Preparation And Analytical Application Of NIR Fluorescent Nanoparticles And Fabrication Of New-Type Biosensing Interfaces For Piezoelectric Immunosensors

Nanoparticle-based bionanotechnology is a rapidly growing field that deals with particulate systems for bioanalytical, biotechnological, and biomedical applications. Recent years have seen proliferated studies of fluorescent organic nanoparticles, among which dye-doped silica nanoparticles attracted research interest due to their potential applications as fluorescence probes for various biological detection. The dye-doped silica nanoparticles entrap a large number of fluorophores in the silica matrix, which produce a strong fluorescence signal when excited properly. Incorporation of dye molecules inside the silica matrix protects the dye from the surroundings, making the fluorescence very stable and thereby offering accurate measurements for bioanalysis. Moreover, the silica surface serves as a universal biocompatible and versatile substrate for the immobilization of biomolecules. Nevertheless, almost all these studies are focused on the entrapment of dyes with excitation in the visible region. These dye-doped silica nanoparticles are susceptible to the interference from autofluorescence and inner filtration effect in cellular or in vivo measurement implementations.Spectrofluorimetry in the near-infrared region (about 650 - 1000 nm) is an area of increasing interest. In comparison to more conventional measurements made in the ultraviolet and visible regions, near-infrared (NIR) fluorescence has many advantages. For instance, NIR fluorescence generally exhibit relatively low levels of background interference, since few naturally occurring molecules undergo electronic transitions in such a low-energy region of the electromagnetic spectrum. Moreover, the penetration of NIR light through skin and overlaying tissue is as deep as a few millimeters, NIR fluorescence holds considerable promise for the development of noninvasive diagnostic techniques. For these reasons, NIR fluorescence imaging is potentially very attractive for in vivo imaging. In addition, as the whole blood shows very weak absorption in the NIR region, there is potential to use NIR fluorescence for direct analysis of whole blood samples without separation steps. The first-part of this thesis focused on the research topics of fluorescent nanoparticles. We synthesized a class of novel core-shell near-infrared fluorescent nanoparticles and used them as highly sensitive and photostable label in the detection of alpha fetoprotein (AFP) and leukemia cancer cells recognition in whole blood samples as well as the detection of single nucleotide polymorphisms (SNPs). Moreover, based on the optical properties of aggregated Au nanoparticles, a colorimetric method for the identification of point mutation based on isothermal rolling-circle amplification has been developed.(1) A class of novel core-shell near-infrared fluorescent nanoparticles was prepared through co-hydrolysis of a hydrophobic silicon alkoxide, hexadecyltrimethoxysilane, and tetraethyl orthosilicate as the dye-doped core followed by the formation of a hydrophilic shell via hydrolysis of tetraethyl orthosilicate in water-in-oil microemulsion. The co-hydrolysis of hexadecyltrimethoxysilane and tetraethyl orthosilicate produced a highly hydrophobic core for the entrapment of a low-cost near-infrared fluorescence dye methylene blue. Experimental investigation of this particular core-shell nanoparticles in comparison with conventional dye-doped silica nanoparticles demonstrated that the hydrophobic core enabled the doped dye to exhibit enhanced fluorescence and show improved stability to dye leaching and exogenous quenchers. In contrast to rhodamine B-doped silica nanoparticles, the near-infrared fluorescent nanoparticles also showed negligible background fluorescence and low inner filtration interference in complex biologic systems such as whole blood. This advantage was utilized for the development of an immunoagglutination assay method based on fluorescence anisotropy measurement for the detection of alpha fetoprotein (AFP) in whole blood samples. The results revealed that the fluorescence anisotropy increases were linearly correlated to the AFP concentration in the range from 1.9 to 51.9 ng/mL.(2) A method for the rapid detection of leukemia cells in whole blood samples has been developed based on a group of aptamers for the specific recognition of leukemia cells and the core-shell near-infrared fluorescent nanoparticles. The results demonstrate the potential application of this method for medical diagnostics.(3) A proof-of-principle has been reported for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on the sensitive fluorescence anisotropy measurement through core-shell fluorescent nanoparticles assembly and the ligase reaction. By incorporating the core-shell fluorescent nanoparticles into the fluorescence anisotropy measurement, this assay provided a convenient and sensitive detection assay that enabled a straightforward single-base discrimination without the need of the complicated operational steps. The assay could be implemented via two steps: firstly, the hybridization reaction that allowed two nanoparticle-tagged probes to hybrid with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the formed duplex, fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.