Synthesis Of Polymethylene Blue Nanoparticles And Its Application In DNA Detection

Sensitivity, selectivity, speediness, accuracy and automatics are the development direction of analytical chemistry. They are also the main drivers for the development of new chemical sensors and biosensors for their use in a wide range of fields. During the last decade, the performance of sensors was greatly improved by the application of nanomaterials in chemical sensors and biosensors. Significant progress has been made in synthetic method of nanomaterials because of the great impact of nanomaterials on sensors. A variety of nanomaterials with highly controllable size, shape, surface charge and physicochemical characteristics have been synthesized. Among them, silica nanoparticles, gold nanoparticles, quantum dot clusters, Semiconductor quantum dots, upconversion nanoparticles, polymer nanoparticles, carbon nanotubes and graphene were widely studied and used in chemical sensors and biosensors. Nanomaterials may be modified with polymers and bioactive molecules in order to enhance biocompatibility and for the achievement of precise targeting. Nanomaterials can also show their unique electrocatalytic properties or be doped by electroactive species. So, they are increasingly being employed in the development of electrochemical DNA biosensors. However, most of the above electrochemical DNA biosensors were developed based on signal amplification of nanoparticles, label or doping of electroactive species, such as methylene blue, ferrocene and nile blue. The electroactivity of electroactive species doped in nanoparticles can be reflected and functionalization of nanoparticles was needed for label DNA sensors.Based on the above issues, the purpose of this thesis is to synthesize a novel type of conductive polymer nanoparticles. The nanoparticles show good electrical activity and can be used as the hybridization indicator and for signal amplification. They also can be used as electrochemical labels.This thesis mainly consists of two parts, review and research work. Review part mainly introduces the characteristics and classification of nanomaterials, the conductive polymers, the electrochemical DNA biosensors and the application of nanomaterials in the DNA electrochemical biosensor; Finally, the purpose and significance this thesis are pointed out. Research work includes two parts as following:1. Synthesis of polymethylene blue nanoparticles (PMBNPs) and their application to label-free DNA detection.In this work, the PMBNPs were first prepared via reverse microemulsion methods using ammonium persulfate (APS) as an oxidant at room temperature. The prepared PMBNPs were characterized by the transmission electron microscopy (TEM) and the cyclic voltammetry. To further investigate the electrochemical behaviors of the prepared PMB nanoparticles, a label-free electrochemical DNA sensor was constructed on account of the direct immobilization of the prepared PMB nanoparticles on glassy carbon electrode. Gold nanoparticles were then assembled onto the PMB nanoparticles modified electrode for immobilizing thiolated DNA. The target DNA was detected by hybridization reaction. The redox current of PMB nanoparticles immobilized on the electrode surface depended on the access of counter-ion in electrolyte solution to the electroactive PMBNPs. Upon the hybridization of DNA, the transfer of counter-ion was blocked partly, which results in the decrease of redox current. On the other hand, redox current of PMBNPs was reduced by hybridization reaction because DNA molecules severely blocks the electron transfer tunnel and reduced effective area of electron transfer. Under the optimum conditions, the difference of current was linear with the concentration of target DNA in the range of 0.2 fM-1 pM. The detection limit was 6.67 fM.2. Electrochemical label-free DNA sensor based on absorption between DNA and polymethylene blue nanoparticles.In this work, the polymethylene blue nanoparticles (PMBNPs) were synthesized via reverse microemulsion system and characterized with transmission electron microscopy (TEM), cyclic voltammetry (CV) and zeta potential. The results indicated that the PMBNPs showed good electrochemical activity and their zeta potential was positive. Based on the absorption between the negatively charged DNA and the positively charged PMBNPs, a DNA sensor was developed. Because the difference in the magnitude of charge between ssDNA and dsDNA resulted in the different amount of PMBNPs absorbed by DNA, DNA was detected by the differential pulse voltammetry of PMBNPs.