Novel Bioassays Based On Functionalized Nanoparticles And Bionic Materials

Use of immunoassay techniques for high sensitive and selective detection of target analytes in complex samples has been one of attractive and hot topics in modern biochemistry and biomedical studies. In particular, the measurement of trace biomarkers is of particular importance for the early diagnosis, therapy, prognosis and pathogeny of corresponding diseases. Up to now, many immunoassay methods have been developed. However, these conventional methods are generally complicated, time-consuming, low-throughout, and necessary for expensive instrumentation, large volumes of reagents and samples and technical skills. Accordingly, exploring some novel immunoassays that are simple, rapid, cost-effective, high-throughout and easy to be clinically popularized and implemented for field analysis is still of considerable interest. In additional, the electrochemical enzyme-based biosensors hold great promise in various bioapplications due to their advantages including their simple-design, high-sensitivity and low-cost. However, the method for enzyme immobilization on the surface of transducers still represents the"bottle-neck"to restrict their rapid development and practical applications. Focused on these mentioned topics in the current immunoassays and the fabrication of enzyme immobilization platforms, several new single-/multi-analyte immunoassays and several new strategies for immobilizing biomolecules to construct electrochemical enzyme biosensors have been developed in the present dissertation and described as follows:(1) Seeking for an effective route for reducing the applied volumes of costly reagents and rare samples would help to reduce the analysis cost of the (magnetic) suspension immunoassays with many advantages and in turn allow them to find more application. In Chapter 2, lotus-leaf-inspired bionic superhydrophobic surface has been creatively introduced into the design of analytical platforms for (magnetic) suspension immunoassays. Firstly, a new phase separation methodology has been initially proposed for creating super-hydrophobic coatings onto the surfaces of various material substrates simply by using a common polymer, i.e., polycarbonate, and its solvent and non-solvents suitably selected. The developed protocol can allow the super-hydrophobic coatings to be fabricated at ambient room temperature within 21 min, showing the water contact angle up to 160°and a sliding angle of less than 5°. They might well retain super-hydrophobicity in whole pH range and have long-term mechanic stability. Subsequently, the polycarbonate coatings were modified onto the inner walls of a set of common detection tubes, which were further assembled together to achieve a multi-well superhydrophobic surface-based suspension immunoassay platform (SSBSIP). Taking human IgG as the model analyte, the analytical advantages of as-prepared SSBSIP have been demonstrated by performing a novel magnetic suspension colorimetric immunoassay based on the gold-enhanced gold nanoparticle label amplification. The experimental results show that under slightly shaking, the SSBSIP can allow for the maximum uniform mixing of reaction suspension (e.g., immunomagnetic probe and sample), resulting in a"rotative flowing reactive system"with high antigen-antibody binding efficiency and rapid immunoreaction kinetic. The SSBSIP-based new technique just required low volumes of reagents and sample (10μL for each immunoreaction step) to achieve rapid (20 min for each immunoreaction step), highly sensitive colorimetric detection of human IgG. The limit of detection for IgG is about 25 ng/mL. Moreover, SSBSIP is self-cleaning, and thus can be reusable.(2) Schistosomiasis, an ancient endemic infectious disease, is a serious threat to the general health of the people in infected area, and has become a global serious public health problem that needs to be urgently solved. In Chapter 3, the newly reported SSBSIP was combined with the copper-enhanced gold nanopartilce label application strategy to develop a novel magnetic electrochemical immunoassay for quantifying Schistosoma japonicum antibodies (SjAb) in infected rabbit serum. It was found that the new method favored the specific determination of SjAb in the dynamic concentration range of 2 ng/mL ~ 15μg/mL, with the limit of detection of about 1.3 ng/mL. It could provide far better performance over the recently reported SjAb detection approaches in terms of volumes applied of reagents and samples, analytical time in single immunoreaction step, and limit of detection, etc. When it was used for the analysis of real samples, the proposed immunoassay system might rapidly quantify the infection degrees of infected rabbit serum samples and show comparable analytical ability to that of the ELISA method, indicating a new powerful tool for clinical diagnoses of Schistosomiasis.