Fluorescent/Magnetic Nanospheres:from Preparation To Biological Applications

Infectious diseases and cancers are great threats to human health, and developing rapid, sensitive and reliable detection method will contribute to the early diagnosis and timely treatment, which will better ensure the safety of human life, health and property. In recent years, with the rapid progress of nanoscience and nanotechnology, various nanomaterials have showed great superiority and potential in the biological detection and medical diagnosis. Among them, quantum dots (QDs), magnetic nanoparticles, and functional materials based on the two, which own unique and excellent properties compared with the traditional materials, have been playing a more and more important role in the biomedicine field. In the past decade, our group has always been devoting ourselves to the construction, research, and application of these nanomaterials, and has made a series of innovative and distinctive achievements. In particular, the fluorescent-magnetic-biotargeting multifunctional nanospheres have been successfully applied to the visual recognition and efficient isolation of cancer cells and proteins. In this thesis, we have fabricated nanobioprobes based on the fluorescent/magnetic nanospheres, and applied them to the detection of bacteria, virus, and tumor cells, which has been summarized as follows:(1) Nanobioprobes had been fabricated based on the fluorescent/magnetic nanospheres, and their basic properties were investigated in detail:①The number concentration of the nanospheres and the number of groups on the surface of each nanosphere were accurately calculated.②Fluorescent/magnetic functional nanospheres were constructed respectively by embedding and assembling nanoparticles (QDs or magnetic nanoparticles) with nanospheres as templates, and the comparison of the two methods was made in detail.③The stability of the functional nanospheres were monitored, including their fluorescence and magnetic properties, dispersibility, and usability.④Nanobioprobes had been fabricated based on the functional nanospheres, and the active biomolecule number on each nanobioprobe was calculated. Moreover, their bioactivity in different storage conditions was monitored over a long period of time. This part of the work lays a sound foundation for the application of the functional nanospheres.(2) With the magnetic nanospheres (MNs) and fluorescent nanospheres (FNs), a convenient, highly sensitive, and reliable strategy for Salmonella typhimurium detection was developed. The process could be accomplished with only one step, in which the immunomagnetic nanospheres (IMNs) and immunofluorescent nanospheres (IFNs) were used to capture and identify the target bacteria simultaneously. With this method, ca.10CFU/mL S. typhimurium could be detected specifically and reproducibly in1h. Moreover, this method was easy to manipulate and time-saving, and its successful application to the synthetic samples (milk, fetal bovine serum, and urine) had validated its potentiality in real, complex sample detection. The concept described herein can be expanded to other pathogen detection and can be modified for simultaneous detection of multiple pathogens with multiantibody-coated IMNs and multicolor IFNs.(3) Inspired by the one-step detection of S. typhimurium, multiplex DNA sequences, including complementary DNA (cDNA) sequences of HIV and HCV, DNA sequence of HBV, were simultaneously detected with MNs and QDs-encoded FNs. MNs-labeled capture DNAs and FNs-labeled probe DNAs were used to hybridize with the corresponding targets at the same time in one pot. By measuring fluorescence intensity of the sandwich complexes, quantitative detection of multiplex DNAs was achieved in1h. This method was very convenient, time-saving, and could reach a detection limit of-100pM. Moreover, the introduction of magnetic separation made the detection process easy to manipulate and increased its anti-interference ability. It had been successfully used to detect DNAs in serum samples, indicative of potential applications.(4) MNs were applied to rapid, efficient capture and sensitive detection of circulating tumor cells (CTCs). The MNs were nanosized with fast magnetic response, and they were very stable and easy to manipulate in a complex matrix. Modified with anti-epithelial-cell-adhesion-molecule (EpCAM) antibody, the obtained IMNs successfully captured extremely rare tumor cells in whole blood with efficiency of more than94%via only5min incubation. Moreover, the isolated cells remained viable at (90.5±1.2)%, and they could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification without disassociating IMNs. Furthermore, the IMNs were successfully applied to the isolation and detection of CTCs in cancer patient peripheral blood samples, which suggested they would be a promising tool for CTC enrichment and detection.