基于近红外上转换纳米探针的固相免疫检测研究

基于近红外上转换纳米探针的固相免疫检测研究 Studies of Solid Phase Immunoassay Based on Near Infrared Upconversion Nanoprobe 周进 ZHOU Jin 2 1 first-author 张美玲 ZHANG Mei-ling 2 1 张俐 ZHANG Li 3 李翠霞 LI Cui-xia 2 赵慧颖 ZHAO Hui-ying 3 张友林 ZHANG You-lin 2 夏安东 XIA An-dong 4 孔祥贵 KONG Xiang-gui 2 中国科学院大学 北京,100049 中国科学院大学 北京,100049 University of Chinese Academy of Sciences University of Chinese Academy of Sciences 发光学及应用国家重点实验室 中国科学院长春光学精密机械与物理研究所, 吉林 长春 130033 发光学及应用国家重点实验室 中国科学院长春光学精密机械与物理研究所, 吉林 长春 130033 State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China 吉林大学 长春,130012 吉林大学 长春,130012 Jilin Unieversity, Changchun 130012, China Jilin Unieversity, Changchun 130012, China 中国科学院 化学研究所, 北京 100190 中国科学院 化学研究所, 北京 100190 Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China 以氯化物为原料通过溶剂热法合成了尺寸均匀的NaYF 4 ：Yb 3+ ，Tm 3+ 上转换纳米粒子（UCNPs），利用聚丙烯酸（PAA）取代UCNPs表面的油酸配体，引入羧基和人IgG的氨基共价结合（H端），再用蛋白G（protein G）作"桥"将兔抗羊IgG（r-a-g IgG）修饰在硅片表面（R端），以消除由于硅片"刚性"表面与r-a-g IgG分子直接偶联而导致其结构或构象的变化，从而实现硅片表面与r-a-g IgG的"柔性"生物偶联。利用抗原抗体的特异性免疫反应，将H端和R端通过羊抗人IgG（g-a-h IgG）结合，建立了简单高效和快速灵敏检测溶液中g-a-h IgG的新型免疫分析方法。实验结果表明：g-a-h IgG在5~400 nmol/L浓度范围内与上转换荧光强度具有良好的线性关系，该传感器检测限为1.82 nmol/L，并表现出优秀的检测特异性。本项研究为临床各种重大疾病的免疫分析诊断提供了一种宽线性范围和高特异性的新型免疫方法和平台。 Uniformed NaYF 4 :Yb 3+ ,Tm 3+ nanoparticles were synthesized through solvent thermal method in chloride. PAA-based ligand replace strategies were carried out to introduce free carboxylic acid groups which allowed further conjugation with NH 2 -containing human IgG. The silicon wafer was modified with rabbit anti-goat IgG by using protein G as "bridge", which contributed to eliminate structural or conformational changes of IgG. Owing to the specific antibody recognition to the antigenic, a simple, rapid, efficient and sensitive immunoassay method was developed. The experimental results demonstrate that there is a very good linear correlation between the upconversion luminescence intensity and the amount of goat anti-human IgG in the range of 5~400 nmol/L. The detection limit is as low as 1.82 nmol/L. Our developed detection system shows excellent detection specificity and sensitivity, which provides a new detection method and platform with wide linear range and high sensitivity for diagnosis of major clinical diseases in the future. 上转换 夹心免疫 生物传感器 特异性 upconversion sandwich immunoassay biosensor specificity CAMARGO M E, MOURA M E, LESER P G. Toxoplasmosis serology:an efficient hemagglutination procedure to detect IgG and IgM antibodies[J]. Revista Do Instituto De Medicina Tropical De So Paulo, 1989, 31(4):279-285. LI C, JING Z, LI Q, et al.. One-step in situ, solid-substrate-based whole blood immunoassay based on FRET between upconversion and gold nanoparticles[J]. Biosens. Bioelectron., 2017, 92:335-341. WU J, FU Z, YAN F, et al.. Biomedical and clinical applications of immunoassays and immunosensors for tumor markers[J]. TrAC Trends Anal. Chem., 2007, 26(7):679-688. CHERMONT Q L M D, SEGUIN J, TREJEAN, et al.. Nanoprobes with near-infrared persistent luminescence for in vivo imaging[J]. Proc. Nation. Acad. Sci. Unit. States Am., 2007, 104(22):9266-9271. SCHENKELAYLAND K, RIEMANN I, DAMOUR O, et al.. Two-photon microscopes and in vivo multiphoton tomographs-powerful diagnostic tools for tissue engineering and drug delivery[J]. Adv. Drug Deliv. Rev., 2006, 58(7):878. 