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A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application
(2023-01-01)
Khemthongcharoen, Numfon
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Uawithya, Panapat
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Yookong, Nutthapon
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Chanasakulniyom, Mayuree
;
Jeamsaksiri, Wutthinan
Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose–response curve of the MOG transfected cell lysate coating plate can be 2–9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance.
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Development of a Serological Dilution Microfluidic Chip for Immunoassay Applications
(2022-07-01)
Thienthong, Therdthai
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Juntasaro, Ekachai
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Khemthongcharoen, Numfon
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Sripumkhai, Witsaroot
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Houngkamhang, Nongluck
This work aims to develop a multiple dilution microfluidic chip that is capable of diluting the human serum by means of two-fold dilution for seven levels from 1:2 to 1:128 with phosphate-buffered saline (PBS) buffer. The dilution in this work is processed in parallel in order to reduce the accumulated errors that the standard pipetting technique generates in the micro-well plate. The serum and PBS buffer are precisely delivered to the micromixers by controlling their flow rates. The dilution is achieved by the passive mixing process for which the serpentine geometry is designed in order to continually generate the Dean vortices along the serpentine microchannel to effectively mix serum and PBS buffer in the microfluidic chip. The prototype of this multiple dilution microfluidic chip is fabricated by using polydimethylsiloxane (PDMS). The dilution-in-parallel capability of this prototype is validated by using the UV-vis absorption method. The results reveal that the measured values of the seven dilution ratios obtained are in good agreement with the exact values. Finally, this prototype is evaluated for serological MOG-IgG detection in order to verify the reliable operation of this multiple dilution microfluidic chip. The prototype can successfully detect MOG-IgG at all volume concentration ratios.
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Microfluidic system evaluation for the semi-automatic detection of MOG-IgG in serum samples
(2021-12-01)
Khemthongcharoen, Numfon
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Uawithya, Panapat
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Yookong, Nutthapon
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Chanasakulniyom, Mayuree
;
Jeamsaksiri, Wutthinan
Myelin oligodendrocyte glycoprotein reactive immunoglobulin G antibody (MOG-IgG) is in patients with central nervous system demyelination. Reliability of the conventional detection method relies on technician skills and pipetting error accumulation. This work develops a microfluidic system for semi-automatic MOG-IgG detection using cell-based immunofluorescence (IF) assay. The polydimethylsiloxane (PDMS) microfluidic was modified by poly-L-lysine to enhance the adhesion of Human embryonic kidney (HEK) cell. The untransfected and GFP-MOG transfected HEK cells were cultured, fixed, and stained in the microfluidic with the feeding reagents regulated by a syringe pump. Cell characterization, limit of detection (LOD), and turnaround time of the IF assay operation in microfluidic were compared to those in standard microplate. In microfluidic, cell-clumping formation can be avoided and thus signal variations that are caused by cell overlapping can be significantly reduced. LOD of MOG-IgG detection in the microfluidic is at least 2.5 times better than that in the microplate. Signal intensities of the IF staining for 1 h in microfluidic are comparable to those stained for overnight in the standard microplate. By integration with a serial dilution microfluidic, the optimal cutoff titer for MOG-IgG positivity in the patient samples was determined by Receiver operating characteristic curve (ROC) analysis.
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Mixing-performance evaluation of a multiple dilution microfluidic chip for a human serum dilution process
(2021-09-30)
Thienthong, Therdthai
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Juntasaro, Ekachai
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Sripumkhai, Witsaroot
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Houngkamhang, Nongluck
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Chanasakulniyom, Mayuree
This paper is aimed to propose a numerically designed multiple dilution microfluidic chip that can simultaneously deliver several serum dilutions in parallel. The passive mixing scheme is selected for dilution and achieved by the serpentine mixing channel in which Dean vortices are induced to increase the contact area and time for better diffusion. The mixing performance at the exit of this dilution chip is numerically evaluated using five commonly-used mixing indices with the goal that the homogeneity of the mixture over the exit cross-sectional area of the mixing channel must be greater than 93.319% to fulfill the six-sigma quality control.
