A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application.

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.

Semi-Automated Microfluidic Device Combined with a MiniPCR-Duplex Lateral Flow Dipstick for Screening and Visual Species Identification of Lymphatic Filariae.

Lymphatic filariasis (LF) is a leading cause of permanent disability worldwide that has been listed as a neglected tropical disease by the World Health Organization. Significant progress made by the Global Program to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decline in the population of the worm that causes LF infection. Diagnostic assays capable of detecting low levels of parasite presence are needed to diagnose LF. There is also a need for new tools that can be used in areas where multiple filarial species are coendemic and for mass screening or for use in a point-of-care setting. In the present study, we applied our previously developed semi-automated microfluidic device in combination with our recently developed mini polymerase chain reaction (miniPCR) with a duplex lateral flow dipstick (DLFD) (miniPCR-DLFD) for rapid mass screening and visual species identification of lymphatic filariae in human blood. The study samples comprised 20 Brugia malayi microfilariae (mf) positive human blood samples, 14 Wuchereria bancrofti mf positive human blood samples and 100 mf negative human blood samples. Microfilariae detection and visual species identification was performed using the microfluidic device. To identify the species of the mf trapped in the microfluidic chips, DNA of the trapped mf was extracted for miniPCR amplification of W. bancrofti and B. malayi DNA followed by DLFD. Thick blood smear staining for microfilariae detection was used as the gold standard technique. Microfilariae screening and visual species identification using our microfluidic device plus miniPCR-DLFD platform yielded results concordant with those of the gold standard thick blood smear technique. The microfluidic device, the miniPCR and the DLFD are all portable and do not require additional equipment. Use of this screening and visual species identification platform will facilitate reliable, cost-effective, and rapid surveillance for the presence of LF infection in resource-poor settings.

Application of a novel rectangular filtering microfluidic device for microfilarial detection.

The rectangular filtering microfluidic chip was invented using microfluidics device fabrication technology and can separate living microfilariae from blood samples without a syringe pump. The diagnostic results are highly effective. The device is based on the principle of separating millions of blood cells from microfilariae using a rectangular filter structure. It disperses fluid evenly into the flow-passage channel, and its rectangular filter structure is the key to success in reducing the pressure and separating blood cells from microfilariae effectively. The flow rate and blood cell concentration were optimized in our study. The chip is intended to be a point-of-care device that can reduce the use of superfluous instrumentation in the field. The technology is designed to be rapid, accurate, and easy-to-use for all users, especially those in remote areas.

Effects of Porous Size and Membrane Pattern on Shear Stress Characteristic in Gut-on-a-Chip with Peristalsis Motion.

Dynamic gut-on-a-chip platform allows better recreation of the intestinal environment in vitro compared to the traditional static cell culture. However, the underlying mechanism is still not fully discovered. In this study, the shear stress behavior in a gut-on-a-chip device with porous membrane subjected to peristalsis motion is numerically investigated using CFD simulation for three different pore sizes and two pattern layouts. The results reveal that, in the stationary microchannel, the average shear stress on the porous membrane is approximately 15% greater than that of the flat membrane, regardless of the pore size. However, when subjected to cyclic deformation, the porous membrane with smaller pore size experiences stronger variation of shear stress which is ±5.61%, ±10.12% and ±34.45% from its average for the pore diameters of 10 µm, 5 µm and 1 µm, respectively. The shear stress distribution is more consistent in case of the staggered pattern layout while the in-line pattern layout allows for a 32% wider range of shear stress at the identical pore size during a cyclic deformation. These changes in the shear stress caused by peristalsis motion, porous size and membrane pattern could be the key factors that promote cell differentiation in the deforming gut-on-a-chip model.

Tuning the Dielectric Constant and Surface Engineering of a BaTiO3/Porous PDMS Composite Film for Enhanced Triboelectric Nanogenerator Output Performance.

