‐drug interactions, requires very small sample amounts to perform analyses, and requires no modifications to or labeling of ligands. This review focuses on surface plasmon resonance–based schemes for screening viral entry inhibitors.     

一种检测血清心肌肌钙蛋白I的生物传感器

目的 :建立一种新的血清心肌肌钙蛋白I(cTnI)定量检测法。方法 :用亲和层析法提纯cTnI,免疫BALB C鼠及新西兰兔 ,并用杂交瘤技术及膜渗滤亲和层析法 ,制备了特异性抗cTnI单克隆抗体和多克隆抗体。以葡萄球菌蛋白A作基底膜 ,特异性多克隆抗体作捕捉抗体 ,单抗 9F5作第二抗体 ,制成了表面等离子体共振生物传感器。比较了直接法和夹心免疫法检测血清cTnI的性能。结果 :夹心免疫法的最低检测限 (0 8μg L)是直接法的 5倍 (4 μg L) ,检测范围为 (0 8～ 2 0 ) μg L ,批内及批间精密度分别达 3 8%～ 5 1% ,6 3%～ 8 1%。用该夹心法及国外试剂盒分别检测 4 0名健康献血队员和 2 8例急性心肌梗死患者血清cTnI水平 ,两者符合率分别为 97 5 %和 94 6 %。结论 :所建立的夹心免疫法操作简单 ,特异性强 ,灵敏度高 ,符合临床诊断要求。     

Molecularly imprinted based surface plasmon resonance nanosensors for microalbumin detection

Human serum albumin (HSA) is a major blood plasma protein also found in urine where its existence may be a marker of some types of liver or kidney dysfunction. Herein, we fabricated a novel surface plasmon resonance (SPR) nanosensor for selective, sensitive, and label-free microalbumin detection both in aqueous and urine sample solutions. First, HSA-imprinted nanoparticles were synthesized, which consist of ethylene glycol dimethacrylate and N-methacryloyl-L-leucine methyl ester as a cross-linker and functional monomer. The nanoparticles were characterized by zeta-size and scanning electron microscope analyses and were dropped onto the SPR chip surface to make HSA sensitive nanosensor. Characterization studies of HSA-imprinted SPR chip were carried out by atomic force microscopy, Fourier-transform infrared spectroscopy, contact angle, and ellipsometer. The limit of detection and limit of quantification values of HSA-imprinted SPR nanosensor were calculated as 0.7?pM and 1.9?pM for the concentration range of 0.15–500?nM. Selectivity studies of HSA-imprinted SPR nanosensor were achieved with hemoglobin and transferrin proteins which were chosen as competitor molecules. HSA-imprinted SPR nanosensor was displayed highly selective and sensitive to HSA.     

Detection of ciprofloxacin through surface plasmon resonance nanosensor with specific recognition sites

The novel nanosensor based on Surface Plasmon Resonance (SPR) was developed for sensitive and selective detection of ciprofloxacin via molecularly imprinted nanoparticles (MIP/NPs). NPs were synthesized through miniemulsion polymerization technique with methacrylic acid as a functional monomer. FTIR, SEM, zetasizer and contact angle measurements were used for the characterization of MIP/NPs. After modification the SPR chip surface, the nanosensor was used for detection of ciprofloxacin in aqueous solution. According to selected concentration range, the correlation coefficient and limit of detections were obtained as 0.993 (R²) and 3.21 and 7.1 ppb in ultrapure water and SWW, respectively. Association kinetic analysis, Scatchard, Freundlich, Langmuir and Freundlich-Langmuir isotherms were also performed on the data to investigate adsorption behaviour of ciprofloxacin on the surface of nanosensor. Tetracycline and enrofloxacin were used as competitor agents to examine the selectivity of nanosensor. Performance of the SPR nanosensor was also investigated by using synthetic wastewater (SWW) for detection of CPX. The reusability of nanosensor was investigated and good repeatability was obtained with 5.81% RSD. As a result, selective, simple and low-cost method to detect ciprofloxacin in aqueous solution was developed by combining MIP/NPs and SPR.     

