抗体和寡核苷酸双标记纳米金生物探针的制备及性能分析

基于纳米金粒子与抗体静电吸附作用,与硫醇修饰的寡核苷酸共价结合,建立一种新的双标记纳米金生物探针的制备方法.通过透射电镜（TEM）、紫外光谱、斑点免疫金渗滤法、免疫金银染色光镜观察法、荧光标记法等检测探针表征,及表面抗体活性情况和寡核苷酸的覆盖率,同时采用变性聚丙烯酰胺凝胶电泳（PAGE）检测寡核苷酸的存在.结果表明,纳米金粒子同时连接抗体和寡核苷酸后生物性能良好,且每个纳米金粒子（10±3）nm表面可覆盖寡核苷酸(92±20)条,双标记纳米金生物探针的制备具有简捷、稳定的特点.可作为一种新型探针应用于超微量蛋白质检测.     

壳聚糖-有机累托石/海藻酸钠复合纳米粒的制备

目的:以BSA作为模型药物,制备壳聚糖季铵盐-OREC复合物纳米微粒,建立一种安全有效的药物控释传递系统。方法:超声条件下,制备不同质量比的具有壳聚糖硅酸盐插层结构的复合物纳米微粒,观察其形态学特征、进行红外光谱分析。同时,测定OREC对BSA包封率和载药量的影响。结果:成功制备了不同质量比的OREC-HTCC纳米粒子。电镜结果显示纳米粒呈圆球形,均匀,平均粒径约为30nm。红外图谱分析证实,HTCC插入了OREC插层中,BSA成功地包裹入HTCC-ALG/OREC混合材料制备的纳米微粒。加入OREC后,纳米粒子的包封率及载药量均明显提高,但随着加入量的增加,包封率及载药量逐渐减少。结论:OREC-HTCC纳米粒子是良好的蛋白药物载体,具有粒径小、包封率高、缓释效果好等优点,为CS-OREC作为潜在的药物给药系统的进一步应用提供科学依据。     

蛋白冠与纳米粒子的相互作用

近年来,尽管纳米粒子在生物医学领域的研究中取得了巨大的进展,但很少能进入临床试验阶段,其中,很大程度取决于人们缺乏对纳米粒子与生理环境之间相互作用的认知,对纳米粒子进入体内后的生物学特性了解有限。在生理环境下,蛋白质会吸附于纳米粒子表面,从而形成蛋白冠,这种纳米粒子-蛋白冠复合物的形成严重影响纳米粒子的生物学特性,限制了纳米粒子的临床应用,因此,蛋白质与纳米粒子之间的相互作用应该被深入研究。目前,对纳米粒子-蛋白冠复合物的研究属于一个相对较新的研究领域。概括了蛋白冠的研究现状,对蛋白冠与纳米粒子相互作用所产生的影响进行了重点阐述,也介绍了预防和减少蛋白冠形成的方法,为纳米粒子的进一步研发提供了思路。     

纳米金对PCR扩增效率影响的机制探讨

近年来,纳米金粒子对聚合酶链式反应（polymerasechainreaction,PCR）的增效作用倍受关注,但是其具体的机制仍未明确提出。研究发现在PCR反应中,纳米粒子的增效作用是存在最佳浓度的,增加DNA聚合酶或者小牛血清蛋白（BSA）可以消除纳米金粒子导致的抑制。我们认为,纳米金粒子可能起到了类似于聚合酶B亚基的作用,提高了DNA聚合酶的延伸能力;而过量纳米金对PCR的抑制作用可能与纳米金结合单链DNA产生的位阻效应有关。     

RGD/FA双靶纳米金棒的制备及其在肿瘤细胞协同靶向成像与光热治疗中的应用

目的:通过制备RGD/FA双靶纳米金考察其与高表达整合素与叶酸受体B16细胞的协同靶向成像与热疗作用;方法:采用功能化PEG分子将靶向小分子RGD与叶酸通过强健Au-S键连接至纳米金棒表面,利用激光共聚焦与808 nm近红外激光器评价修饰纳米金的协同靶向作用;结果:RGD与叶酸分子被成功连接于纳米金表面,且双靶纳米金对小鼠黑色素瘤细胞具有较好的协同靶向作用;结论:同时靶向同一肿瘤细胞的不同表位,可克服单一靶向功能化纳米粒子难以在肿瘤位点有效积累的问题,本研究为多功能纳米金棒在临床肿瘤早期诊断与光热治疗中的应用提供研究基础。     

