Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface‐enhanced Raman spectroscopy

Carcinoembryonic antigen (CEA) is a wide‐spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10^?12 M.     

Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 × 10⁵. The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10⁴ CFU (colony forming units) L⁻¹. The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method.     

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL⁻¹ in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL⁻¹ was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80–110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level).     

Preparation of a multi-hapten antigen and broad specificity polyclonal antibodies for a multiple pesticide immunoassay

A multi-determinant artificial antigen was prepared by haptens of four pesticides (chlorpyrifos, triazophos, carbofuran and parathion methyl) conjugating to the carrier protein BSA in turn. Male New Zealand white rabbits were immunized with this multi-determinant immunogen to produce the polyclonal antibodies (PAbs), which can recognize the four pesticides. The PAbs displayed high level for each relative hapten-OVA conjugate, with the favorable titers of 4.49 × 10⁴, 8.98 × 10⁴, 2.24 × 10⁴ and 1.86 × 10⁴, for CHBu-OVA, THHe-OVA, BFNB-OVA and MP5-OVA, respectively. Characterization studies of the PcAbs showed that it has high affinity and specificity to the four relative pesticides. An indirect competitive ELISA was developed for multi-residue determination. The I₅₀ value for the four pesticides was 0.290, 0.065, 0.582 and 2.824 μg mL⁻¹, with the detection limit (I₁₀) of 0.022, 0.005, 0.015 and 0.115 μg mL⁻¹ for carbofuran, triazophos, chlorpyrifos and parathion methyl, respectively. The linear rang was 0.016-2.000, 0.005-0.500, 0.010-2.000 and 0.063-5.000 μg mL⁻¹, respectively, for carbofuran, triazophos, chlorpyrifos and parathion methyl. Results indicated that, this study provided a new strategy to develop immunoassays through artificial antigen design for pesticides multi-residue determination.     

固态纳米孔对蛋白质易位的实验研究

蛋白质因其多样性和功能性,是生物体内一类非常重要的分子.通常蛋白质的表征需要借助荧光或者酶的标记.而纳米孔技术,得益于免标记、单分子检测等优势,为蛋白质的表征提供了新方向.我们使用固态纳米孔完成了单个蛋白质分子及蛋白质-蛋白质结合物的检测.可以发现,外部电压和电解质溶液的酸碱度会直接影响蛋白质分子表面带电量,从而加快或延迟其在孔内的易位时间.抗原、抗体本质上都是蛋白质,两者之间具有高度特异性.通过比较抗体溶液在添加特异性抗原前后的易位事件,实现了单个蛋白质分子和蛋白质-蛋白质结合物的区分.未来,纳米孔技术有望应用于多蛋白质分子的辨识、蛋白质分子相互作用机制等方面的研究.     

三唑磷的免疫分析技术研究

将合成的三唑磷半抗原采用活性酯法分别与牛血清白蛋白和卵清蛋白共价偶联制备突出三唑磷分子结构特征的人工抗原与包被抗原。以人工抗原免疫新西兰白兔获得抗血清,采用硫酸铵分步盐析和DEAE纤维素反相吸附法从抗血清中分离纯化对三唑磷具特异性亲合力的抗体,以辣根过氧化物酶采用混合酸酐法标记半抗原。在此基础上,首次成功建立了对三唑磷具高特异性的间接竞争、包被抗体直接竞争酶联免疫吸附分析（ELISA）技术。在优化条件下,三唑磷测定的线性浓度范围为0．001～1．0mg／L,检出限0．11μg/L,其他类似结构的常用有机磷酸酯类杀虫剂和苯唑醇不干扰三唑磷的测定。     

农药甲胺磷人工抗原的光谱鉴定研究

对农药甲胺磷人工抗原BSAM的光谱进行了鉴定 ,鉴定结果表明 :人工抗原BSAM的紫外光谱图与原载体和半抗原的紫外光谱相比均发生变化 ;人工抗原BSAM的红外光谱图上具有与甲胺磷分子相似的P—O—C和PO特征吸收峰 ;人工抗原BSAM具有与标准甲胺磷一致的3 1 PNMR化学位移峰 ,磷钼蓝分光光度法测定人工抗原BSAM中的磷浓度后计算出人工抗原BSAM的偶联率为 1 1。4种光谱方法测定结果综合表明甲胺磷人工抗原BSAM合成成功。     

白血病细胞酶联免疫酶催化银沉积于插指电极阵列电化学免疫分析新方法

建立了一种检测白血病细胞表面抗原的细胞酶联免疫电化学分析新方法. 该方法兼有细胞酶联免疫分析抗原、抗体结合的特异性和插指电极阵列酶催化银沉积电化学分析的灵敏性. 在聚苯乙烯微孔板中包被白血病细胞, 先后加入鼠抗人抗体及碱性磷酸酶(ALP)标记的马抗鼠抗体, ALP催化抗坏血酸磷酸酯(AAP)水解成抗坏血酸(AA), AA使银离子还原成银单质并沉积到插指电极阵列表面, 导致插指电极阵列上相邻两个梳齿导通. 通过对电导率的测定, 可实现对细胞表面抗原的高灵敏分析. 此分析方法灵敏度高(可检测出50个左右的HL-60细胞)、特异性好, 且可用于大量样品的分析, 为白血病等肿瘤疾病的早期诊断和免疫分型提供了新技术. 此外, 该方法也可用于细胞表面分子基因工程抗体活性的检测.     

Simple fabrication of a smart microarray of polystyrene microbeads for immunoassay

We describe a simple method to fabricate an array of polystyrene microbeads (PS μbeads) conjugated with an elastin-like polypeptide (ELP) on a glass surface using a removable polymer template (RPT). A thin layer of adhesive was spun-cast on glass and cured by UV radiation. Micropatterns of an RPT were then transferred onto the surface by microcontact printing. The adhesion of PS μbeads on the surface depended on the adhesion performance of the adhesive layer, which could be adjusted by irradiation time. An array of PS μbeads conjugated with ELP was used for a smart immunoassay of prostate-specific antigen (PSA), a cancer marker. By controlling the phase transition of ELP molecules, PSA molecules were selectively adhered or released from the bead surface. The selective and reversible binding of PSA molecules on the bead surface was characterized with fluorescence microscopy.     

Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.