Assawapong Sappat

Ultrasensitive detection of Vibrio cholerae O1 using microcantilever-based biosensor with dynamic force microscopy

This work presents the first demonstration of a cantilever based cholerae sensor. Dynamic force microscopy within atomic force microscope (AFM) is applied to measure the cantilevers resonance frequency shift due to mass of cell bound on microcantilever surface. The Vibrio cholerae O1, a food and waterborne pathogen that caused cholera disease in human, is a target bacterium cell of interest. Commercial gold-coated AFM microcantilevers are immobilized with monoclonal antibody (anti-V. cholerae O1) by self-assembled monolayer method. V. cholerae O1 detection experiment is then conducted in concentrations ranging from 1×10(3) to 1×10(7) CFU/ml. The microcantilever-based sensor has a detection limit of ∼1×10(3) CFU/ml and a mass sensitivity, Δm/ΔF, of ∼146.5 pg/Hz, which is at least two orders of magnitude lower than other reported techniques and sufficient for V. cholerae detection in food products without pre-enrichment steps. In addition, V. cholerae O1 antigen-antibody binding on microcanilever is confirmed by scanning electron microscopy. The results demonstrate that the new biosensor is promising for high sensitivity, uncomplicated and rapid detection of V. cholerae O1.

Flow injection based microfluidic device with carbon nanotube electrode for rapid salbutamol detection

A microfabicated flow injection device has been developed for in-channel electrochemical detection (ECD) of a beta-agonist, namely salbutamol. The microfluidic system consists of PDMS (polydimethylsiloxane) microchannel and electrochemical electrodes formed on glass substrate. The carbon nanotube (CNT) on gold layer as working electrode, silver as reference electrode and platinum as auxiliary electrode were deposited on a glass substrate. Silver, platinum, gold and stainless steel catalyst layers were coated by DC-sputtering. CNTs were then grown on the glass substance by thermal chemical vapor deposition (CVD) with gravity effect and water-assisted etching. 100-microm-deep and 500-microm-wide PDMS microchannels fabricated by SU-8 molding and casting were then bonded on glass substrate by oxygen plasma treatment. Flow injection and ECD of salbutamol was performed with the amperometric detection mode for in-channel detection of salbutamol. The influences of flow rate, injection volume, and detection potential on the response of current signal were optimized. Analytical characteristics, such as sensitivity, repeatability and dynamic range have been evaluated. Fast and highly sensitive detection of salbutamol have been achieved. Thus, the proposed combination of the efficient CNT electrode and miniaturized lab-on-a-chip is a powerful platform for beta-agonists detection.

On-Chip Immunoassay for Determination of Urinary Albumin

An immunoassay performed on a portable microfluidic device was evaluated for the determination of urinary albumin. An increase in absorbance at 500 nm resulting from immunoagglutination was monitored directly on the poly(dimethylsiloxane) (PDMS) microchip using a portable miniature fibre-optic spectrometer. A calibration curve was linear up to 10 mg L–1 (r2 = 0.993), with a detection limit of 0.81 mg L–1 (S/N = 3). The proposed system showed good precision, with relative standard deviations (RSDs) of 5.1%, when evaluated with 10 mg L–1 albumin (n = 10). Determination of urinary albumin with the proposed system gave results highly similar to those determined by the conventional spectrophotometric method using immunoturbidimetric detection (r2 = 0.995; n = 15).

Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter

In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.

High-sensitivity humidity sensor utilizing PEDOT/PSS printed quartz crystal microbalance

In this work, quartz crystal microbalance humidity sensor was fabricated by inkjet printing technique. Poly (3, 4-ethylenedioxythiophene)/poly-styrene-sulfonic acid (PEDOT/PSS), one of the most widely used polymer composites, was printed on QCM electrode as sensing layer using Dimatrix material inkjet printer. The main advantage of this coating method is its high precision of solution coating with accurately controlled volume and area. The printed layer was varied from 1 to 20 layers. With 20 PEDOT/PSS printed layers, the humidity sensitivity is found increased by more than three orders of magnitude compared to uncoated QCM. In addition, the PEDOT/PSS coated QCM exhibits fast humidity detection with short response and recovery times. Thus, the PEDOT/PSS printed on the QCM electrode is an effective way to improve humidity-sensing characteristic of QCM.

