基于微流控芯片SERS生物传感器的发展与应用

表面增强拉曼光谱(surface enhanced Raman spectroscopy,SERS)作为一种新兴的、具有超高灵敏度无标记指纹的高级痕量分析技术,与微流控芯片相结合用于光流体检测可以实现快速、无损伤检测分析。稳定、重现、高强度、高灵敏度的SERS信号是获取有效生物传感信息的首要条件。大量研究表明,可控性金属纳米粒子聚集体或纳米颗粒阵列对产生上述SERS信号至关重要。本文综述了利用电场、磁场形成可控性金属纳米粒子聚集体、固体SERS活性基底对高灵敏度SERS信号的增强和重现作用,固态SERS活性基底对其体表面积的增加作用,以及SERS生物传感器在临床免疫测定中的应用等方面的研究进展,为进一步深入研究贵金属纳米结构对SERS信号的增强、重现机制,特别是SERS生物传感器的应用提供参考,同时也为通过优化设计固态SERS活性基底进而实现便携、智能的SERS生物传感器系统提供一定的科学依据。     

网状纤维染色中氨银溶液配制的再认识

传统组织病理学技术理论中,用于病理切片组织网状纤维染色的氨银溶液有多种配制方法,本文基于相关化学原理对其进行比较优选. 1 传统氨银溶液配制方法的比较分析 1.1 James法、Lillie法和Lillie-Fullmer法的异同 Lillie法和Lillie-Fullmer法氨银溶液几乎是一样的,James法氨银溶液虽配制工艺不同,但试剂成分也是相同的,3种方法都只用硝酸银水溶液与氨水直接反应制得氨银溶液,而且都认为溶液略有混浊为好.3种方法的试剂用量较大.     

草鱼鱼皮胶原蛋白与NIH3T3成纤维细胞的相容性

体外检测草鱼鱼皮酸溶性胶原蛋白(FASC)的细胞相容性。采用MTT实验,检测不同浓度的FASC溶液或FASC涂层胶对NIH3T3成纤维细胞增殖能力的影响(n=6);利用transwell趋化实验,检测不同浓度的FASC溶液对成纤维细胞的趋化作用(n=3);利用transwell侵袭实验,检测不同浓度FASC涂层胶的细胞通透性(n=3)。结果表明,FASC溶液浓度依赖性地促进NIH3T3成纤维细胞增殖,16 μg/mL的FASC溶液在48和96 h的增殖率分别为63.7%±7.9%和87.3%±8.7%,较对照组有显著性差异(P<0.001)。NIH3T3细胞能够在FASC涂层胶上生长,各实验组与对照组相比,增殖率均无显著性差异(P > 0.05)。趋化实验显示,FASC溶液各浓度梯度组与阴性对照组的趋化指数均有显著性差异(P < 0.05),提示FASC对成纤维细胞具有趋化作用。侵袭实验结果显示,成纤维细胞可以通过FASC涂层胶。实验证实,FASC与NIH3T3细胞具有良好的相容性。     

梅毒压电石英晶体免疫传感器的研制

分别用聚乙烯亚胺(PE I)和戊二醛(G lu)溶液在压电石英晶体银电极上固定梅毒抗体来检测不同浓度的标准抗原溶液。抗体在晶体表面固化后通过原子力显微镜(AFM)显示依然保持Y型分子结构。石英晶体的响应线性范围5×10-5~1.25×10-4g/mL,相关系数为0.9976,最佳响应pH值为7.5。在检测时通过和BSA相对照,传感器有良好的选择性。最后,又对传感器重复性作了一定探讨。     

尿酸钠对BALB/c小鼠抗体应答及树突状细胞和迟发型超敏反应的影响

目的:探讨尿酸钠作为佐剂对BALB/c小鼠体液和细胞免疫应答的影响。方法:利用尿酸钠悬浮液为佐剂、天花粉蛋白(TCS)为免疫原对BALB/c小鼠进行免疫,以酶联免疫测定法检测特异性抗体IgG的效价。体外诱导小鼠树突状细胞(DC),流式细胞术分析DC表型,评价尿酸钠体外对DC成熟的效应。以二硝基氟苯建立迟发型超敏反应(DTH)模型,分析尿酸钠在体内对细胞免疫应答的影响。结果:传统弗氏佐剂可极大地增强小鼠对TCS的抗体应答,尿酸钠佐剂对抗体应答不但没有促进,与单独使用免疫原相比,抗体应答反而明显降低。流式细胞术分析显示,尿酸钠对DC表达CD11c和CD83没有影响,但可明显提高MHCII的表达水平。DTH模型中,尿酸钠增强致敏原引发的耳廓肿胀程度,并促进DTH小鼠淋巴细胞的体外增殖能力。结论:尿酸钠悬浮液作为佐剂,对细胞免疫有显著增强作用,而对体液免疫应答却有一定的抑制作用,提示该佐剂在疫苗研究中有潜在应用前景。     