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Polydimethylsiloxane (PDMS) microfluidic modifications for cell-based immunofluorescence assay
(2021-01-01)
Khemthongcharoen, Numfon
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Uawithya, Panapat
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Chanasakulniyom, Mayuree
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Yasawong, Montri
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Jeamsaksiri, Wutthinan
Polydimethylsiloxane (PDMS) is a hydrophobic elastomer commonly used for microfluidic fabrication. PDMS has to be modified to improve its hydrophilicity and thus inhibits non-specific protein adsorption. This work evaluates the modification materials for the development of microfluidic cell-based immunofluorescence (IF) assay. In cell-based IF assay, PDMS is modified not just to inhibit the adsorption of non-specific florescent-conjugated protein that causes the elevation of background signal, but also to firmly support cell adhesion for subsequent immunostaining procedure. PDMS materials modified by three regular modification materials consisting of an extracellular matrix (poly-L-lysine; PLL), a hydrophilic polymer (polyvinyl alcohol; PVA) and a non- ionic surfactant (pluronic F127) were compared with each other based on hydrophilicity improvement, minimization of non-specific background signal, and enhancement of human embryonic kidney (HEK) cell adhesion. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) analysis confirms that all modification materials are successfully immobilized on the PDMS surfaces. Due to its antifouling mechanism, pluronic modification greatly improves the hydrophilicity of the PDMS and inhibits non-specific protein adsorption. Even though the hydrophilicity and non-specific protein adsorption resistivity of the PDMS modified with PLL did not significantly differ from those of the unmodified PDMS, PLL modification obviously promotes HEK cell adhesion. Negative control and Myelin Oligodendrocyte Glycoprotein (MOG) expressing HEK cells were immobilized in microfluidics for IF assay evaluation. Results demonstrate that positive MOG expressing cells can be selectively stained by anti-MOG IgG antibody within 1 h at room temperature. Microfluidic platforms also enhance immobilized cell distribution, which compatibly supports single-cell analysis technique.
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Effect of amino-, mercapto-silane coupling as a molecular bridge of polyvinyl chloride ion-selective membrane on silicon nitride for nitrate ISFET sensors
(2020-06-01)
Chaisriratanakul, Woraphan
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Bunjongpru, Win
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Pankiew, Apirak
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Srisuwan, Awirut
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Jeamsaksiri, Wutthinan
This paper presents a modification of a Si<inf>3</inf>N<inf>4</inf> Polyvinyl chloride (PVC) membrane on a surface of an ion selective field effect transistor (ISFET) using silane with two different functional groups for comparison of nitrate sensing characteristics. Effects of amino (NH<inf>2</inf>) and thiol groups (SH) toward structures and characteristics of nitrate sensors were analyzed using the following techniques: ellipsometry, scanning electron microscope (SEM), infrared spectroscopy (FTIR), zeta potential and electrochemical impedance spectroscopy (EIS). After surface modification, the thickness of the silane layers for both cases were similar with uniform surface topology. Absolute zeta potentials of PVC ion-selective membrane (7.65 mV) and Si<inf>3</inf>N<inf>4</inf> surface modification with MPTMS (-51.46 mV) reflected the surface functional group and showed that membranes could be bonded to the PVC ion-selective membrane. Since APTMS produced higher EIS than MPTMS, the resulting nitrate sensors modified by MPTMS had higher sensitivity than the sensors with APTMS. Finally, the modified sensors had a useful life time of 3 months.
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Modification of polyvinyl chloride ion-selective membrane for nitrate ISFET sensors
(2020-05-15)
Chaisrirattanakua, Woraphan
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Bunjongpru, Win
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Pankiew, Apirak
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Srisuwan, Awirut
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Jeamsaksiri, Wutthinan
In this work, a Polyvinyl Chloride (PVC) ion-selective membrane was modified to detect nitrate based on Ion Selective Field Effect Transistor (ISFET) sensing technology in order to eliminate chloride interference. A modification was done with ethylenediamine solution to achieve animated PVC, which was then oxidized with sodium tungstate to form nitrone coated PVC membrane. The modified PVC was characterized using FTIR, 1H NMR, GPC, FE-SEM and EIS. The FTIR spectrum of animate PVC demonstrated -NH<inf>2</inf> bending at 1580 cm−1, NO<inf>2</inf> asymmetric stretching at 1650 cm−1 and N[sbnd]O out-of-plane deformation vibration at 837 cm−1. GPC analysis of the modified PVC showed that the PVC molecular weight was shifted to a high molecular weight. The PVC ion-selective membrane was immobilized on the ISFET to create nitrate sensors with the following characteristics. The Nitrate-Nitrogen detection limit is 1.20 ppm with linear range from 3−20 ppm at sensitivity of 56 ± 2 mV/dec. The sensor is useful for detecting small amount of nitrate in a mixed solution, which usually is the case in most situations. This sensor could be applied in many applications, such as agricultural industry, water reserve management and medical field as well.