(4) Using the distance-dependent optical properties of aggregated Au nanoparticles functionalized with oligonucleotides, we developed a detection method for identification of point mutation based on the isothermal rolling-circle amplification and the agglutination aggregation of Au nanoparticles.The remaining sections of the present thesis repart the research results concerning fabrication of new-type biosensing interfaces for pizeoelectric immunosensor. Pizeoelectric immunosensor are widely used for the assay of biological analytes due to the advantages of this approach including simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be improved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent property is the immobilization of bio-species on the surface of transducers. Focused on these topics in the formation of the biosensing interface, several new procedures for immobilizing bio-species to construct biosensor have been developed.(5) Electropolymerized films (Eps) and the self-assembled technology are combined for a novel immobilization of biomolecules applied to an immunosensor for detecting the antitrypsin (α1-AT) in human serum. The o-phenylenediamine electropolymerized films are immobilized on the surface of quartz crystal microbalance (QCM). After self-assembling a nano-gold layer on the Eps, the antibodies of the antitrypsin (Anti-α1-AT) are electrostatically adsorbed onto the QCM for immunoreacting Antitrypsin. The surface morphologies of the QCM are investigated by scanning electron microscopy (SEM) after being modified with o-phenylenediamine Eps and nano-gold layer. The conditions of immobilizing anti-α1-AT are optimized in detail. Compared to the glutaraldehyde binding approach, the antibodies immobilized by the nano-gold self-assembly procedure present higher bioactivity and greater frequency response to immunoreaction. It is concluded that this immunosensing system provides the advantages of improved sensitivity, selectivity and reusability.(6) A piezoelectric immunosensor based on Nafion membrane interface with a simple immobilization procedure has been developed for the determination of complement C4 in human serum. The polyanionic perfluorosulfonated Nafion polymer was used to modify the electrode surface of QCM as a platform for the immobilization of complement C4 antibodies. The surface morphology of QCM modified with Nafion membrane was investigated using scanning electron microscopy. The system was optimized with regard to parameters involved in the preparation of immunosensor and the assay process. The proposed immunosensor possesses nice response to C4 in the range of 0.08-1.6μg/mL with a relative standard deviation of below 5.3%. Moreover, the proposed immunosensor shows the advantage in terms of the speed and ease in the immobilization procedure as well as the simple and advantageous regeneration process. Experimental results obtained with regards of non-specific adsorption and recoveries indicated that the proposed immunosensor offers a promising alternative tool for clinical diagnosis of complement C4.(7) A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time-output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.(8) A piezoelectric immunosensor based on a novel immobilization strategy combining cysteamine self-assembled monolayer and chitosan/alginate adsorption procedure has been developed for the determination of factor B in human serum. Cysteamine SAM was first applied to the gold electrode surface of the crystal, and a strong positively-charged chitosan monolayer was posed on the cysteamine SAM by using glutaraldehyde cross-linking. Alginate-factor B was then electrostatically immobilized on the crystal with the aid of the negatively-charged alginate layer. The conditions of immobilizing antibodies were optimized in detail. Comparing with the conventional glutaraldehyde covalent immobilization, the immunosensor with the proposed immobilization procedure shows an improved performance in terms of the magnitude of the response and sensitivity. Moreover, the proposed immobilization procedure results in immobilized entities with relatively high biological activity and favorable immunosensing characteristics. The QCM devices are readily regenerated. Analytical results obtained from the tests of non-specific adsorption and recoveries indicated that the immunosensor prepared by use of the chitosan/alginate adsorption procedure is a useful tool for the determination of factor B in plasma in clinical diagnosis.(9) A quartz crystal microbalance sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) in binding avidin. Avidin formed a metastable complex with HABA immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM and enhances the amount of the immobilized molecules on the QCM, as a longer"space arm"in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017– 1.67μg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.