荧光免疫及磁免疫层析法检测莱克多巴胺的研究

分别将量子点和超顺纳米磁珠作为荧光探针和磁信号探针应用于免疫反应中,构建了检测莱克多巴胺的荧光免疫和磁免疫层析的分析方法,并成功应用于尿液中莱克多巴胺的检测。两种方法均基于免疫竞争模式,在荧光免疫分析方法中,量子点偶联上识别莱克多巴胺的抗体,样品中莱克多巴胺和包被在ELISA板上莱克多巴胺的完全抗原竞争结合量子点,样品中莱克多巴胺的浓度越高,ELISA板上吸附的量子点越少,所测荧光强度值越低,该方法的检出限为1 ng·mL-1,检测时间为4 h。在磁免疫层析方法中,检测线上特异性捕获的纳米磁珠颜色的深浅和尿液中莱克多巴胺浓度成反比例关系,即莱克多巴胺的浓度越高,检测线的颜色越浅,该方法的定性检出限为10 ng·mL-1,检测时间为15 min。两种方法各有优缺点,基于量子点的荧光免疫分析法在痕量检测和定量分析方面具有优势,而磁免疫层析法更适合于现场快速检测。     

基于免疫磁分离和酶促信号放大传感器的构建及应用研究

甲胎蛋白(AFP)作为肿瘤标志物,其检测在肝癌早期诊断过程中有着重要意义。磁分离技术能够实现快速无损分离,可以有效简化实验过程。本实验用免疫磁珠作为固相载体连接捕获抗体,并与酶标二抗通过AFP连接形成夹心结构,酶催化外加底物使其显色,最终用紫外分光光度法检测底物浓度间接实现对溶液中AFP的定量检测。体系对AFP的检测范围为10~80ng·mL-1,检出限为3.6ng·mL-1。该方法灵敏度高,重现性好,为AFP的检测提供了新的思路和方法。     

水溶性量子点荧光探针用于帕珠沙星的含量测定

文章采用荧光光谱和紫外光谱研究了CdTe量子点(CdTe QDs)与广谱抗菌药物帕珠沙星的相互作用。结果表明,随着帕珠沙星浓度的增加,CdTe QDs荧光强度有规律的降低,但通过透射电镜图对QDs及加入帕珠沙星后的QDs进行比较,发现QDs仍然均一单分散,表明反应的作用机理可能是帕珠沙星促使QDs表面键合的有机分子发生变化,在Cd空位表现出的表面缺损上形成了碲氧复合物,致使荧光猝灭。因此该反应可作为一种新颖的快速检测帕珠沙星含量的方法。在一定条件下,帕珠沙星溶液的浓度与量子点荧光强度成线性关系,线性范围为10.0～850 μg·mL-1,相关系数r为0.995 4,检测限(S/N=3)为3.254×10-3 μg·mL-1。药物对量子点的猝灭常数为2.188×104 L·mol-1。应用到冻干粉针剂和氯化钠注射液中帕珠沙星的含量测定,所得结果与标示量一致。该方法较常用检测方法具有简便、快捷、灵敏、线性范围宽的优点,并有望进一步开展发药物体内显像及作用机理的研究。     

量子点荧光免疫和磁弛豫时间传感检测沙门氏菌的研究

基于量子点和超顺磁纳米颗粒分别构建了沙门氏菌的荧光免疫和磁驰豫时间(magnetic resonance switch,MRS)免疫传感的分析方法,并应用于水样中沙门氏菌的检测。在荧光免疫分析方法中,利用偶联有沙门氏菌捕获抗体的磁探针对样品中沙门氏菌进行富集,然后加入偶联有沙门氏菌检测抗体的量子点荧光探针,基于双抗夹心的模式对水样中沙门氏菌的含量进行了测定。量子点的荧光强度和样品中沙门氏菌的浓度呈正相关,检出限是102 cfu·mL-1,检测时间为2h。在MRS免疫反应过程中,偶联有沙门氏菌捕获抗体的超顺纳米磁珠会特异性地结合沙门氏菌,导致超顺纳米磁珠由原来的分散状态转变为聚集状态,从而引起其相邻水分子的质子横向弛豫时间(spin-spin relaxation time,T2)的改变,通过测定T2的改变量ΔT2实现水样中沙门氏菌的检测。结果表明MRS传感器检测沙门氏菌的检测限是103 cfu·mL-1,检测时间是0.5h。比较了这两种方法在检测沙门氏菌方面的优劣及应用潜力。     

