Wu Nengying, Wei Yuxi, Pan Lanlan, Yang Xiaolin, Qi Honglan, Gao Qiang, Zhang Chengxiao
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062, People's Republic of China.
Mikrochim Acta. 2020 Jun 9;187(7):377. doi: 10.1007/s00604-020-04349-w.
Lateral flow immunostrips were newly designed and a sensitive and rapid fluorometric method for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a model target of small biomarker molecules was developed. The upconversion nanoparticles (UCNPs, NaYF4:Yb/Er core, and polyacrylic acid (PAA)-modified shell, size ~ 39 nm, excitation wavelength = 980 nm; emission wavelength = 540 nm) were employed as fluorescence signal material. The 8-OHdG antibody (Ab) was taken as the recognition probe while UCNP-labeled Ab was taken as the signal probe. Bovine serum albumin (BSA) was designed as carrier protein for 8-OHdG to form 8-OHdG-BSA conjugate as the capture probe. The lateral flow immunostrips were prepared by laminating a sample pad (glass fiber membrane), a test pad (nitrocellulose membrane), and adsorption pad (filter paper) on PVP backing. The capture probe was immobilized on the test zone while an IgG antibody taken as the control probe was immobilized on the control zone. When the signal probe and the sample were in sequence loaded on the sample pad, 8-OHdG analyte bound with the signal probe, and then the excess of the signal probe move along the strip and is collected by the capture probe on the test zone while the remnant signal probe is collected by the control probe on the control zone. The signal probe and capture probe were synthesized and characterized. The fluorescence intensity on the test zone was inversely proportional to the concentration of 8-OHdG for the quantitative determination while the fluorescence emission on the control zone was observed to validate the assay. The developed method showed a wide linear range from 0.10 to 10 nM, a quite low detection limit of 0.05 nM, small sample volume requirement (100 μL), short assay time (15 min), and good method reproducibility (RSD = 4.4%, nine immunostrips). Graphical abstract Schematic illustration of the configuration and measurement principle of lateral flow fluorescence immunostrip for 8-OHdG: (a) configuration; (b) preparation: load of capture probe (BSA-8-OHdG, 2 μL) on test zone; load of control probe (IgG Ab, 2 μL) on control zone; load of signal probe (UCNP-Ab, 16 μL) on sample pad; (c) measurement: load of sample (8-OHdG, 100 μL) on sample pad, collection, and measurement.
新设计了侧向流动免疫试纸条，并开发了一种灵敏且快速的荧光法来测定作为小生物标志物分子模型靶标的8-羟基-2'-脱氧鸟苷（8-OHdG）。采用上转换纳米颗粒（UCNPs，NaYF4:Yb/Er核，聚丙烯酸（PAA）修饰壳，尺寸约39nm，激发波长 = 980nm；发射波长 = 540nm）作为荧光信号材料。将8-OHdG抗体（Ab）用作识别探针，而将UCNP标记的Ab用作信号探针。设计牛血清白蛋白（BSA）作为8-OHdG的载体蛋白，形成8-OHdG-BSA缀合物作为捕获探针。通过将样品垫（玻璃纤维膜）、测试垫（硝酸纤维素膜）和吸附垫（滤纸）层压在PVP背衬上来制备侧向流动免疫试纸条。将捕获探针固定在测试区，同时将用作对照探针的IgG抗体固定在对照区。当信号探针和样品依次加载到样品垫上时，8-OHdG分析物与信号探针结合，然后过量的信号探针沿试纸条移动并被测试区的捕获探针收集，而剩余的信号探针被对照区的对照探针收集。对信号探针和捕获探针进行了合成和表征。测试区的荧光强度与8-OHdG浓度成反比用于定量测定，同时观察对照区的荧光发射以验证测定。所开发的方法显示出宽线性范围为0.10至10 nM，相当低的检测限为0.05 nM，小样品体积要求（100μL），短测定时间（15分钟）以及良好的方法重现性（RSD = 4.4%，九条免疫试纸条）。图形摘要 8-OHdG侧向流动荧光免疫试纸条的结构和测量原理示意图：（a）结构；（b）制备：在测试区加载捕获探针（BSA-8-OHdG，2μL）；在对照区加载对照探针（IgG Ab，2μL）；在样品垫加载信号探针（UCNP-Ab，16μL）；（c）测量：在样品垫加载样品（8-OHdG，100μL），收集并测量。