基于纳米金修饰的核酸适体生物传感器用于环孢素浓度的体外测定

利用环孢素(CsA)的两条高亲和力核酸适体生物传感器用于环孢素浓度的体外测定,在柠檬酸三钠为还原剂条件下,以氯金酸为原料,制备纳米金(Au)溶液,并对其进行表征,将aptamer-Ⅰ经酰胺化反应共价结合到磁性纳米颗粒表面,以纳米金(Au)修饰aptamer-Ⅱ,以固定在磁性纳米颗粒表面的aptamer-Ⅰ与环孢素孵育,再与Au-aptamer-Ⅱ作用,形成磁性纳米颗粒/环孢素/纳米金三明治夹心结构,磁分离技术分离三明治复合物,210 nm处检测分离前后紫外吸收强度的变化,对环孢素进行定量检测。结果紫外检测结果显示,制备的纳米金溶胶在520 nm处呈现典型的吸收峰,JEM-4 000EX电镜结果显示,纳米金溶胶颗粒粒径分布均匀、形貌、分散度较好,粒径大小在30 nm左右,此法对环孢素的响应范围是50~1 500 ng/m L,其线性回归方程为Y=4×10~(-3)X+5.769×10~(-1),R=0.997 3。此法可在1~2 h内完成检测,有望用于血液环孢素浓度的测定。     

基于纳米金的比色分析法在核酸检测中的应用

随着纳米技术的发展,运用纳米粒子检测核酸成为研究的热点.在众多检测方法中,基于纳米金的比色分析法操作较为简便,只需普通光学仪器甚至肉眼即可观察结果,从而表现出广阔的市场及临床应用前景.基于纳米金的比色分析法有多种,不同检测原理的方法在灵敏度和实用性上存在差异.根据纳米金是否经寡核苷酸探针修饰可将其分为基于功能化纳米金的比色分析法和基于未功能化纳米金的比色分析法,前者又分为利用纳米金颜色变化的聚集反应体系以及利用纳米金特殊氧化-还原能力的银染增强体系.     

纳米金标记核酸探针检测小反刍兽疫 病毒核酸的研究

试验旨在探讨利用纳米金标记寡核苷酸探针快速检测小反刍兽疫病毒核酸的方法。针对小反刍兽疫病毒N基因的高度保守区设计两条特异寡核苷酸探针,一条探针5’端修饰生物素,另一条探针3’端修饰巯基。巯基化的探针通过Au-S键连接到纳米金颗粒上。靶核酸两端分别与两条探针结合,形成"生物素化探针-靶核酸-纳米金探针"复合物,该复合物通过生物素-亲和素系统,固定在固相载体上,最后银染放大信号。通过肉眼观察法、光镜观察法、分光光度法分析银染灰度,从而间接测定靶核酸的量。初步检测PPRV核酸最低浓度达10fmol/L,所需时间约为1.5h。该方法灵敏度高、操作简单,为临床检测小反刍兽疫病毒核酸提供实验数据和技术支持。     

基于纳米金光学性质的分子检测与应用

纳米金颗粒是近年研究最为广泛的纳米材料之一,它具有良好的生物相容性、化学稳定性以及独特的光学性质,在生物分子检测、诊断和治疗方面具有很大的发展潜力。尤其是纳米金显示出特殊的表面等离子体共振现象,导致了粒子表面产生强电磁场,并最终增强了诸如吸收和散射的辐射特性,其散射光强与粒子的尺寸和团聚状态有密切关系。而由于共振现象而产生的纳米金对光的强烈吸收并高效转换为热效应也被用于检测和治疗。此外,与纳米金尺寸相关的局域表面等离子体共振光学特性,能够在粒子附近产生很强的电磁场增强,从而构成表面增强拉曼散射的基础。纳米金在强光照射下也表现出良好的抗光漂白的荧光现象,其特有的荧光寿命也成为检测的一种有效手段。与其他荧光物质作用时,又表现出表面增强荧光特性以及荧光共振能量转移。综述中,在介绍纳米金这些特殊光学性质的基础上,回顾了其在生物分子检测方面的应用进展。     