基于免疫传感器和激光诱导击穿光谱的痕量农药测量方法

农药在农作物病虫害及农作物高产稳产等方面起到了相当重要的作用,但是农药的长期大量使用,对生态和人类健康造成了极大的危害。根据相关文献查阅,基于免疫传感器和激光诱导击穿光谱的分析方法对农药残留的检测未见相关报道。提出了激光诱导击穿光谱对免疫传感器捕获待测目标物的探针进行检测,从而间接计算出待测目标物的浓度。使用免疫层析试纸条对痕量农药进行检测,虽然免疫层析试纸条可以实现对痕量农药的测量,但是仅能定性且检测的范围很窄,为了拓宽免疫层析试纸条对痕量农药的测量范围,运用激光诱导击穿光谱（laser induced breakdown spectroscopy,LIBS）对免疫层析试纸条上捕获痕量农药的金属纳米颗粒进行光谱测量,构建免疫层析试纸条与LIBS的检测方法。以毒死蜱农药为研究对象,因农药残留属于小分子抗原检测,所以免疫层析试纸条运用竞争法对毒死蜱进行检测。滴加了低浓度毒死蜱的免疫层析试纸条上控制线和检测线的颜色差异不明显,难以用人眼分辨出是否检测到了毒死蜱。对添加了毒死蜱的免疫层析试纸条的控制线和检测线分别选取点,测量Au Ⅰ 242.733 nm处的光谱,用控制线的平均光谱强度减去检测线的平均光谱强度即为毒死蜱的信号,此方法可以检测出免疫层析试纸条观察不到的信号,还规避了LIBS检测限高的问题。随着毒死蜱浓度从0增加到106 ng·mL-1,LIBS光谱数据差值逐渐增大。为了消除随机误差的影响,使用ΔLIBS强度（测量样品强度减去空白样品强度）与毒死蜱浓度取Lg绘制校准曲线。ΔLIBS强度与毒死蜱浓度在10～106 ng·mL-1范围内呈线性相关,Y=6.14X+31.85,R2=0.969,毒死蜱的检测限为0.39 ng·mL-1。研究表明,免疫层析试纸条与LIBS结合实现了对痕量农药的测量,能有效的扩宽毒死蜱的检测范围。同时,免疫层析试纸条与LIBS的结合对其他物质的检测也值得进一步研究。     

基于固相抗体模式时间分辨荧光免疫分析测定雌三醇

采用二步标记法,借助二乙烯三胺五乙酸酐,将Eu3+标记到雌三醇完全抗原上,基于固相抗体竞争反应模式,建立了时间分辨荧光免疫分析测定雌三醇的新方法。在优化的条件下,方法的线性范围为0.005—1000ng·mL-1,检出限为4pg·mL-1。该方法用于检测血清中雌三醇的含量,结果令人满意。     

基于InP@ZnS QDs/Dured纳米荧光探针的DNA检测

利用巯基丙酸包覆的In P@Zn S量子点(QDs)与Dured构建了一种检测DNA的荧光探针。在该探针中,以环境友好型带负电的In P@Zn S量子点为荧光团,与带正电的Dured通过静电结合,构建了In P@Zn S QDs/Dured纳米荧光探针。通过荧光共振能量转移(FRET)机理,量子点荧光被猝灭;当DNA存在时,Dured与DNA的特异性结合使Dured从In P@Zn S QDs表面脱附,FRET过程被打断,In P@Zn S QDs荧光恢复,以荧光"关-开"方式检测DNA。该探针检测DNA的线性范围为2.0~275.0 ng·L-1,检测限为1.0 ng·L-1,并可用于模拟生物生理条件下的DNA检测。     