In this work, synergistic effects derived from surface engineering and dielectric property tuning were exploited to enhance the output performance of a triboelectric nanogenerator (TENG) based on an inorganic/porous PDMS composite in a contact-separation mode. BaTiO3 (BT)/porous PDMS films with different BT weight ratios were fabricated and evaluated for triboelectric nanogenerator (TENG) application. Maximum output signals of ca. 2500 V, 150 µA, and a power density of 1.2 W m-2 are achieved from the TENG containing 7 wt % BT, which is the best compromise in terms of surface roughness, dielectric constant, and surface contact area as evidenced by SEM and AFM studies. These electrical signals are 2 times higher than those observed for the TENG without BT. The 7BT/porous PDMS-based TENG also shows high stability without a significant loss of output voltage for at least 24 000 cycles. With this optimized TENG, more than 350 LEDs are lit up and a wireless transmitter is operated within 9 s. This work not only shows the promoting effects from porous surfaces and an optimized dielectric constant but also offers a rapid and template/waste-free fabrication process for porous PDMS composite films toward large-scale production.

A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection.

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Investigation on the Prevalence of Canine Microfilaremia in Thailand Using a Novel Microfluidic Device in Combination with Real-Time PCR.

We conducted a survey of canine microfilaraemia in 768 dogs in Chanthaburi, Samut Sakhon, and Narathiwat provinces of Thailand using a novel semi-automated, microfluidic device that is easy and rapid to perform. Microfilariae species were identified using High Resolution Melting real-time PCR (HRM real-time PCR). The prevalence of canine microfilaremia was 16.2% (45/278) in Chanthaburi and 5.5% (12/217) in Samut Sakhon. The prevalence of canine microfilaremia in Narathiwat was 22.7% (67/273). Brugia pahangi and Dirofilaria immitis were the predominant species of filariae found in the infected dogs from Chanthaburi and Narathiwat, respectively. The low prevalence of canine microfilaremia of Samut Sakhon may reflect the success of the Soi Dog foundation's efforts and the establishment of veterinary control programs. An effective disease control and prevention strategies is needed in Chanthaburi and Narathiwat to reduce the risks of zoonotic transmission of the parasites. An appropriate drug treatment should be given to infected dogs and prophylactic drugs are suggested to be given to dogs age ≤1-year-old to prevent filarial infection. The novel microfluidic device could be implemented for surveillance of filariae infection in other animals.

Fabrication of High Aspect Ratio Micro-Structures with Superhydrophobic and Oleophobic Properties by Using Large-Area Roll-to-Plate Nanoimprint Lithography.

Bio-inspired surfaces with superamphiphobic properties are well known as effective candidates for antifouling technology. However, the limitation of large-area mastering, patterning and pattern collapsing upon physical contact are the bottleneck for practical utilization in marine and medical applications. In this study, a roll-to-plate nanoimprint lithography (R2P NIL) process using Morphotonics' automated Portis NIL600 tool was used to replicate high aspect ratio (5.0) micro-structures via reusable intermediate flexible stamps that were fabricated from silicon master molds. Two types of Morphotonics' in-house UV-curable resins were used to replicate a micro-pillar (PIL) and circular rings with eight stripe supporters (C-RESS) micro-structure onto polycarbonate (PC) and polyethylene terephthalate (PET) foil substrates. The pattern quality and surface wettability was compared to a conventional polydimethylsiloxane (PDMS) soft lithography process. It was found that the heights of the R2P NIL replicated PIL and C-RESS patterns deviated less than 6% and 5% from the pattern design, respectively. Moreover, the surface wettability of the imprinted PIL and C-RESS patterns was found to be superhydro- and oleophobic and hydro- and oleophobic, respectively, with good robustness for the C-RESS micro-structure. Therefore, the R2P NIL process is expected to be a promising method to fabricate robust C-RESS micro-structures for large-scale anti-biofouling application.

Formation of double emulsion micro-droplets in a microfluidic device using a partially hydrophilic-hydrophobic surface.

