질병 진단 플랫폼 개발을 위한 다양한 물질의 표면 변화와 결합력에 대한 연구

Abstract

ABSTRACT Background and Purpose: Rapid and sensitive detection of low amounts of pathogen in large volume samples is needed for early diagnosis and treatment of patients and surveillance of pathogen spreading. In bacteria with low abundance in samples, which contain various contaminating factors, sample analyses should be conducted with meticulous care; therefore, emerging technologies can facilitate the characterization od disease-causing bacteria with more sensitivity, rapidity, and ease of use. Surface modification is very important for analyte isolation and detection platforms. The immobilization occurs when the functional groups of analyte react with the functional groups of the modified surfaces. The binding of analyte on the surface is the primary step for enrichment and isolation following later step of detection. The combination of isolation and detection methods would create good diagnostic platforms. There are many reagents that have been using for surface modification. Biological reagents such as carbohydrate binding protein, iron binding protein or antimicrobial peptide give us a benefit in reaction with analyte. Thus, the study of surface modification and covalent coupling of biological reagents onto different materials is highly needed. This research dissertation presents the surface modification of various nanomaterials with different biological reagents followed by functionalization using covalent coupling procedures. The aim of this work is to introduce stable synthesis and modification of various materials that is suitable for diagnostic platforms.

Materials and Methods In this work, fabrication, synthesis, surface modification, characterization and application of microfluidic device based on modified surface of polydimethylsiloxane (PDMS) chip and nanoparticles based on diatom earth (DE) and iron (Fe) were reported. The biological reagents include carbohydrate binding protein (ConA) and antimicrobial peptide (Magainin-1) were immobilized onto different materials of microfluidic PDMS chip and magnetic diatom nanoparticle. Strategy for surface modification is through silanization of surfaces by using covalent coupling. The procedure includes attaching a thio-terminal silane via the hydroxyl groups of the surface. The hydroxyl groups (-OH) on the surface of PDMS (with active silanol groups) were created by plasma treatment that altered the hydrophobic surfaces of PDMS to hydrophilic. The hydroxyl groups on the surface of iron oxide nanoparticles were created by polymerization of bis-MPA dendrimers onto the amine-modified surface (that was formed on iron oxide nanoparticles by sol-gel process). 3-MPS was used to replace surface hydroxyl groups by NH3 groups, following bifunctional cross-linker (GMBS) that allows amide binding to the terminal amino group. This strategy is efficient to react with biological reagent (antibody, protein, peptide etc.). In the first section, a microfluidic device with asymmetric herringbone groove arrays was modified its surface with Concanavalin A (ConA) and Magainin 1 for rapid detection of low amount of pathogen. The surface modification has been done by covalent immobilization, [3-mercaptopropyl]trimethoxysilane (3-MPS) for silanization on the surface of the microfluidic chip and N-(γ-maleimidobutyryloxy)succinimide ester (GMBS), a heterobifunctional cross-linker, were employed to immobilize the ConA through amide bonds. In the second section, ConA and antibodies were coated on the surface of L-arginine modified magnetic DE for bacteria enrichment and exosome isolation. L-arginine was attached to the amine functional group of (3-Aminopropyl)triethoxysilane (APTES) on the magnetic DE via an amide bond. The immobilization of ConA on magnetic DE was followed same process with section 1. The antibodies were coated on the surface of modified magnetic DE following the immobilization of glutamate decarboxylase (GAD) onto the surface by covalent coupling.

Results After optimization of enrichment with Concanavalin A and detection conditions, Salmonella enterica serotype Typhimurium could be detected in urine sample (10 mL) at a concentration as low as 5 CFU/ml in real-time using a label-free method. The detection limit of the platform by using Magainin-1 was found to be 20 times more sensitive in 10 mL urine with Salmonella and 10 times more sensitive in 10 mL urine with Brucella than that of real-time PCR without enrichment process. This platform provides rapid enrichment and detection of pathogens such as Salmonella enterica and Brucella ovis within 100 min. The nano-sized magnetic diatom has been successfully created by the formation of iron oxide on the surface of biosillia porous diatomite via precipitation method. Following the addition of L-arginine-containing amino groups, the magnetic diatom showed a broad absorbance in the visible light spectrum (UV-Vis), and the amine functional groups of 3-aminopropyl triethoxysilane on the DE via the amidine bond were confirmed using Fourier transform infrared analysis. The concanavalin A modified magnetic diatom could be used to enrich a minimum of 50 CFU of Salmonella for small and large scale of volume sample. It also shows a promising ability to capture another bacterial species. The antibody modified magnetic diatom could be used for exosome isolation with the detection of 102 exosome particles as limit of detection. Functionalized mag-DE could be magnetically isolated in a rapid and more efficient manner than using the conventional method, which can be altered for exosome isolation in research and clinical applications.

Conclusions In the current study, rapid and sensitive detection of pathogen diagnosis based on microfluidic enrichment with a label-free nanobiosensing platform is useful as it has potential for better diagnosis of pathogen by increasing the capture efficiency of the pathogen in large volume sample, subsequently enhancing the detection limit of pathogenic DNA. Besides, a new green synthesis strategy for the mag-DE under simple and general conditions has been developed. Fe3O4 formation on the surface of biosilica porous diatomite via precipitation was used to create a magnetic diatom (mag-DE) through the bottom-up approach, which was low-cost, robust, non-toxic, and eco-friendly. The study presents the unique physical and chemical properties of green synthesized mag-DE, whose surface can be easily modified. Thus, it creates a novel potential strategy for several desirable applications for various types of biological samples.

Perspective In a future perspective a long term goal is the rapid, low-cost, accurate and high yield method for laboratory researches and application in multiplexed point-of-care testing.