适配体传感法快速测定啤酒中赭曲霉毒素A

应用自组装方式,构建了金纳米粒子/核酸适体/氨基碳量子点荧光传感赭曲霉毒素A高灵敏检测方法。在pH 3.0酒石酸-HCl缓冲溶液中,巯基修饰的赭曲霉毒素A核酸适体在金纳米粒子表面自组装,形成金纳米粒子/核酸适体复合物,再在pH 7.0的磷酸盐缓冲溶液中,氨基碳量子点在金纳米粒子/核酸适体复合物上自组装,形成金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系。金纳米粒子的摩尔吸光系数大、能带宽使其具有强烈的荧光猝灭功能,氨基碳量子点形成金纳米粒子/核酸适体/氨基碳量子点后发生荧光猝灭, 此时体系的荧光为背景荧光,其强度记为F₀;由于金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系中核酸适体对赭曲霉毒素A具有特异性识别与结合功能,向金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系溶液中加入赭曲霉毒素A后,赭曲霉毒素A则与复合物中核酸适体立即发生特异性结合并释放出氨基碳量子点,体系荧光恢复,其荧光强度记为F。依据体系荧光强度的变化(F-F₀)与赭曲霉毒素A浓度之间的关系,建立赭曲霉毒素A核酸适体荧光传感检测方法。研究了金纳米粒子和核酸适体摩尔比、孵化时间、pH等因素对传感器性能的影响,确定了最优条件为金纳米粒子∶核酸适体t为1∶190、孵化时间为6 min、pH 7.0时;在最优条件下,赭曲霉毒素A浓度在0.005～1.00 ng·mL-1范围与体系荧光强度变化呈良好线性关系,线性回归方程为：F-F₀=6.499+211.6c(c为赭曲霉毒素A的浓度,单位：ng·mL-1),相关系数r为0.995 5,按3倍标准差与工作曲线的斜率的比值(3σ/k)计算,得检测限为3 pg·mL-1。在实际样品中的回收率在93.3%～108.9%,相对标准偏差小于5%,能满足啤酒样品中赭曲霉毒素A快速检测要求。对13个市售啤酒样品进行检测,其中6个样品检出赭曲霉毒素A,污染率为46.15%。受污染样品的赭曲霉毒素A含量在0.008～0.63 ng·mL-1范围。该荧光传感法检测赭曲霉毒素A具有灵敏性好、特异性高、常见真菌毒素无干扰、方法简单、快速,便于大众化推广应用的优点。     

基于CdTe量子点的三通道传感器对蛋白质的同步识别

描述了一种利用CdTe量子点对十三种不同蛋白质进行同步识别检测的三通道传感器。实验发现,不同的蛋白质分别与CdTe量子点结合后,可以使得CdTe量子点的荧光强度和颜色发生不同程度的改变;使用线性判别分析处理(LDA)可以实现这十三种蛋白质的区分识别。同时,使用荧光显微镜进行观察,可以发现与不同的蛋白质混合后,CdTe量子点可以在玻璃基底上呈现出形态各异的微结构。根据CdTe量子点的荧光强度、颜色和在玻璃基底上的形状变化,即可实现该三通道传感器的构建。该方法简单、快速且成本低,可为多通道生化传感器提供新的思路和素材。     

基于氮化碳量子点和金纳米簇的尿液中胰蛋白酶高灵敏度荧光检测研究

胰蛋白酶生产障碍会阻碍消化过程,在胰腺组织以外产生胰蛋白酶可能涉及癌症过程。胰蛋白酶明显增高可能表明胰腺炎或者慢性肾功能衰竭等病症的发生,它的含量与生命活动息息相关,简单并及时监测胰蛋白酶含量对疾病的诊断具有重要的参考价值。因此,该研究构建氮化碳量子点和金纳米簇（CNQDs和AuNCs）的复合纳米探针检测尿液中胰蛋白酶含量。通过煅烧三聚氰胺获得氮化碳粉末,并将氮化碳粉末作为原材料通过溶剂热法合成了发射峰在440 nm的类石墨相氮化碳量子点（CNQDs）。牛血清蛋白（BSA）和CNQDs两者同时作为还原剂和稳定剂合成了金纳米簇（AuNCs）,且AuNCs吸附在氮化碳量子点表面形成具有双发射性质的CNQD-AuNCs复合荧光纳米材料,发射波长分别为440 nm（CNQD的发射波长）和650 nm（AuNC的发射波长）。由于胰蛋白酶能特异性的水解CNQD-AuNCs中的牛血清蛋白,导致牛血清蛋白结构被破坏,从而破坏AuNCs稳定的结构,使得其沉淀聚集,引起荧光猝灭。由于AuNCs产生的650 nm处的荧光被猝灭,而CNQDs产生的440 nm处的荧光不受影响,CNQD-AuNCs复合荧光纳米探针产生比率型荧光信号响应。利用比率型荧光信号的变化情况,可实现胰蛋白酶的定量检测。CNQD-AuNCs探针在650 nm处的荧光强度随着胰蛋白酶浓度的增加而逐渐下降,而440 nm处的荧光强度保持不变。胰蛋白酶在一定浓度下（10～400 ng·mL-1）与荧光强度比值（I₆₅₀/I₄₄₀）呈良好的线性关系,建立的线性方程为y=2.471-0.004x[y为荧光强度比值（I₆₅₀/I₄₄₀）,x为胰蛋白酶的浓度（ng·mL-1）],相关系数（R2）高达0.997 6,检测限为1.5 ng·mL-1（3倍信噪比）。利用建立的荧光法检测尿液中胰蛋白酶（实际含量分别为50,100和150 ng·mL-1）,检测得到的平均含量分别为52.41,103.25和154.39 ng·mL-1。尿液中胰蛋白酶的回收率和相对标准偏差范围分别为102.93%～104.82%和3.57%～4.16%。结果表明,利用荧光强度比值（I₆₅₀/I₄₄₀）作为胰蛋白酶定量检测的信号,能够校正外界影响因素的干扰,克服单一荧光信号易受光漂白、探针浓度、激发光强度以及光程等外界因素的影响的缺点。基于CNQD-AuNCs建立的比率型荧光分析方法能够实现尿液中胰蛋白酶的高灵敏度和高特异性检测,为实际样品中胰蛋白酶的检测提供科学依据。     

Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Leishmania Detection in Sandflies

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (～100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.     

基于石墨相氮化碳量子点直接荧光猝灭法检测碘离子的研究

石墨相氮化碳(g-C₃N₄)荧光纳米材料具有原料便宜、制备容易、荧光量子产率高、光学稳定性好、毒性低等优点,并且避免有机荧光染料复杂的合成步骤或者金属半导体量子点对环境潜在的危害,这些优点使得g-C₃N₄纳米材料成为新兴的荧光探针用于检测金属离子。最近,已有文献报道重金属汞离子能够高灵敏高选择性地猝灭g-C₃N₄量子点的荧光,加入碘离子能够提取被键合的汞离子形成碘化汞(HgI₂)进而恢复g-C₃N₄量子点的荧光,从而建立一种高灵敏检测碘离子的荧光传感器。然而,该方法依然需要重金属汞离子的参与,限制了该方法的推广应用。通过硝酸氧化块体g-C₃N₄并结合水热法处理制备了一种水溶性好、荧光强度高的g-C₃N₄量子点。该量子点的荧光发射波长位于368 nm,且其荧光发射波长不随激发波长的改变而改变,表明该量子点的尺寸比较均一。笔者发现碘离子在220 nm处有一个较强的吸收峰,与该量子点的激发光谱(中心波长245 nm)具有较大的重叠,从而产生内滤效应引起该量子点的荧光发生猝灭。利用这一性质,构建了一种选择性检测碘离子的新型荧光传感器。在最优检测条件下,g-C₃N₄量子点的荧光猝灭强度(ΔF)与碘离子浓度(X,μmol·L-1)在10～400 μmol·L-1之间具有良好的线性关系,线性方程为ΔF=0.325 79X+6.039 05(R2=0.999 5),检出限为5.0 μmol·L-1。通过“混合即检测”并且不需要借助与重金属离子的配位作用就能够检测碘离子,因此该方法具有快速、环保以及操作简便等优点。     

Magnetic Fluorescent Composite Nanoparticles for the Fluoroimmunoassays of Newcastle Disease Virus and Avian Virus Arthritis Virus

A new detection format for multiplexed analysis based on the use of magnetic fluorescent composite nanoparticles was presented in this paper. Two different antigens, Newcastle disease virus (NDV) antigen and Avian virus arthritis virus (AVAV) antigen, were conjugated to two kinds of magnetic fluorescent composite nanoparticles of different luminescent colors, while red-emitting CdTe QDs were attached to the antibody of NDV and AVAV. Both CdTe QDs-labeled antibodies and magnetic fluorescent composite nanoparticles labeled antigens were used to form a typical immunoreaction system for the detection of NDV and AVAV. Also a typical mixed separation format was realized, which showed the outstanding magnetic properties of composite nanoparticles and the broad application in immunoseparation.     