The objective of this paper is to propose a surface modification method for preparing PDMS microfluidic devices with partially hydrophilic-hydrophobic surfaces for generating double emulsion droplets. The device is designed to be easy to use without any complicated preparation process and also to achieve high droplet encapsulation efficiency compared to conventional devices. The key component of this preparation process is the permanent chemical coating for which the Pluronic surfactant is added into the bulk PDMS. The addition of Pluronic surfactant can modify the surface property of PDMS from a fully hydrophobic surface to a partially hydrophilic-hydrophobic surface whose property can be either hydrophilic or hydrophobic depending on the air- or water-treatment condition. In order to control the surface wettability, this microfluidic device with the partially hydrophilic-hydrophobic surface undergoes water treatment by injecting deionized water into the specific microchannels where their surface property changes to hydrophilic. This microfluidic device is tested by generating monodisperse water-in-oil-in-water (w/o/w) double emulsion micro-droplets for which the maximum droplet encapsulation efficiency of 92.4% is achieved with the average outer and inner diameters of 75.0 and 57.7 µm, respectively.

A Potential Application of Triangular Microwells to Entrap Single Cancer Cells: A Canine Cutaneous Mast Cell Tumor Model.

Cellular heterogeneity is a major hindrance, leading to the misunderstanding of dynamic cell biology. However, single cell analysis (SCA) has been used as a practical means to overcome this drawback. Many contemporary methodologies are available for single cell analysis; among these, microfluidics is the most attractive and effective technology, due to its advantages of low-volume specimen consumption, label-free evaluation, and real-time monitoring, among others. In this paper, a conceptual application for microfluidic single cell analysis for veterinary research is presented. A microfluidic device is fabricated with an elastomer substrate, polydimethylsiloxane (PDMS), under standard soft lithography. The performance of the microdevice is high-throughput, sensitive, and user-friendly. A total of 53.1% of the triangular microwells were able to trap single canine cutaneous mast cell tumor (MCT) cells. Of these, 38.82% were single cell entrapments, while 14.34% were multiple cell entrapments. The ratio of single-to-multiple cell trapping was high, at 2.7:1. In addition, 80.5% of the trapped cells were viable, indicating that the system was non-lethal. OCT4A-immunofluorescence combined with the proposed system can assess OCT4A expression in trapped single cells more precisely than OCT4A-immunohistochemistry. Therefore, the results suggest that microfluidic single cell analysis could potentially reduce the impact of cellular heterogeneity.

Investigation of Leukocyte Viability and Damage in Spiral Microchannel and Contraction-Expansion Array.

Inertial separation techniques in a microfluidic system have been widely employed in the field of medical diagnosis for a long time. Despite no requirement of external forces, it requires strong hydrodynamic forces that could potentially cause cell damage or loss during the separation process. This might lead to the wrong interpretation of laboratory results since the change of structures and functional characteristics of cells due to the hydrodynamic forces that occur are not taken into account. Therefore, it is important to investigate the cell viability and damage along with the separation efficacy of the device in the design process. In this study, two inertial separation techniques-spiral microchannel and contraction-expansion array (CEA)-were examined to evaluate cell viability, morphology and intracellular structures using a trypan blue assay (TB), Scanning Electron Microscopy (SEM) and Wright-Giemsa stain. We discovered that cell loss was not significantly found in a feeding system, i.e., syringe, needle and tube, but mostly occurred in the inertial separation devices while the change of cell morphology and intracellular structures were found in the feeding system and inertial separation devices. Furthermore, percentage of cell loss was not significant in both devices (7-10%). However, the change of cell morphology was considerably increased (30%) in spiral microchannel (shear stress dominated) rather than in CEA (12%). In contrast, the disruption of intracellular structures was increased (14%) in CEA (extensional and shear stress dominated equally) rather than spiral microchannel (2%). In these experiments, leukocytes of canine were used as samples because their sizes are varied in a range between 7-12 µm, and they are commonly used as a biomarker in many clinical and medical applications.