尿酸对HBV表位肽S28-39负载DC诱导免疫效应的影响

目的 观察尿酸对由HBV表面抗原表位肽负载的树突状细胞诱导免疫效应的影响.方法 以负载肽S28-39的小鼠骨髓来源树突细胞,联合尿酸(uric acid, UA)尾静脉注射接种小鼠,每周1次,共2次.分为DC对照组,联合尿酸组,尿酸对照组,正常对照组.流式细胞仪法检测特异性CTL活性;ELISA法检测小鼠脾细胞培养上清液IFN-γ水平.结果 体内S28-39特异性CTL,以联合尿酸接种组杀伤活性最强;联合尿酸接种组脾细胞IFN-γ分泌较对照组增高.结论 尿酸能够增强乙肝表位肽负载的树突细胞诱导的S28-39特异性CTL杀伤活性;能促进免疫小鼠脾淋巴细胞分泌IFN-γ.尿酸具有增强负载S28-39树突细胞诱导的细胞免疫效应的活性.     

网状纤维染色关键染液的配制方法

网状纤维的染色方法较多,Gomori银染色法是最常用的方法之一。染色过程需用酸性高锰酸钾溶液、铁明矾溶液、草酸溶液、氨银溶液、甲醛溶液、氯化金溶液、硫代硫酸钠溶液7种[1],其中氨银溶液是提高网状纤维染色力的关键染液,其配制质量至关重要。本实验在配制方法上进行了改良,使网状纤维染色阳性强度增加,且使染液保存期延长,现介绍如下。1材料与方法1.1材料硝酸银、氢氧化钠、氨水。     

拉曼光谱技术及其在中医药鉴定中的应用

目的：拉曼光谱技术（Ramanspectroscopytechnique）是一种非破坏性的指纹成像技术。现已在材料、化工、生物医学、环保等领域有着广泛的应用。而根据拉曼光谱技术所发展起来的表面增强拉曼光谱技术（Surface—EnhanceRamanSpectra,SERS）也在溶剂检测中得到了普遍的应用。然而,拉曼光谱在中医药鉴定方面的应用却并不多见。目前．随着中医药在国际上的发展,对于中医药质量的监控已经越来越受到重视。本文旨在讨论拉曼光谱技术及其在中医药鉴定中的应用。方法：通过调研大量的相关文献,讨论并总结了拉曼光谱技术及表面增强拉曼光谱技术（SERS）在鉴定中医药及中医药煎剂成分、产地、真伪等方面的应用。结果：拉曼光谱技术凭借其样品制备简便、对水的散射小等优点,拉曼光谱成功鉴别出了单味中医药的成分、产地以及真伪。同时,表面增强拉曼光谱技术在中医药煎剂成分的鉴定中也体现出了其优越性。．结论：拉曼光谱技术及表面增强拉曼光谱技术做为一种新的鉴定手段,中医药鉴定方面有着巨大的潜力,并有望成为中医药鉴定的另一强大工具。     

植物性胶黏剂的黏附性及生物相容性

背景：前期研究将从植物中提取的原材料加工制备出了一种黏性较强的可吸收性局部止血胶黏剂。 目的：检测局部止血的植物性胶黏剂对肾脏组织的黏附力,并检测其组织相容性。 方法：将不同浓度的植物性胶黏剂均匀涂抹在家兔肾脏表面,检测同一浓度在双侧肾脏不同部位的黏附力。将KM小鼠分为：实验组在大腿肌群切口内填入植物性胶黏剂,对照组切口未加入胶黏剂。 结果与结论：不同浓度的植物性胶黏剂有不同黏附力,1 g植物性胶黏剂粉末中加入0.6 mL蒸馏水组呈较浓糊状,且黏附力高。将胶黏剂埋植入小鼠体内后,皮肤切口愈合良好,实验组和对照组小鼠血生化、免疫学检测差异无显著性意义(P > 0.05),实验组小鼠手术区肌肉、肝脏和肾脏组织标本苏木精-伊红染色无明显炎症反应。表明植物性胶黏剂有良好的黏附性和组织相容性。     

