Construction Of NIR-excited Nano-biosensors And Their Applications In Whole Blood Detection

Cancer has been a serious threat to human health for the low clinical cure rate. Since the current diagnostic results are almost medium or advanced stage, which misses the optimum treatment and increases the mortality. While, the cure rate of the early-stage cancer is up to 80%. Therefore, developing the early diagnostic techniques of cancer is of great significance for human health.Blood immunoassays are the main approaches for clinical disease diagnosis. Among various methods, Fluorescence-based immunoassay techniques have attracted extensive attention for their simplicity and higher sensitivity. However, the generally applied fluorescent probes are all UV-Vis excited, which can cause strong fluorescence background in whole blood samples and annihilate the fluorescent signal. Thus, it is hard to realize the direct whole blood immunoassay. Advance serum/plasma separation has to be conducted before immunoassay, which may result in the change of the structure or conformation of the biomolecules and fade of the specificity and sensitivity of the immunoassay. The inaccurate diagnosis will undoubtedly result in wrong treatment plan and extremely serious consequence.Recently, NIR-excited nanomaterials have gained significant attention since NIR excitation?lies in “biological window” 700 – 1300 nm? can effectively minimize the auto-fluorescence background from whole blood. It provides new hope for direct whole blood immunoassay.The starting point of the thesis is to solve the challenging problems of immunofluorescent approaches in whole blood immunoassays. Based on the researches about solid-substrate biosensors and fiber-optic biosensors, a series of innovative works are carried out, such as developing novel sensing modes, designing and preparing of NIR-excited nanoprobes and ET pairs, constructing novel solid-based biosensors and further applying for whole blood immunoassays etc. The major objective is to develop a novel technology for the early-diagnosis and early-warning of serious diseases. The main innovative results of the thesis are summarized as follows:1. Intracellular pH?pHi? plays a pivotal role in revealing the cellular activities?such as metabolism, proliferation, apoptosis etc.? and early warning of diseases?such as cancer and Alzheimer's?. Howerver, the accurate quantitative sensing of pHi is still a great challenge no matter in in molecular biology or medicine fields. In the thesis, NaYF4:Yb³⁺,Tm³⁺ UNCPs were used as ET donors and pH-sensitive FITC as acceptors. Under NIR-excitation without the undesired auto-fluorescence background and photo damage, two upconversion emission bands of Tm³⁺?475 nm as the response signal and 645 nm as the inert reference signal? were employed for self-ratiometric p H measurement. A highly-sensitive quantitative measurement of 3.56 per unit change in pH value in a broad linear range of pH from 3.0 to 7.0 was achieved.2. Fluorescence-based immunoassay methods, mainly divided into heterogeneous and homogeneous assays, have been generally applied in current clinics. However, for the commercial heterogeneous approaches, limited by the UV-Vis excitation of fluorescent probes, advance serum/plasma separation and complicated washing steps have to be conducted which will influence the accuracy of immunoassay. For the homogeneous approaches, the aggregation of fluorescent nanoprobes and the light scattering can influence the sensitivity and accuracy. To solve the above challenging problems, we developed a novel in situ immunoassay technology by applying UCNPs-based ET technology?UCNPs as donors, GNPs as acceptors? on a solid-substrate, and realized a one-step in situ immunoassay of whole blood samples. A high sensitivity of 3.4 nM in 20-fold diluted whole blood samples and an unprecedented wide linear range from 5 nM to 0.4 ?M were achieved.3. Highly sensitive immunoassay of whole blood samples is of great significance for early diagnosis and warning of serious diseases, such as cancer and AIDS. However, limited by the inherent property of fluorescent probes and the light scattering, absorption, and autofluorescence from whole blood, the whole blood immunoassay is still a challenging problem. In the thesis, we constructed a novel fiber-optic biosensor based on NIR-excited SERS nano-tags. The SERS signal from 4-MBA and Ag NPs was used as the response signal. A silica shell was adopted to protect SERS nano-tags and increased the stability in whole blood. The SERS nano-tag-based fiber-optic strategy successfully realized an in situ immunoassay of AFP?cancer biomarker? in unprocessed whole blood. Highly sensitive and reproducible response in the range of 50- 500 ng/mL was achieved.A series of exciting results have been achieved on biomedical immunoassay technology by combining NIR-excited nanoprobes with various sensing approaches, which solved the above challenging problems in immunofluorescent immunoassays and developed one-step in situ whole blood immunoassay technologies. The works laid the foundation for early clinical diagnosis of cancer. It will be the main direction to develop multi-channel and ultra-sensitive technologies for whole blood immunoassays in biomedical fields and further in vivo immunoassay technologies.