Novel Methods For Respiratory Syncytial Virus Nano-immunoassay

Respiratory syncytial virus (RSV), a negative sense single-stranded RNA virus in the Paramyxoviridae family, has been long recognised as the primary cause of serious viral lower respiratory tract illness in infants and young children worldwide. In the USA alone, from85000to144000infants with RSV infection are hospitalized for bronchiolitis or pneumonia annually. The Centers for Disease Control and Prevention consider RSV to be the "most common cause of bronchiolitis and pneumonia in children under1year of age in the United States.". In addition, RSV can cause severe illness in the elderly people and those adults with cardiopulmonary diseases or immunocompromised. Globally, disease burden associated RSV infection is estimated at64million cases and160000deaths every year. Since RSV is extremely contagious and since no safe and effective vaccine is available at present, there is a critical need for the development of novel methods to diagnose RSV infection.So far, the diagnosis of RSV infection has been generally made by virus isolation, detection of the viral antigen or viral specific RNA in respiratory secretions. Isolation of RSV using cell culture was considered as "gold standard" for confirmation of RSV infection in a long period of time, because it is effective and often complementary. However, this procedure is time-consuming (3-6days), laborious and may require experienced staff to perform. The need for rapid, sensitive, specific procedures for the detection of RSV has led to the development of methods which do not rely on cell culture techniques, such as reverse transcription and polymerase chain reaction (RT-PCR) for the detection of viral specific nucleic acid sequences. However, most PCR methods require some costly equipment and are not suitable for use outside the laboratory. In this contribution, nanomaterials are introduced to the immunoassay for RSV. The main contents in this thesis are as follows:1. The application of metal nanoparticles in RSV immunoassay, including the following three parts:(1) Sensitive detection of RSV using surface-enhanced Raman scattering of enzyme reaction product. By using the reaction product as a Raman probe, we have developed a simple and sensitive method for an immunoassay based on surface-enhanced Raman scattering (SERS). In the proposed assay, capture antibody was immobilized on a solid substrate reacting with antigen, then binding with another antibody labeled with peroxidase. If this complex was reacted with3,3’,5,5’-tetramethylbenzidine (TMB) and hydrogen peroxide, a radical cation was generated. This compound was adsorbed on a silver colloid easily. The concentration of virus via the intensity of a SERS signal of reaction product could be determined. Furthermore, TMB is one of the most widely used substrate for HRP-based commercial ELISA test kits, indicating that this thechnique is applicaple to a large number of other targets.(2) Gold nanoparticles-based enhanced ELISA for RSV. In this work, we described a highly sensitive enzyme-linked immunosorbent assay (ELISA) assay utilizing gold nanoparticles (AuNPs) for the detection of RSV. Gold nanoparticles were used as carriers of the signalling antibody anti-RSV-HRP (horseradish peroxidase) in order to achieve an amplification of the signal. Compared to conventional ELISA procedures, this assay resulted in higher sensitivity and shorter assay time in the range between0.5and50pg/mL. The application of AuNPs to the enhanced ELISA showed acceptable reproducibility, stability, and could be readily extended to other ELISA systems.(3) Combined gold nanoparticle layer and tyramide signal amplification for ultrasensitive detection of RSV. Tyramide signal amplification is an enzyme-mediated detection method. When H2O2and biotinyl-tyramide (a biotin-phenolic compound) were added to an assay system containing immobilized HRP or ALP, the enzyme could catalyze the activation of the phenolic group resulting in covalent binding to electron rich moieties on the solid phase. Subsequent reaction with steptavidin-HRP caused the binding of additional HRP to the now biotinylated solid phase, resulting in signal amplification. Additionally, a stable gold nanoparticle layer (GNPL)-modified plate was proven to be more efficient in binding proteins while mataining their activity. GNPL-based ELISA demonstrated that significantly amplified the ELISA signal. In this work, a dual signal amplification strategy was developed by combining a tyramide signal amplification system with the GNPL for highly sensitive detection of RSV. By measuring the absorbance at450nm on a microplate reader, we observed that the intensity increased gradually as the amount of RSV added was increased. The calibration curve suggested that the linear detection range was from0.05to30pg/mL. Furthermore, RSV was spiked in the culture medium,2%bovine and human samples, respectively. The recovery values ranged from90.6%to98.1%, demonstrating the potential clinical applicability of the immunoassay.2. The application of gold/graphene nanocomposites in RSV immunoassay, including the following two parts:(1) Gold nanoparticles/graphene composites with enhanced peroxidase-like activity for colorimetric immunoassay of RSV. Nanomaterial-based enzyme mimics have attracted great interest because they are more stable under harsh conditions, cost-effective to produce and do not suffer from denaturation compared to natural enzymes.However, their applications in biological systems are relatively rare because their catalytic activity decreases after adsorption of biomolecules such as serum albumin, DNA. In this study, we prepared the AuNPs/GO composites through a facile and efficient strategy with the use of tannic acid (TA) as a reducing and stabilizing agent. And we have developed a novel colorimetric immunoassay for virus detection based on mercury-stimulated peroxdase-like activity of gold nanoparticles/graphene composites. Semi-quantization of RSV could be done by the naked eye, which is identical to the spectrophotometric detection of RSV in the range between0.1and10pg/mL.(2) AuNPs/graphene hybrids-based ultrasensitive plasmonic immunoassay for pathogen detection with the naked eye. In this report, we constructed an ultrasensitive sandwich plasmonic immunoassay by combing AuNPs/graphen hybrids with the AuNPs-catalyzed enlargement without using enzyme to avoid the false negative result from enzyme denature in the assay. The AuNPs/graphene hybrid was prepared by a facile one-step hydrothermal reduction of a mixture of HAuCl4and graphene oxide (GO) in aqueous solution. The very high surface area of graphene contributed to the growth of a large amount of AuNPs. Subsequently, antibody (Ab) against target pathogen was biogonjugated to the AuNPs/graphene through simple adsorption. As-prepared Ab-AuNPs/graphene could specifically recognized pathogen which was captured by the antibody immobilized on the polystyrene (PS) substrate in a96-well plate, forming sandwich complex. To increase the optical signal, enlargement of AuNPs was carried out using the AuNPs-catalyzed reduction of Au3+by NH2OH based on the seeding method. Combining graphene and gold enhancement, extremely low detection limit would be achieved in this plasmonic immunoassay. Meanwhile, the colour change could be easily monitored by the naked eye, making it perfectly suited for detecting pathogens in resource-poor settings.In summary, we have developed a serial of RSV detection methods based on the unique property of metal nanoparticles (gold and silver) or gold/graphene nanocomposites. Overall, this thesis does not only broaden the applications of nanomaterials in immunoassay chemistry, but also provide the scientific basis for RSV diagnosis in clinical applications.