Establishment Of Testing Platform For Tumor Marker Detection With Quantum Dot-based Immunochromatography Test Strip

Immunochromatography test strip(ICTS) as a new diagnostic method had been developed rapidly in recent years for their convenient and fast testing. However, the fundamental limitation of quantitative and sensitive analysis severely hampers the application of ICTS as a reliable medical testing in detection of cancer. In order to combing the advantage of sensitivity detection based on the fluorescence and rapid detection based on the ICTS, quantum dots(QDs) a new fluorescence labeling was introduced in the ICTS as the signal-generating particle. This combination enables rapid, highly sensitive, specific and low-cost detection for quantitative point-of-care diagnostics, which is of great significance and application value for major diseases especially in cancer.Firstly, the high quality CdSe core nanocrystals were sythesised by organometallic methods, then multi-shell structure QDs were synthesised by SILAR method with high fluorescence efficiency and stability, because the nanocrystals were well electronically passivated by the radial increased of the respective valence-and conduction-band offsets. Secondly, octadecylamine(ODA) was grafted to poly(tertbutyl acrylate-co-ethyl acrylate-co- methacrylic acid)(ABC triblock copolymer) as the hydrophobic side chains. This modified ABC triblock copolymer was used to encapsulating QDs and form the high performance QD nanobeads. Thirdly, the QDs based IC TS were established by intergrating QD nanobeads with IC TS. At the same time, corresponding test strip reader was developed. Finally, the QDs based ICTS were applied to detect the tumor markers. Then the effective reaction time, sensitivity, specificity and capability of quantitative detection of these QD nanobeads based ICTS were investigated to comprehensively display its performance.The HRTEM images of CdSe demonstrated that the high crystallinity, uniform particle size distribution and regular shape. We found that the nucleation and and crystal growth were influenced by the ratio of Cd and Se precursors, the reaction temperature and the reaction time. Increase the amount of the Se precursors would conduce to form CdSe core nanocrystals with uniform size, narrow FWHM and high quantum efficiency. Meanwhile, the crystal growth could also be controlled by the reaction temperature or time. Then the inorganic shell materials were used to synthesis multi-shell structure QDs by SILAR method. The HRTEM images, UV-vis spectrum, PL spectroscopy and XRD spectrum indicate d that the the multi-shell structure QDs were obtained and the shells contributed to increase the quantum yield of and stability of the QDs. The QD nanobeads were fabricated by encapsulating QDs within ABC-g-ODA and their structure and properties were fully evaluated by TEM analysis, DLS and surface zeta potentials. All the results were demonstrated that the QD nanobeads were monodisperse with good sphericity and distributions. There were lots of carboxyl groups on the surface of the QD nanobeads, which was befinicial to the functionalization of the QD nanobeads. Furthrmore, compared with water-soluble quantum dots obtained by SiO2, MAA and MUA encapsulation, the QD nanobeads revealed higher stability. The particle size, zeta potential and PL intensity of QD nanobeads remained the same in PBS buffer at least 20 days. More importantly, the fluorescence intensity of QD nanobeads could remand at least 30% in the strong acid solution. These results manifested the outstanding stability of the QD nanobeads we prepared.The QD nanobeads were then combined with ICTS to estabilish QDs based ICTS for a sensitive and quantitative analysis. The materials of the conjugated pad and the sample pad were optimazied as well as the kind and work concentrations of the blocking reagent such as bovine serum albumin(BSA), hydrophilic polymer synthesis, Tween-20 and the surgars. After that, quantitative detection of cancer protein biomarkers(alpha- fetoprotein(AFP), prostate specific antigen(PSA) and human chorionic gonadotropin(HCG)) were bulid on the foundation of the QD nanobeads based ICTS. Under optimal conditions, the concentrations of tumor markers could be determined within 10 ~ 20 min with high sensitivity and specificity. At the same time, the possibility of clinical utility was demonstrated by the measurements in PSA positive patient specimens. Then the QD-based fluorescence encoding ICTS was established to detection two tumor markers for one time, which could identify the type and concentration of the tumor markers according to the fluorescence colors and intensity. The results revealed that there was no cross- link between the two markers.Our data suggests that the QD nanobeads based IC TS platform is a rapid and low-cost, yet still highly sensitive and specific platform for quantitative point-of-care diagnostics, which holds promise to become part of routine medical testing for cancer detection.