Low cost hot embossing process for plastics microfluidic chips fabrication

In this work, we develop plastic microfluidic chips based on low cost hot embossing process with metal micromold. Metal micromold was formed on aluminum substrates by CNC milling machine and high precision micromachine. The hot embossing system is in-house made with computer aid design by Solid Work program. The system consists of four main parts, structural body, heating system, compressive system and control electronics. The compressive system consists of two top hydraulic single-stage pistons and four middle-stage pistons. Polymethyl methacrylate (PMMA) microfluidic chips were then produced by hot embossing under different applied temperatures and time. It was found that optimum temperature and time for minimum contraction and depth error were 80 degree C and 2–5 minutes. The developed technique offer advantages for microfluidic chip fabrication in term of quality, complexity and cost.

Development of traveling wave dielectrophoretic (twDEP) microfluidic system

In this work, we present a microfluidic system consisting of 16 parallel electrodes array for cell manipulation by traveling wave dielectophoretic force and electronic circuit design for creating driving signal. Polystyrene microspheres suspensions in water were used as the tested cells. Cells respond to the electric field in various mechanisms depending on the frequency of applied AC signals. When the frequency of applied AC fields is in the range where dielectrophoresis (DEP) is negative, cells experience twDEP force in such a way that they were repelled from the electrode rather than being trapped by positive DEP. The driving signals in the system are created by economical electronic circuit. As the frequency of the applied signals is in the range of 50-700 kHz, cells were move under the influence of twDEP force. As the frequency of the applied signals is more than 700 kHz, cells started moving out of the center between electrodes. These results are consistent with the theory. Because of the fact that twDEP force depends on the effective polarizability and size of particle, it gives us a chance to make the device for cells fraction and separation which can be further applied in biological and medical application such as motion control and cell selectivity.

Turbidity detection of shrimp Taura Syndrome Virus by loop-mediated isothermal amplification reaction

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid, which can be applied for disease diagnosis in shrimp aquaculture. During the LAMP reaction, the white precipitate of magnesium pyrophosphate (Mg2P2O7) is formed correlates with the amount of synthesized DNA. So, the turbidity can be measured. In this study, a portable turbidimeter has been developed for field to detection of Taura Syndrome Virus (TSV) that causes large economic losses to most major shrimp-producing countries including Thailand. The device could maintain an optimal temperature (63 °C) for 25 µl of LAMP sample solution contained in a 0.2 ml commercial PCR tube. We also applied the spectroscopic measurement technique to monitor a by-product of LAMP reaction, light emitting diode (LED) was used as a light source. Light dependent resistance (LDR) was used as detector. The results obtained from turbidity measurement revealed the same detection limit to those from agarose gel electrophoresis method.

Multi-channel turbidity detection of shrimp virus by loop-mediated isothermal amplification reaction

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid, which can be applied for disease diagnosis in shrimp aquaculture. During the LAMP reaction, the white precipitate of magnesium pyrophosphate (Mg2P2O7) is formed correlates with the amount of synthesized DNA. So, the turbidity can be measured. In this study, a portable turbidimeter has been developed for field to detection of Taura Syndrome Virus (TSV) that causes large economic losses to most major shrimp-producing countries including Thailand. The device could maintain an optimal temperature (63 °C) for 25 µl of LAMP sample solution contained in a 0.2 ml commercial PCR tube. We also applied the spectroscopic measurement technique to monitor a by-product of LAMP reaction, light emitting diode (LED) was used as a light source. Light dependent resistance (LDR) was used as detector. The results obtained from turbidity measurement revealed the same detection limit to those from agarose gel electrophoresis method.

Fabrication and characterization of traveling wave dielectrophoretic (twDEP) microfluidic devices

In this work, we present a microfluidic device with 16 parallel electrodes array and microchamber for cell manipulation by traveling wave dielectrophoretic force. Polystyrene microspheres suspensions in water were used as the tested cells. Cells respond to the electric field in various mechanisms depending on the frequency of applied AC signals. When the frequency of applied AC fields is in the range where dielectrophoresis (DEP) is negative, cells experience twDEP force in such a way that they were repelled from the electrode rather than being trapped by positive DEP. As the frequency of the applied signals is in the range of 50 -700 kHz, cells were move under the influent of twDEP force. As the frequency of the applied signals is more than 700 kHz, cells started moving out of the center between electrodes. These results are consistent with the theory. Because of the fact that twDEP force depends on the effective polarizability and size of particle, it gives us a chance to make the device for cells fraction and separation which can be further applied in biological and medical application such as motion control and cell selectivity.