高压微胶囊成型装置制备用于成囊的海藻酸钙胶珠

利用高压微胶囊成型装置进行了制备用于成囊的海藻酸钙胶珠的试验研究 ,从而为用于药物控制释放的聚精氨酸基微胶囊的制备提供基础制备参数。将海藻酸钠溶液经试验装置滴入氯化钙溶液中固定化成海藻酸钙凝胶珠 ,考察了海藻酸钠浓度对制得的胶珠性能的影响及推进速度、针头内径、电压等对制得的胶珠直径的影响。得到较佳的海藻酸钠浓度为1.8% (W /V) ,发现推进速度提高 ,胶珠直径增大 :针头内径越大 ,胶珠直径越大 ;电压增大 ,胶珠的直径先减少后增大 ;不同针头内径制备出胶珠的平均直径最小时所对应的电压也随针头内径的增大而增大     

Label-free serum ribonucleic acid analysis for colorectal cancer detection by surface-enhanced Raman spectroscopy and multivariate analysis

Studies with circulating ribonucleic acid (RNA) not only provide new targets for cancer detection, but also open up the possibility of noninvasive gene expression profiling for cancer. In this paper, we developed a surface-enhanced Raman scattering (SERS), platform for detection and differentiation of serum RNAs of colorectal cancer. A novel three-dimensional (3-D), Ag nanofilm formed by dry MgSO(4) aggregated silver nanoparticles, Ag NP, as the SERS-active substrate was presented to effectively enhance the RNA Raman signals. SERS measurements were performed on two groups of serum RNA samples. One group from patients, n=55 with pathologically diagnosed colorectal cancer and the other group from healthy controls, n=45. Tentative assignments of the Raman bands in the normalized SERS spectra demonstrated that there are differential expressions of cancer-related RNAs between the two groups. Linear discriminate analysis, based on principal component analysis, generated features can differentiate the colorectal cancer SERS spectra from normal SERS spectra with sensitivity of 89.1 percent and specificity of 95.6 percent. This exploratory study demonstrated great potential for developing serum RNA SERS analysis into a useful clinical tool for label-free, noninvasive screening and detection of colorectal cancers.     

A sandwich substrate for ultrasensitive and label-free SERS spectroscopic detection of folic acid / methotrexate

A highly sensitive surface enhanced Raman scattering (SERS) substrate with particle-film sandwich geometry has been developed for the label free detection of folic acid (FA) and methotrexate (MTX). In this sandwich structure, the bottom layer is composed of a copper foil decorated with silver nanoparticles synthesized by the galvanic displacement reaction, and top layer is constituted by silver nanoparticles. The FA and MTX molecules are sandwiched between the silver nanoparticles decorated copper film and the silver nanoparticles. The plasmonic coupling between the two layers of the sandwich structure greatly enhances the SERS spectra of FA and MTX. SERS activity of the substrate was studied and optimized by adjusting the time of galvanic displacement reaction. The SERS spectra of the FA and MTX showed the minimum detection concentration of 100 pM. The identification of methotrexate and folic acid analogs was also carried out by SERS spectra analysis.     

An amperometric biosensor for uric acid determination prepared from uricase immobilized in polypyrrole film

In order to prepare a biosensor for the determination of uric acid, electropolymerization of pyrrole on Pt surface was carried out with an electrochemical cell containing pyrrole, ferrocene (as a electron mediator) and tetrabutylammonium tetrafluoroborat in acetonitrile by cyclic voltammetry between 0.0 and 1.0 V (vs. Ag/AgCl) at a scan rate of 50 mV/s upon Pt electrode. Uricase was immobilized by a glutaraldehyde/gelatine croslinking procedure on to polypyrrole film after the electropolymerization processes. The response of the biosensor against uric acid was measured after 330 seconds following the application of a constant potential of +0.7 V (vs. Ag/AgCl). The resulting biosensor exhibits excellent electrocatalysis for the uric acid. The amperometric determination is based on the electrochemical detection of H2O2, which is generated in enzymatic reaction of uric acid. The sensor responds to uric acid with a detection limit of 5.0 x 10(-7) M. The sensor remains relatively stable for 5 weeks. Interference effect were investigated on the amperometric response of the biosensor. Determination of uric acid was carried out in the biological fluids by biosensor.     

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of excitatory amino acids

The release of excitatory amino acids (EAAs) from injured neurons has been associated with secondary injury following head trauma. The development of a rapid and sensitive method for the quantification of EAAs may provide a means for clinical management of patients affected by head trauma. We explore the potential application of surface-enhanced Raman spectroscopy (SERS) for rapid quantification of the concentration of EAAs in aqueous silver colloids. The EAAs glutamate (Glu) and aspartate (Asp) are released following head injury and have been observed to exhibit SERS spectra that should enable them to be distinguished in a complex aqueous media. Of the two EAAs, the concentration of Glu has been shown to be more indicative of injury to the central nervous system. Using 30-s scans and a 50-mW argon laser, aqueous Glu is quantifiable from 0.4 to 5 micromol/L and is spectrally distinguishable from Asp. In addition, initial in vivo microdialysis experiments suggest that this SERS system is capable of measuring chemical changes following head trauma in the rat brain. Compared with current high-performance liquid chromatography (HPLC) techniques for amino acid detection, the short scanning and processing time associated with the SERS approach enables measurement on a near-real-time basis, providing clinical information in anticipation of pharmaceutical intervention.     