靶向金纳米棒对隐球菌活性影响的体外实验研究

目的：设计对隐球菌荚膜特异性标记的靶向金纳米棒,研究靶向金纳米棒的体外光热作用对隐球菌活性的影响。方法晶种生长法制备金纳米棒,偶联隐球菌荚膜抗体,检测表征,与隐球菌体外孵育,近红外激光照射,检测隐球菌活性变化。结果成功制备与荚膜抗体偶联的金纳米棒,体外近红外照射后,隐球菌活性较未偶联抗体的金纳米棒组显著降低。结论靶向性金纳米棒显著增强了近红外激光对隐球菌的光热效应,可用于治疗隐球菌感染的新尝试。     

Resonance Rayleigh-scattering method for the determination of proteins with gold nanoparticle probe

Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

Nanoparticle-based electrochemiluminescence immunosensor with enhanced sensitivity for cardiac troponin I using N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles as labels

A novel nanoparticle-based electrochemiluminescence (ECL) immunosensor was designed for highly sensitive and selective detection of human cardiac troponin I (cTnI), an important Acute Myocardial Infarction (AMI)-related biomarker, by using N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles (ABEI-AuNPs) as labels. ABEI-AuNPs were successfully synthesized via a simple seed growth method. A great number of luminescence molecules ABEI as stabilizers were coated on the surface of the AuNPs, which exhibited better ECL activities than previously reported luminol functionalized gold nanoparticles. ABEI-AuNPs were used as new ECL labels to build bio-probes by conjugation with secondary antibodies, which showed good ECL activity, immunological activity, and stability. Another kind of AuNPs functionalized with streptavidin was modified on the electrode surface for biotinylated antibodies capture through the specific interaction of biotin/streptavidin and enhancing the electrical connectivity. By combining with the novel ECL labels and amplification of AuNPs and biotin-streptavidin system, a high sensitive sandwich-type electrochemiluminescence immunoassay was developed for detecting human cTnI with a low detection limit of 2 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of cTnI in real samples, which was of great potential application in clinical analysis. Importantly, the sensitive detection would have far more diagnostic value than would absolute measurements during the early stage of AMI.     

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.     

Characterization of DNA chips by nanogold staining

DNA microarray is an important tool in biomedical research. Up to now, there are no chips that can allow both quality analysis and hybridization using the same chip. It is risky to draw conclusions from results of different chips if there is no knowledge of the quality of the chips before hybridization. In this article, we report a colorimetric method to do quality control on an array. The quality analysis of probe spots can be obtained by using gold nanoparticles with positive charges to label DNA through electrostatic attraction. The probe spots can also be detected by a simple personal computer scanner. Gold nanoparticles deposited on a glass surface can be dissolved in bromine-bromide solution. The same microarray treated with gold particles staining and destaining can still be used for hybridization with nearly the same efficiency. This approach makes quality control of a microarray chip feasible and should be a valuable tool for biomarker discovery in the future.     

Single-molecule enzymology based on the principle of the Millikan oil drop experiment

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.     

Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles

A simple, cost-effective and rapid colorimetric method for any or all of Hg(2+), Pb(2+) and Cu(2+) detection using papain-functionalized gold nanoparticles (P-AuNPs) has been developed. Papain is a protein with seven cystein residues, which can selectively bind with Hg(2+), Pb(2+) and Cu(2+). We functionalized gold nanoparticles with papain. The P-AuNPs could be used to simultaneously detect Hg(2+), Pb(2+) and Cu(2+), and showed different responses to the three ions in an aqueous solution based on the aggregation-induced color change of gold nanoparticles. The P-AuNPs displayed the most obvious response to mercury ions in water in contrast to lead and copper ions, and the real water sample analysis verified the conclusion. The sensitivity of the detection system was influenced by the pH of the P-AuNPs solution, the concentration of P-AuNPs and the size of gold nanoparticles, and we found that larger gold nanoparticles contributed to more sensitive results. The detection system can detect as low as 200 nM Hg(2+), Pb(2+) or Cu(2+) using 42 nm gold nanoparticles. We expect our approach to have wide-ranging applications in the developing region for monitoring water quality in some areas.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.