자성 나노입자 클러스터를 기반으로 하는 신속 간편한 바이오센서의 개발

Abstract

In this thesis, the development of rapid and facile biosensors based on magnetic nanoparticle clusters was introduced to detect food-borne pathogenic bacteria, biomarker protein and antibiotics molecules. They provide highly simple and sensitive detection by combining magnetic nanoparticle clusters and functional materials or using magnetic nanoparticle clusters itself. This thesis consists of 6 chapters. Chapter 1 describes general introduction about biosensors, adaptation of functional nanoparticle, and function of magnetic nanoparticle. Chapter 2 introduces a facile and sensitive analytical method that uses gold-coated magnetic nanoparticle clusters (Au/MNCs) and magnetophoretic chromatography with a precision pipette for the detection of Salmonella bacteria. Antibody-conjugated Au/MNCs are used to capture the Salmonella bacteria in milk and are then separated from the milk by applying an external magnetic field. The Salmonella-containing solution is sucked into a precision pipette tip to which a viscous polymer solution is then added. Once the magnetophoretic chromatography process has been carried out for 10 min, the presence of 100 cfu/mL Salmonella bacteria can be detected with the naked eye because the bacteria have become concentrated at the narrow pipette tip. The performance of this method was evaluated by using dynamic light scattering and light absorption spectroscopy. Chapter 3 presents a colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL-1 E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min. Chapter 4 introduces a sensitive and easy method for the detection of the cardiac marker, troponin I, using magnetic immunoassay and ubiquitous pH meters. Monoclonal antibody-functionalized Fe3O4 magnetic nanoparticle clusters (MNCs) were synthesized to capture troponin in human serum, and MNC-troponin complexes were magnetically isolated using a permanent magnet. These complexes were subsequently conjugated to polyclonal antibody-functionalized acetylcholinesterase (AchE) and dispersed in acetylcholine (Ach) solution. As the Ach was hydrolyzed to choline and acetic acid, the pH of the solution decreased, and the resulting pH change was measured in real time using a pH meter. The sensitivity of detection of this assay was found to be 10 pg/mL troponin in human serum after 10 min of the hydrolysis reaction. Further, the pH change could be determined with the naked eye from the color change of a pH indicator strip. Chapter 5 reports a facile and sensitive method for the detection of Salmonella bacteria in milk using a personal glucose meter (PGM). Monoclonal antibody-functionalized magnetic nanoparticle clusters (MNCs) were used to capture Salmonella bacteria in milk, and MNC-Salmonella complexes were magnetically separated from the sample using a permanent magnet. The complexes were further conjugated to polyclonal antibody-functionalized invertase and dispersed in a 0.5 M sucrose solution. After the hydrolysis of sucrose to glucose and fructose, the concentration of glucose was measured using the PGM. The hydrolysis reaction was conducted at various temperatures, and the optimal temperature and activation energy were determined. The detection limit of Salmonella in milk was found to be 10 cfu/mL. Chapter 6 describe the development of a rapid and facile method for the colorimetric detection of penicillin antibiotics, including penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin and dicloxacillin, using hybrid magnetic nanoparticle clusters (HMNCs) and penicillin class-selective, antibody-functionalized platinum nanoparticles (Ab-Pt). Amine-functionalized HMNCs were used to chemically capture antibiotics from sample solution through the formation of amide bonds between amine groups of HMNCs and carboxyl groups in the antibiotics. Ab-Pt was then allowed to bind to antibiotic-HMNC complexes, after which the resulting Pt-antibiotic-HMNCs were added to a 3,3’,5,5’-tetramethylbenzidine (TMB) solution. The color change caused by platinum nanoparticle-mediated oxidation of TMB enabled antibiotics to be identified with the naked eye and quantified by measuring light absorption. The use of the penicillin class-selective antibody allowed the simultaneous detection of penicillin class antibiotics, thereby reducing the cost, time, and effort associated with prescreening antibiotic residues in sample specimens.