便携式表面等离子共振传感器在血样检测中的应用

近年来,基于表面等离子共振（surfaceplasmonresonance,SPR）而开发的生物传感器（biosensor）在基础生物医学研究中被广泛应用,并向高精度和便携式方向发展。SPR技术本身具有实时性、高精度、可重复使用的优点。尽管SPR技术本身特异性不高,但随着表面化学的发展,更多的受体（receptor）可被结合在SPR传感器的表面,使SPR传感器能够只对目标分子响应。本文主要介绍目前主流SPR仪的工作原理及SPR在血样检测中的临床应用。     

表面等离子共振检测金属表面不同相对分子质量的壳聚糖对质粒DNA的保护作用

目的研究采用表面等离子共振（SPR）技术，检测不同相对分子质量壳聚糖对质粒DNA（pDNA）的保护作用，以防止核酸酶（DNaseⅠ）的降解。方法用具有羧基化葡聚糖表面的CM5芯片固定在Biacore3000生物大分子相互作用系统中制成壳聚糖芯片，采用SPR检测在核酸酶存在下，不同相对分子质量壳聚糖对pDNA的保护作用。结果在人体温度（37℃）下，浓度5U／μ1的DNaseⅠ在lmin内即可降解没有保护的质粒DNA，而相对分子质量为200000以上的壳聚糖对质粒具有良好的保护作用。结论表面等离子共振技术可以作为体外研究金属表面高分子对于质粒DNA保护作用的一种新方法。     

SPR传感器光学参数的确定及应用

本文从菲涅尔公式出发,给出了SPR传感器中金属膜光学参数的确定方法,并以此为基础测定了水、乙醇,氯仿,甘油的折射率。     

表面等离子体共振传感技术及其在临床检验中的应用

表面等离子体共振(SPR)传感技术具有实时、快速、无需标记、无背景干扰和样品无损等优点,被广泛应用于生物技术、医学、环境科学及药物检测等领域.着重介绍了SPR传感相关技术的研究进展,特别总结了SPR芯片的表面修饰技术和SPR传感器联用技术,其中联用技术重点从分子印迹联用技术、免疫分析联用技术及核酸联用技术3个方面及在医疗等临床检验领域中的应用进行了综述.     

Detection of the ΔF508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology

In the present paper, we applied surface plasmon resonance (SPR) and biosensor technologies for biospecific interaction analysis (BIA) to detect ΔF508 mutation (F508del) of the cystic fibrosis transmembrane regulator (CFTR) gene in both homozygous as well as heterozygous human subjects. The proposed method is divided into three major steps. The first step is the immobilization on a SA5 sensor chip of two biotinylated oligonucleotide probes (one normal, N‐508, and the other mutant, ΔF508) that are able to hybridize to the CFTR gene region involved in F508del mutation. The second step consists of the molecular hybridization between the oligonucleotide probes immobilized on the sensor chips and (1) wild‐type or mutant oligonucleotides, as well as (2) single‐stranded DNA obtained by asymmetric polymerase chain reaction (PCR), performed using genomic DNA from normal individuals and from F508del heterozygous and F508del homozygous patients. The third, and most important, step consists of the evaluation of differential stabilities of DNA/DNA molecular complexes generated after hybridization of normal and ΔF508 probes immobilized on the sensor chips. The results obtained strongly suggest that the proposed procedure employing SPR technology enables a one‐step, nonradioactive protocol for the molecular diagnosis of F508del mutation of the CFTR gene. This approach could be of interest in clinical genetics, as the hybridization step is oftenly required to detect microdeletions present within PCR products. Hum Mutat 13:390–400, 1999. © 1999 Wiley‐Liss, Inc.     

Self-assembled monolayer for toxicant detection using nucleic acid sensor based on surface plasmon resonance technique

Double stranded calf thymus deoxyribonucleic acid (dsCT-DNA) has been covalently immobilized onto self-assembled monolayer (SAM) of beta-merceptoethanol (MCE) on gold substrates via N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide chemistry for fabrication of the surface plasmon resonance (SPR) based biosensing device. The dsCT-DNA-MCE-SAM/Au bioelectrode has been characterized using electrochemical impedance spectroscopy, cyclic voltammetry, contact angle measurements and atomic force microscopy, respectively. This biosensing device has been utilized for detection of cypermethrin (0.0005 ppm) using electrochemical and SPR techniques, respectively. The mechanism of interaction of cypermethrin with dsCT-DNA immobilized onto MCE-SAM has been proposed.     