Diagnosis of feline filariasis assisted by a novel semi-automated microfluidic device in combination with high resolution melting real-time PCR.

BACKGROUND: The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. METHODS: The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were 'spiked' with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. RESULTS: In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi, 15 as Dirofilaria immitis and 7 as| D. repens. CONCLUSIONS: We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

Chemiluminescence detection with microfluidics for innovative in situ measurement of unbound cobalt ions in dynamic equilibrium with bound ions in binding study with polyethyleneimine and its functionalized nanoparticles.

This work reports a novel method for in situ measurement of binding of cobalt ions to polyethyleneimine (PEI) and polyethyleneimine-functionalized poly (methyl methacrylate) nanoparticles (PEI-NPs) using simple microfluidics with a chemiluminescence detection system. The catalytic effect of free cobalt ion in solution on the luminol-hydrogen peroxide chemiluminescence was employed for the detection of unbound cobalt in dynamic equilibrium with cobalt bound to PEI or PEI-NPs. Many binding measurements lead to incorrect estimation of free metal ions due to insufficient separation of bound and free ions. The catalytic activity of only unbound cobalt ion on the luminol reaction was demonstrated by observing that PEI and PEI-NPs alone did not give chemiluminescence. Also, both Co-PEI and Co-PEI-NPs complexes gave no chemiluminescence when cobalt ion is fully bound with excess PEI or PEI-NPs. In addition diethylenetriamine (dien) as a model ligand to completely bind the cobalt ions was also employed as further confirmation. The chemiluminescence measurement employing microfluidics was then successfully applied for the measurement of binding cobalt ion to PEI and PEI-NPs. This in situ measurement of binding does not require filtration of the two species. As there is no perturbation of equilibrium, an accurate binding measurement can therefore be successfully performed. Experimental parameters, such as concentrations of polymers and cobalt ions, and equilibration time were investigated. Analysis of the experimental data employed the binding equation derived assuming independent and equivalent binding sites of the polymer for the metal ions. Also the binding constant of cobalt ions with PEI-NPs is first reported employing chemiluminescence detection. This work provides quantitative determination of the binding constant and total binding capacity of PEI and PEI-NPs with cobalt ions using chemiluminescence detection and microfluidics as an innovative in situ measurement of the unbound cobalt ions.

The Viability of Single Cancer Cells after Exposure to Hydrodynamic Shear Stresses in a Spiral Microchannel: A Canine Cutaneous Mast Cell Tumor Model.

Our laboratory has the fundamental responsibility to study cancer stem cells (CSC) in various models of human and animal neoplasms. However, the major impediments that spike our accomplishment are the lack of universal biomarkers and cellular heterogeneity. To cope with these restrictions, we have tried to apply the concept of single cell analysis, which has hitherto been recommended throughout the world as an imperative solution pack for resolving such dilemmas. Accordingly, our first step was to utilize a predesigned spiral microchannel fabricated by our laboratory to perform size-based single cell separation using mast cell tumor (MCT) cells as a model. However, the impact of hydrodynamic shear stresses (HSS) on mechanical cell injury and viability in a spiral microchannel has not been fully investigated so far. Intuitively, our computational fluid dynamics (CFD) simulation has strongly revealed the formations of fluid shear stress (FSS) and extensional fluid stress (EFS) in the sorting system. The panel of biomedical assays has also disclosed cell degeneration and necrosis in the model. Therefore, we have herein reported the combinatorically detrimental effect of FSS and EFS on the viability of MCT cells after sorting in our spiral microchannel, with discussion on the possibly pathogenic mechanisms of HSS-induced cell injury in the study model.

A silicon nitride ISFET based immunosensor for Ag85B detection of tuberculosis.

A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 µg ml-1. This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 µg ml-1 without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization.

Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface.