Boonsong Sutapun

SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification

In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.

Hemoculture and Direct Sputum Detection of mecA‐Mediated Methicillin‐Resistant Staphylococcus aureus by Loop‐Mediated Isothermal Amplification in Combination With a Lateral‐Flow Dipstick

This study reports loop‐mediated isothermal amplification (LAMP) for rapid detection of methicillin‐resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences—femB to identify S. aureus and mecA to identify antibiotic‐resistant gene. Reference strains including various species of gram‐positive/gram‐negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost‐effective method in a routine diagnostic laboratory.

Application of surface plasmon resonance biosensor for the detection of Candida albicans

In this study, surface plasmon resonance imaging (SPR imaging) was developed for the detection of Candida albicans which is a causal agent of oral infection. The detection was based on the sandwich assay. The capture antibody was covalently immobilized on the mixed self assemble monolayers (SAMs). The ratio of mixed SAMs between 11-mercaptoundecanoic acid and 3-mercaptopropanol was varied to find the optimal ratio for use as a sensor surface. The results showed that the suitable surface for C. albicans detection was SAM of carboxylic (mixed SAMs ), even though mixed SAMs had a high detection signal in comparison to mixed SAMs , but the non-specific signal was higher. The detection limit was 107 cells/ml for direct detection, and was increased to 106 cells/ml with sandwich antibody. The use of polyclonal C. albicans antibody as capture and sandwich antibody showed good selectivity against the relevant oral bacteria including Escherichia coli, Streptococcus mutan, Staphylococcus aureus, β-streptococci, and Lactobacillus casei. SPR platform in this study could detect C. albicans from the mixed microbial suspension without requirement of skillful technician. This SPR imaging biosensor could be applied for Candida identification after cultivation.

Microfluidic PMMA-based microarray sensor chip with imaging analysis for ABO and RhD blood group typing

Solid phase microarrays have been described for use in blood typing; red blood cells (RBCs) captured on immobilized antibodies were detected using surface plasmon resonance or fluorescence. We present antibody microarray on Poly (methylmethacrylate) (PMMA) surface coupled with microfluidic system for ABO and RhD blood typing. After immobilized by antigen–antibody interaction, the RBCs were detected by image recognition.

Label-Free Detection of Meca-Mediated Methicillin-Resistant Staphylococcus Aureus (MRSA) Using Surface Plasmon Resonance Spectroscopy

The DNA hybridization of methicillin-resistant staphylococcus aureus (MRSA) was investigated by using surface plasmon resonance (SPR). 3 dimensional (3D) surface, carboxymethyl dextran hydrogel, were used as the sensor surface. The surface was functionalized with streptavidin where the biotinylated DNA probe can be specifically bound. Loop-mediated isothermal amplification (LAMP) products of MRSA were hybridized with the biotinylated probe and the biding was followed by SPR. The real time monitoring of hybridization kinetics of unlabeled mecA Lamp amplicons product was successfully carried out. The optimal condition of the probe density was 0.1 μM for MRSA at 0.06 nM. Non-specific absorption was not observed.

Fiber-optic confocal probe with an integrated real-time apex finder for high-precision center thickness measurement of ball lenses

This paper describes the development of a fiber-optic confocal probe suitable to measuring the central thickness of highcurvature small-diameter optical ball lenses. The confocal probe utilizes an integrated camera that functions as a realtime apex-sensing device. An additional camera is used to monitor the shape of the reflected light beam. Placing the instrument sensing spot off-center from the apex will produce a non-circular image at the camera plane that closely resembles an ellipse for small displacement. By analyzing the shape of the reflected light spot, we are able to precisely determine the focus point of the confocal probe relative to the apex point to better than 2-μm precision for ball lenses with diameters in the range of 3 – 10 mm. The proposed confocal probe offers a low-cost alternative technique for quality control of ball lenses during the manufacturing process.

Multichannel microfluidic chip for rapid and reliable trapping and imaging plant-parasitic nematodes

Faster and reliable testing technique to count and identify nematode species resided in plant roots is therefore essential for export control and certification. This work proposes utilizing a multichannel microfluidic chip with an integrated flow-through microfilter to retain the nematodes in a trapping chamber. When trapped, it is rather simple and convenient to capture images of the nematodes and later identify their species by a trained technician. Multiple samples can be tested in parallel using the proposed microfluidic chip therefore increasing number of samples tested per day.