Real-Time Dark-Field Light Scattering Image Of Gold Nanoparticles-Lablled Respiratory Syncytial Virus Infecting Cell

Viruses, especially the infectious virus, are a great threat to human health. So understanding the infection and pathogenic mechanism of virus is crucial for the prevention and treatment for viral disease. Now, single virus tracking is an imaging method which is focused on tracking the critical steps of virus infection process in real-time to explore the complicated dynamic interaction between single virus particle and host cell, and finally reveal the mechanism of virus infection. In recent years, in order to successfully trace viruses, fluorescent protein molecules or fluorescent dyes are used to label viruses and cells. However, the usefulness of these tags over comparative long periods is limited owing to their photobleaching properties and low emission efficiency. Quantunm dots (QDs), although have been successfully applied in imaging owing to their bright emissions and greater photostability than organic dyes, are easily aggregated and potential toxic. Therefore, it is worth to find out alternative method to avoid the shortage of the tags. Herein, gold nanoparticles (AuNPs) with good biocompatibilities are used to label respiratory syncytial virus (RSV) to real-time visualize and track the dynamic processes to understand the mechanism of virus invasion. Extensional research contents include the following three aspects:1. Sensitive quantification of RSV by direct dark-field microscopic scattering imaging of the surface of targeted host cells. Virus are targeted and labelled by anti-RSV antibody modified AuNPs, and the host cells are used to test viruses. The viruses on the cell membrane surface will be quantitative detected through the statistics for the percentage of the AuNPs scattering signal. Studies have found that anti-RSV antibody modified AuNPs, which are added into cells, attach onto the cell membrane surface after a certain amount of viruses is added and the extra viruses are moved. And with the increased amount of viruses, the scattering signal of AuNPs on the cell membrane surface gradually enhances under the dark-field imaging. The results of imaging are analyzed, and we easily find that with the increased amount of viruses, the percentage of the scattering signal of AuNPs on the cell membrane surface gradually enhances. In this experiment, the visualization quantity for virus is accomplished owing to the cells as the substrate and AuNPs scattering signal as detecting signal for testing virus. Thus the method of the visualization quantity for virus is builded, which appears same superiorities, such as high selectivity, high sensitivity and simple operation.2. Real-time tracking of gold nanoparticles-bioconjugated RSV infecting HEp-2 cells. The AuNPs modified with streptavidin (SA) are used to label virus. Under the dark-field microscope, the invasion dynamics of virus will be observed for long time through the scattering signal of virus labelled with AuNPs. SA is a protein, AuNPs modified with SA are easy to obtain owing to the formation of Au-S bond between AuNPs and SA. Viral membrane protein are labelled with biotin to get biotin modified virus. A SA molecular bind with four molecules of biotin through strong affinity, which induces SA modified AuNPs to easilylabel biotin-modified virus. Due to the unique localized surface plasmon resonance (LSPR) properties of AuNPs, the viral infection is monitored and observed over long periods by observing the scattering signal of virus labelled with AuNPs. The results of research find that viral infection is rapid and short about 1 min, and it may be endocytosis process. Compared with other methods of fluorescence modified virus, the good biocompatibility and stability of AuNPs do not exist the problems of low emission efficiency and photobleaching, and the marking strategy is simple, gentle and low effects on viral infection.3. Fluorescence and dark-field dual-mode imaging of nucleic acid of RSV. In this paper, the optical probe of hairpin DNA functionalized AuNPs is builded. AuNPs are used as energy receptors and luminous element, the hairpin DNA is element for detecting viral nucleic acid RNA, which realizes fluorescence and dark-field dual-mode imaging of viral nucleic acid RNA after viruses infect and get into cells. Hairpin DNA is coupled to the gold colloid surface via a 5’thiol, thereby quenching fluorescence emission of the 3’TAMRA and weakening light sacettering signal of AuNPs (dark green). The stem regions maintain the hairpin conformation through complementary hydrogen bonding. Hybridization between the loop region and the viral nucleic acid RNA sequence results in opening of the hairpin DNA, fluorescence emission and light scattering singal enhancement of AuNPs (bright green). It realizes fluorescence and dark-field of dual-mode imaging for intracellular nucleic acid RNA of viruses. The results of research also find that the large number of replication of nucleic acid RNA of viruses is accomplished around the cell membrane after the viruses invade into the cells. The preparation of hairpin DNA functionalized AuNPs is simple, and the combination of the fluorescence imaging and the dark-field one is successful. And it can be widely used in the field of biomedical imaging.In conclusion, AuNPs are used to easily and quickly label virus in this paper, with the technology of dark-field light scattering image, viral infection process can be tracted for a long time through the scattering signal of AuNPs. In this paper, virus is labelled without effects on viral infection. And AuNPs are chosed as labeling material, avoiding that the fluorescent dyes and fluorescent proteins fluorescence signal is weak and easily bleached and QDs is low biological compatibility and stability. This article also realizes some imaging research which includes detecting and quantitating viruses on the cell membrane surface, viral infection process and replication process of viral nucleic acid. This paper makes imaging analysis for viral infection process from outer to inner, which provides the opportunity to further understand the mechanism of viral infection. In addition, this article uses the strategy of imaging analysis through AuNPs scattering signal, which is expected to expand other good biocompatibility nanometer materials used for biological markers. And these good biocompatibility nanometer materials will be used widely in the field of biomedical imaging.