基于表面等离子体共振传感技术检测小分子物质的研究进展

表面等离子体共振（sPR）传感技术是近年发展起来的一种新的光学检测技术,它将生物学、高分子化学及传感技术结合,形成具有快速、灵敏、特异以及操作简便的检测技术。概述了应用SPR传感技术进行小分子物质检测的原理、主要方法及研究进展,分析了该方法的优势与缺陷,并对其发展前景进行了展望。     

应用表面等离子共振生物传感器分型检测HPV及其应用评价

目的评价表面等离子共振(SPR)生物传感器分型检测女性生殖道人类乳头瘤病毒(HPV)的临床应用价值。方法采集女性宫颈脱落细胞504例,按病理学结果分为炎症组、宫颈上皮内瘤变(CIN)Ⅰ组、CINⅠ-Ⅱ组、CINⅡ组、CINⅢ组、宫颈癌组。应用SPR生物传感器对各组进行检测,一次性分型检测16种HPV高危型和8种低危型,同时采用克隆测序作平行对照,结合病理诊断结果,对SPR生物传感器进行应用评价。结果SPR生物传感器和克隆测序结果的一致率为0.994,Kappa指数为0.987,(P=0.0000.05)。SPR生物传感器测得各病理组别HPV阳性率、高危型阳性率及多重感染率依次为总体(64.7%、62.1%、15.5%),炎症(41.9%、36.6%、11.8%),CINⅠ(44.6%、41.2%、13.5%),CINⅠ-Ⅱ(51.9%、48.1%、11.1%),CINⅡ(74.2%、74.2%、9.7%),CINⅢ(94.3%、93.5%、23.6%)及宫颈癌组(98.2%、98.2%、16.4%)。24种HPV基因型检出21种,阳性率依次为:16、58、33、52、66、11、18、53、6、31、45、39、81、59、70、68、51、54、56、35、40。SPR生物传感器检测HPV DNA诊断CIN III和宫颈癌的灵敏度为95.5%、特异度为52.1%、阳性预测值为52.1%、阴性预测值为95.5%。结论随宫颈病变严重程度的增高,HPV感染率和高危型感染率呈升高趋势,多重感染率无明显升高趋势;SPR生物传感器检测HPV DNA与克隆测序一致性良好,可以实现HPV分型检测,其诊断CINⅢ和宫颈癌具有较高的灵敏度和阴性预测值,在宫颈病变的临床诊断和流行病学调查中具有重要意义。     

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six–eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F_2P) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3ζ1 ITAM with a non-peptidic linker made up of γ-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.     

Vaccination for birch pollen allergy: comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance

BACKGROUND: Allergen-specific immunotherapy (SIT) is associated with increased levels of allergen-specific IgG in serum. However, it is not clear to what extent qualitative changes in the allergen binding capacity of IgG may be induced as well. OBJECTIVE: The purpose of this study was to investigate the influences of SIT on antibody affinity. METHODS: The binding affinity of purified serum IgG1, IgG4 and IgE to the major allergen in birch (Betula verrucosa) pollen, Bet v 1, was analysed by surface plasmon resonance. The antibodies were obtained from 10 birch pollen-allergic patients receiving SIT and from 10 patients with no SIT. RESULTS: The patients having received SIT have a significant higher titre of anti-Bet v 1 antibodies in their blood, but the affinity to Bet v 1 of allergen-specific IgE, IgG1 and IgG4 does not differ between the two groups. For IgG1 and IgG4, correlations between less allergic symptoms and affinity of the antibodies were observed both in the SIT group and to a smaller extent in the non-SIT group. CONCLUSION: SIT has no effect on antibody affinity of allergen-specific IgE, IgG1 or IgG4. Allergic patients with high-affinity IgG1 and IgG4 antibodies report less symptoms than patients with low-affinity antibodies.