基于氢化物发生技术的CdSe量子点水相制备新方法研究及其用于银的高灵敏传感分析

报道了一种新型的利用氢化物发生技术水相合成高质量硒化镉量子点(CdSe QDs)的方法。通过将硼氢化钾与亚硒酸混合产生H_2Se气体,并将其可调控地引入到含镉溶液中,从而制备出化学性质稳定、荧光性能良好的CdSe量子点。所合成的量子点被成功应用于环境水样及细胞样品中痕量银的分析,分析检出限为0.005μg·mL~(-1),相对标准偏差小于2.7%(n=7),分析结果令人满意。该方法具有操作简单、稳定性好、灵敏度高、绿色环保等优点。     

Highly sensitive colorimetric and fluorescent sensor for cyanazine based on the inner filter effect of gold nanoparticles

Cyanazine residue poses a great threat to human health and its derivatives would remain in soils, natural waters, and other environmental domains for a long time. Herein, a simple, rapid, and ultra-sensitive analytical method for the determination of cyanazine (CZ) based on inner filter effect (IFE) of Au nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (QDs) is first described in this study. With the presence of citrate-stabilized AuNPs, the fluorescence of GSH-capped CdTe QDs was remarkably quenched by AuNPs via IFE. The fluorescence of the AuNP–CdTe QD system was recovered upon addition of CZ. CZ can adsorb on to the surface of AuNPs due to its cyano group that has good affinity with gold, which could induce the aggregation of AuNPs accompanying color change from red to blue. Thus, the IFE of AuNPs on CdTe QDs was weakened, and the fluorescence intensity of CdTe QDs was recovered accordingly. A good linear correlation for detection of CZ was exhibited from 0.05 to 9 μM, and the detection limit reached 0.1568 μM, which was much lower than the safety limit required by the USA, the UK, and China. In order to probe into the selectivity of AuNPs towards CZ over other pesticides, various frequently used pesticides were mixed with AuNPs. AuNP composite solution shows good selectivity towards CZ among other pesticides. This method was successfully carried out for the assessment of CZ in real samples with satisfactory results, which revealed many advantages such as high sensitivity, low cost, and non-time-consuming compared with traditional methods.     

巯基乙胺稳定的CdTe量子点荧光猝灭法测定间苯二酚

以CdTe量子点作为荧光探针,基于荧光猝灭法对间苯二酚进行了定量检测,考察了缓冲溶液体系、量子点浓度、反应时间等多种因素的影响.实验结果表明,在pH 6.5的0.2mol/LNa<,2>HPO<,2>-NaH<,2>PO<,4>缓冲液中,反应时间为15min.间苯二酚浓度为1.6×10<'-6>-2.0×10<'-5>...     

CdTe QDs-based prostate-specific antigen probe for human prostate cancer cell imaging

l-glutathione (GSH) stabilized CdTe quantum dots (QDs) were directly prepared in aqueous solution. The as-prepared QDs were linked to prostate-specific antigen (PSA) for the direct labeling and linked to immunoglobulin G (IgG) for the indirect labeling of fixed prostate cancer cells. The results indicated that QD-based probes were ideal fluorescent markers with excellent spectral properties and photostability and much better than organic dyes making them very suitable in target detection. Meanwhile, the indirect labeling showed much better specificity than the direct labeling. Furthermore, the prepared CdTe QDs did not show detectable effect on cell growth after having cultured for three days, which suggested that the l-glutathione capped CdTe had scarcely cytotoxicity.     

Room-Temperature Phosphorescence Turn-on Detection of DNA Based on Riboflavin-Modulated Manganese Doped Zinc Sulfide Quantum Dots

A sensitive phosphorescent sensor based on riboflavin (RF)-modulated mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs) was developed and utilized as room-temperature phosphorescence (RTP) sensor for DNA detection. The RTP of the MPA-capped Mn-doped ZnS QDs was stored via photoinduced electron transfer by RF, and formed an electrochemically nonactive QDs/RF nanohybrids through electrostatic attraction. In the presence of DNA, RF could bind with DNA, which has a double helical structure, via electrostatic interaction and intercalation. RF can be removed from the surface of the QDs, thus releasing the RTP of the QDs. On the basis of this principle, an RTP sensor for DNA detection was developed. Under optimal conditions, the detection limit for DNA was 15 μg mL^?1, the relative standard deviation was 1.9 %, and the method recovery ranged from 97 % to 103 %. The proposed method was applied to biological fluids, in which satisfactory results were obtained.