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Fabrication of MEMS-based capacitive silicon microphone structure with staircase contour cavity using multi-film thickness mask
(2019-02-01)
Jantawong, Jirawat
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Atthi, Nithi
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Leepattarapongpan, Chana
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Srisuwan, Awirut
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Jeamsaksiri, Wutthinan
In this work, a silicon capacitive microphone structure with a three-dimensional staircase style contour cavity (S-CTC) is developed using a newly developed Multi Film Thickness photo lithography process. This newly developed Multi Film masking process overrides the conventional wisdom in which the staircase style cavity is formed using multiple exposures of mask and multiple etchings of silicon. With this newly developed photo lithographic technique, a Multi Film Thickness (MFT) mask is fabricated with varying chromium film thicknesses such as 0, 4, 14, and 114 nm thick. The mask was then evaluated to fabricate a microphone structure with a three-dimensional staircase style cavity. Once the mask was patterned onto a substrate, a single dry etching of silicon was carried out and a desired three-dimensional stair cavity pattern was achieved. Surprisingly, our results show that a capacitive sensor with an S-CTC structure has an increased absolute capacitance value by an average of 30% in comparison to a conventional box cavity (BC) structure. This may indeed improve SNR as we anticipated. The capacitance value changed from 2.2pf to 2.9pf for the same dimension of devices. This promising technology renders an opportunity to improve the next generation of ultra-low-pressure sensors for microphone applications.
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Fabrication of Contour Cavity Using Multi-Exposure Lithography for MEMS Capacitive Microphone
(2018-07-02)
Jantawong, Jirawat
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Atthi, Nithi
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Leepattarapongpan, Chana
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Jeamsaksiri, Wutthinan
;
Austin, Anu
This paper presents a novel technique to optimize a capacitive pressure sensor with multi-exposure dose lithography. As part of achieving high signal to noise ratio (SNR) in microphone, a new design was implemented. The new design defies the conventional wisdom of parallel plate theory and implement a concept of contour cavity (backplate) to follows the diaphragm deflection. This 3-dimensional contour cavity (CC) is fabricated by multi-exposure dose lithography with single silicon etching process. The CC structure with different etched depth of 0.65, 1.15, and 1.57 micron were fabricated by multi-exposure dose of 92, 120, and 210 mJ/cm2on 5.0 micron thick photoresist and single step etching with CF<inf>4</inf>/O<inf>2</inf>plasma (Si/PR etch selectivity: 1.3). Due to this novel contour cavity design, polysilicon membrane with 0.8 micron thick and a diameter of 930 micron structure produces the capacitance value of 2.88 pF, which is 28.6% higher than that of conventional parallel plate capacitive sensor.
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Effects of Annealing Process on the Electrical Properties of MEMS Capacitive Microphone
(2018-07-02)
Jantawong, Jirawat
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Leepattarapongpan, Chana
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Chaowicharat, Ekalak
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Jeamsaksiri, Wutthinan
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Austin, Anu
In this paper, the effects of annealing temperature after pre-bonding device wafer and cover wafer during wafer fusion bonding process on the electrical properties of MEMS-based capacitive microphone with 0.53 μm thick poly-Si membrane were investigated. By the annealing in N2 ambient (14 SLM) at 900°C for 30 min, the device exhibited capacitance of 2.0 pF with pull-in voltage of 12 V. The significantly improvement of electrical properties is related to the reducing of residual stress in poly-Si membrane to near-zero level by using suitable annealing condition.

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