(3) Multi-analyte immunoassays have attracted increasing attention in recent years, mainly because of their outstanding advantages including high-throughout, small volumes of reagents and samples required, low-cost and so on. In Chapter 4, a renewable, site-selective immobilization platform of microelectrode array for multiplexed immunoassays has been initially developed using pencil graphite particles coated with gold layers as microelectrodes. Based on the gold nanoparticle-based signal amplification, the developed microelectrode array sensor can allow for the detection of human IgG model analyte in the linear range of 0.05 ~ 100 ng/mL, with the limit of detection of about 36 pg/mL. Its sensitivity is over 103 larger than that of the conventional, bulk gold electrode. The rapid regeneration of the used microelectrode array platform can additionally be realized by a simple electrochemical treatment. The high selectivity of four individually addressable microelectrode array platforms for multiple antigens in a single sample has been further demonstrated in the multiplexed immunoassay experiments.(4) The nitrocellulose membrane-based immunogold staining methods present a series of advantages and are highly suited for the screening of infectious diseases in low-income area and developing countries and point-of-care testing and filed-based analysis of target bio-species such as proteins. However, most of the existing methods may still suffer from some problems associated with very low use efficiency of nitrocellulose membrane, un-suitability for multi-analyte detection and serious"coffee-ring effect", etc. In Chapter 5, an aqueous solution diffusion-localized platform (ASDLP) has been initially fabricated by assembling nitrocellulose membrane array onto the superhydrophobic polycarbonate surface with the use of adhesive tape. This new platform was then explored for the development of multiplexed immunogold staining technique for the simultaneous colorimetric detection of multiple proteins in a single sample. Due to the excellent"water (aqueous solution)-repellent"property of the polycarbonate supporting coating, the diffusion of reagents and samples could be localized on the superhydrophilic nitrocellulose membrane. Thus, the ASDLP-based immunogold staining method proposed are endowed with various advantages, such as 100% use efficiency of nitrocellulose membrane, the ability to be multiplexed for the simultaneous colorimetric detection of multi-analyte, the ability to minimize the"coffee-ring effect", low background colour signal on the non-testing area, and the good reusability of ASDLP, etc.(5) The effective immobilization of enzyme onto the electrodes is considered as one of the key steps, since the performances of biosensors mainly depend on the amount and bioactivity of enzyme immobilized. In chapter 6, a novel immobilization platform has been developed for fabricating electrochemical enzyme biosensors by using flower-like zinc oxide particles and gold nanoparticles. Zinc oxide particles with micro-nano hierarchical structure were synthesized and casted on electrode with chitosan to provide a large surface area for embeeding gold nanoparticle-labelled with horseradish peroxidase enzyme. The enzyme biosensor obtained was then tested for the detection of H2O2. Experimental results showed that the horseradish peroxidase could be immobilized into the composite matrix with well-retained activity and large loading amount. Moreover, the gold nanoparticles would allow for rapid and direct electron transferring between the active sites of the enzyme immobilized and the electrode surface. The direct electric detection of H2O2 could thus be achieved. The developed enzyme biosensor determined H2O2 in a concentration range of 1.5μM ~ 45 mM, with the limit of detection of about 0.7μM.(6) The polymer-embedding-based enzyme immobilization methods can offer many advantages. But most of the widely used polymer materials such as chitosan commonly show low adhesive ability to the surface of the supporting electrode, thus leading to the leak of the immobilized enzyme in washing and testing procedures. In Chapter 7, we synergistically used mussel-inspired adhesive polydopamine bionic film and gold nanoparticles to develop a highly robust enzyme immobilization platform for electrochemical biosensors. Horseradish peroxidase was chosen as an enzyme model. The as-preapred platform has been successfully adopted for developing a novel enzyme biosensor of direct electrochemical for the detection of H2O2. Experimental results showed that the nanocomposite matrix can enable the immobilization of horseradish peroxidase on the electrode in high loading amount, high stability, and well-retained activity. In addition, it can allow for the rapid, direct electron transfer between the active sites of the horseradish peroxidase immobilized and the supporting electrode surface. Quick, direct electrochemical determination of of H2O2 was expected. In comparison with the widely-used chitosan, the polydopamine-based biosensor developed shows much better analytical performances, and can quantitatively determine H2O2 in the range of 0.4μM ~ 45 mM with the limit of detection of about 0.37μM. It may also possess good detection reproducibility and storage stability.