何奇, 樊君, 胡晓云, 等. NaYF4:Er3+的水热合成及其紫外上转换发光性能[J]. 发光学报, 2012, 33(2):122-127. HE Q, FAN J, HU X Y, et al.. Hydrothermal synthesis of NaYF4:Er3+ and its ultraviolet up-conversion light emitting property[J]. Chin. J. Lumin., 2012, 33(2):122-127. (in Chinese). 于放达, 陈欢, 赵丹, 等. 利用核壳结构实现纳米颗粒的多色上转换发光[J]. 发光学报, 2014, 35(2):165-171. YU F D, CHEN H, ZHAO D, et al.. Multicolor upconversion luminescence from core-shell structured nanoparticles[J].Chin. J. Lumin., 2014, 35(2):165-171. (in Chinese). 张喜生, 晏春愉, 郑海荣. 光谱学方法研究Tm3+离子的上转换发光影响因素[J]. 光子学报, 2010, 39(8):1515-1518. ZHANG X S, YAN C Y, ZHENG H R. Spectroscopic study of the upconversion effect of Tm3+ ions[J]. Acta Photon. Sinica, 2010, 39(8):1515-1518. (in Chinese). 何春凤, 赵丹, 秦冠仕, 等. Tm3+/Yb3+共掺杂ZBLAN玻璃的多光子紫外上转换发光[J]. 光子学报, 2011, 40(1):61-63. HE C F, ZHAO D, QIN G S, et al.. Multi-photon UV upconversion luminescence of Yb3+ and Tm3+ co-doped ZBLAN glass[J]. Acta Photon. Sinica, 2011, 40(1):61-63. (in Chinese). 李慧, 杨魁胜, 祁宁, 等. Yb3+/Er3+掺杂氟氧化物微晶玻璃的制备与发光性能[J]. 中国光学, 2011, 4(6):672-677. LI H, YANG K S, QI N, et al.. Preparation and luminescence properties of Yb3+/Er3+-codoped oxyfluoride glass ceramics[J]. Chin. Opt., 2011, 4(6):672-677. (in Chinese). CHATTERJEE D K, RUFAIHAH A J, ZHANG Y. Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals[J]. Biomaterials, 2008, 29(7):937-943. CORSTJENS P, ZUIDERWIJK M, BRINK A, et al.. Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences:a rapid, sensitive DNA test to identify human papillomavirus type 16 infection[J]. Clinic. Chem., 2001, 47(10):1885-1893. QIAN H S, GUO H C, HO P C L, et al.. Mesoporous-silica-coated up-conversion fluorescent nanoparticles for photodynamic therapy[J]. Small, 2009, 5(20):2285-2290. SHE W, LUO K, ZHANG C, et al.. The potential of self-assembled, pH-responsive nanoparticles of mPEGylated peptide dendron-doxorubicin conjugates for cancer therapy[J]. Biomaterials, 2013, 34(5):1613-1623. KIM S, KIM C A M, PYO H B, et al.. Synthesis of anionic sulfate functionalized polystyrene latex and protein adsorption for application as a biosensor[J]. J. Nanosci. Nanotechnol., 2009, 9(12):7171-7176. 10.3788/fgxb20183908.1059 O482.31 国家自然科学基金（61575194，11474278，11504371，11674316，11604331，11374297）;中国科学院长春光学精密机械与物理研究所发光学及应用国家重点实验室开放课题（SKLA-2016-03）资助项目 Supported by National Natural Science Foundation of China(61575194,11474278,11504371,11674316,11604331,11374297);Open Projects of State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences(SKLA-2016-03) 2018-01-13 2018-02-24 2017-12-04 2018-08-05 2020-08-11 8 39