A study of dose-response relationships of allpurinol in the presence of low or high purine turnover

Summary Effects of allopurinol (125–500 mg/m² body surface) were studied in normal subjects during periods of 18 days both during a purine-free, isoenergetic liquid formula diet and additional intake of ribonucleic acid, 4 g/day. Plasma uric acid and renal excretion of uric acid, oxypurines (hypoxanthine plus xanthine) and orotic acid were measured and total purine excretion calculated. Effects of allopurinol were evaluated by comparison of the results obtained in the steady state during diet alone (average of days 7–10) with those during allopurinol administration (days 16–18).During the purine-free diet, plasma uric acid was lowered more than urinary uric acid by allopurinol on doses of 250–500 mg/m² (44%–54% of control values on 500 mg/m²), demonstrating an increase in renal clearance. At the same dose, the uric acid lowering effect of allopurinol was more pronounced with than without purine loads (plasma 41%, urine 32% of control on 500 mg/m² during purine intake), while renal uric acid clearance was decreased. The more pronounced reduction of uric acid excretion during purine administration was balanced to the greater part by a more pronounced increased in oxypurine excretion. Total purine excretion was reduced by about 20% during the purine-free diet irrespective of dose. The size of this purine deficit was doubled, but was also independent of dose during addition of purines. Orotic acid excretion increased with dose during allopurinol treatment and was reduced by addition of purines.With respect to uric acid lowering effects, these results are in accordance with findings in patients overproducing uric acid endogenously and suggest that the uric acid lowering effect of allopurinol is enhanced with increasing concentrations of purine bases, presumably due to the tight binding of oxipurinol to xanthine oxidase. The small uricosuric effect of allopurinol seen during ingestion of the purine-free diet possibly is attributable to drug-induced orotic aciduria. The increase in size during purine intake of the purine deficit may result from reduced absorption of dietary purines during allopurinol treatment. Apparently, maximum effects of allopurinol on endogenous synthesis and/or absorption of purines from the gut are exerted by low doses.Abbreviations HPRT Hypoxanthine phosphoribosyl transferase - PRPP Phosphoribosyl pyrophosphate - RNA Ribonucleic acid This study was supported in part by grants of the Deutsche Forschungsgemeinschaft and Deutsche Wellcome GmbHDedicated to Prof. Nepomuk Zöllner on the occasion of his 65th birthday     

Polyhedral silver mesocages for single particle surface-enhanced Raman scattering-based biosensor

Surface-enhanced Raman scattering-based signal detection and molecular identification faces the lack of reproducibility and reliability thus hampers its practical applications. Here, we demonstrate a facile particle mediated aggregation protocol to synthesize highly roughened mesosuperstructure--silver polyhedral mesocages. The individual silver octahedral mesocage, owing to highly-roughed surface topography, anisotropic growth as well as intraparticle effect, creates homogenously distributed multiple effective hot spots on the surface of single mesoparticle, hereby exhibits a high reproducibility and an unusual SERS enhancement, i.e., ～ 10(8)-10(9) magnitude. As such, the current protocol opens avenues for the fabrication of structurally reproducible mesosuperstructure-based SERS sensors.     

人类精液中一氧化氮和尿酸含量检测的关系

目的探讨人精液中一氧化氮(N0)与尿酸含量的关系,对精了质量的影响。方法参照WHO标准方法,进行精液常规分析,按精子密度、活动率不同分为(正常、<20、20~40、>40)4个组。采用镀铜镉还原荧光法检测NO代谢产物硝酸盐(NO3-)。采用尿酸酶一过氧化物酶偶联法检测精液尿酸含量。结果70例不育组精液中尿酸含量和NO含量为(236.4±47.8)μmol/L、(78.7±1.6)μmol/L与正常生育组(398.6±52.3)μmol/L、(41.84±1.6)μmol/L呈显著性差异(P<0.01)。将尿酸含量与NO含量进行相关性分析,两者呈显著性负相关(r=-0.96,P<0.05)。不育各精子密度和活动率组精液尿酸含量随精子密度及活动率增加而上升,N0含量随之下降(P<0.01),结论精液尿酸含量测定可作为评价精子受活性氧损害的重要指标,证明尿酸对活性氧尤其在医学领域极为重视的NO损害精子具有保护性作用。     

Surface‐Enhanced Raman Scattering on Molecularly Imprinted Polymers in Water

Molecularly imprinted polymer layers were prepared on SERS‐active surfaces in order to directly monitor the uptake and release of certain substances to this polymer layer by surface enhanced Raman‐scattering (SERS). The imprinting system consisted of either (2S,3S)‐(+)‐di‐O‐benzoyl‐tartaric acid ( 1 ) or N‐benzyloxycarbonyl‐(L )‐aspartic acid ( 2 ) as templates. As binding site N,N′‐diethyl‐4‐vinylbenzamidine ( 3 ) was used which binds in a 2:1 complex to the templates. The release and uptake of the templates to the imprinted polymer could be followed in aqueous solution under physiological conditions (aqueous buffer, ambient temperature). The recorded SERS bands can be unequivocally assigned to the substances taken up, and it is therefore possible to directly detect a certain substance in the imprinted layer. Release and uptake are quick processes occurring within minutes. This method also offers the opportunity to use these layers in chemosensors in which case the stability has to be further increased.

SERS spectra of polymer coatings containing the template N‐benzyloxycarbonyl‐(L )‐aspartic acid ( 2 ) on gold and on silver surfaces, Raman spectra of pure 2 (template) and of a HEPES buffer solution (0.1 M , pH 7.3) which was used for the adsorption measurement.     

A novel screen-printed microfluidic paper-based electrochemical device for detection of glucose and uric acid in urine

A novel screen-printed microfluidic paper-based analytical device with all-carbon electrode-enabled electrochemical assay (SP-ACE-EC-μPAD) has been developed. The fabrication of these devices involved wax screen-printing, which was simple, low-cost and energy-efficient. The working, counter and reference electrodes were screen-printed using carbon ink on the patterned paper devices. Different wax screen-printing processes were examined and optimized, which led to an improved method with a shorter heating time (~5 s) and a lower heating temperature (75 °C). Different printing screens were examined, with a 300-mesh polyester screen yielding the highest quality wax screen-prints. The carbon electrodes were screen-printed on the μPADs and then examined using cyclic voltammetry. The analytical performance of the SP-ACE-EC-μPADs for the detection of glucose and uric acid in standard solutions was investigated. The results were reproducible, with a linear relationship [R²?=?0.9987 (glucose) or 0.9997 (uric acid)] within the concentration range of interest, and with detection limits as low as 0.35 mM (glucose) and 0.08 mM (uric acid). To determine the clinical utility of the μPADs, chronoamperometry was used to analyze glucose and uric acid in real urine samples using the standard addition method. Our devices were able to detect the analytes of interest in complex real-world biological samples, and have the potential for use in a wide variety of applications.     

颈动脉斑块、血同型半胱氨酸及血尿酸对短暂性脑缺血 发作进程的影响研究

目的 探讨不同病程短暂性脑缺血发作（TIA）患者血清同型半胱氨酸（HCY）、血清尿酸（UA）含量的变化及临床意义。方法 选取2013年6月~2017年6月在我院神经内科住院的患者80例,根据TIA最终是否进展为脑梗死分为A组（TIA未进展为脑梗死者）和B组（TIA在24~48 h经头颅CT或MRA检查证实有脑梗死发生者）,每组40例。另选取同期我院40例健康体检者作为对照组。所有患者及健康体检者均行血清同型半胱氨酸及血清尿酸水平并进行分析,其中A、B两组分别行颈动脉超声检查,并对两组患者颈动脉斑块性质进行比较。结果 B组患者血同型半胱氨酸和血尿酸水平均高于A组和对照组,差异具有统计学意义（P＜0.05）;A组血同型半胱氨酸和血尿酸水平高于对照组,差异具有统计学意义（P＜0.05）。A组检出硬斑块15例,软斑块10例;B组检出硬斑块10例,软斑块25例;B组颈动脉软斑块检出率为62.50%,高于A组的25.00%,差异有统计学意义（P＜0.05）。结论 血同型半胱氨酸和血尿酸水平升高及颈动脉斑块尤其是不稳定斑块可能是TIA患者的危险因素,也是TIA患者早期展为脑梗死的重要危险因素。早期发现血同型半胱氨酸、血尿酸水平升高及颈动脉斑块可作为TIA患者的病情评估依据,并对TIA患者是否进一步进展为脑梗死具有一定的预测作用,故可进行早期干预,进而预防TIA患者进一步进展为脑梗死。