Development of advanced lateral flow immunosensors using electrochemiluminescence and metal-enhanced fluorescence

Abstract

Point of care testing (POCT) devices has become the most used tool, allowing rapid testing at, or near, the site of patient care for healthcare system, food safety, and environment monitoring. One of the fundamental aims of POCT devices is the development of a cost-effective, portable, sensitive, and user-friendly system. Lateral flow immunosensor (LFI) has led the innovative advent of POCT along with microfluidic paper-based analytical devices (µPADs). Especially, LFI has become a worldwide platform for the identification of analytes and pathogens, which operated by minimally trained personnel in areas where no sophisticated instruments are available. However, the two main drawbacks of the LFI have often limited the detection of targets requiring high-sensitivity detection due to its poor limit of detection (LOD) and detection range. To address this, various signal amplification strategies have been studied for the improvement of the detection sensitivity of conventional LFI. In this study, we grafted electrochemiluminescence (ECL) and metal-enhanced fluorescence (MEF) onto LFI format for signal amplification. In chapter 2, we reported an electrochemiluminescent LFI (ECL-LFI) for sensitive detection of C-reactive protein (CRP) with wide dynamic range. In order to apply the ECL reaction, slight modification of LFI strip was required to insert an electrode for an electrochemical reaction. Ru(bpy)32+-immobilized gold nanoparticles (AuNPs) was used as an ECL signal generator and CRP-targeting probe. ECL-LFI allowed sensitive detection of CRP in spiked serum, which is two orders of magnitude broader than that of conventional colorimetric LFI. Furthermore, the clinical usability of the ECL-LFI was evaluated using clinical serum samples, which showed good linear correlation (R2 = 0.9896) with a clinical chemistry analyzer. In chapter 3, we reported an ECL-LFI for the detection of troponin I (TnI), using Ru(bpy)32+-loaded mesoporous silica nanoparticles (RMSNs) as a ECL signal generator. Since mesoporous silica nanoparticles (MSNs) has excellent textural properties, large amount of Ru(bpy)32+ were loaded into the MSNs, resulting in strong ECL signals. We optimized loading condition of Ru(bpy)32+ for the effective synthesis of RMSNs and explored the application of antibody-immobilized RMSNs for the detection of TnI. The ECL-LFI enabled highly sensitive detection of TnI spiked into human serum within 20 min at femtomolar levels (ca. 0.84 pg/mL), which is 3-order more sensitive than with fluorescence detection. Furthermore, we successfully quantified TnI concentration in 35 clinical serum samples across low range (0.01–48.31 ng/mL) with good linear correlation (R2 = 0.9897) with a clinical immunoassay analyzer. In chapter 4, we reported a highly sensitive MEF-based LFI (MEF-LFI) for the detection of troponin I (TnI) and influenza A nucleoprotein (NP). MEF nanoparticles was synthesized via a core-shell structure, realizing the employment of inorganic (mesoporous silica) and organic (bovine serum albumin and streptavidin) shells to synthesize two type of fluorescent gold nanorods (GNRs). These shells enabled to load large amount of fluorophores and improve assay performance via an effective immobilization of antibody to the shell. The fluorescence of fluorophores was significantly enhanced 2-3-fold due to the strong surface plasmon resonance coupling. Through the optimization of assay condition, we successfully detected both TnI and NP at 1 pg/mL. In this dissertation, the improved performance of LFI, which was based ECL and MEF phenomenon, has been demonstrated for the detection of various target protein. These results showed the efficacy of these systems as high-performance sensing strategies capable of capitalizing on future POCT markets for biomolecule detection.