Showing papers by "Gwo-Bin Lee published in 2018"

A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis

Abstract: Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that rapidly amplifies specific DNA molecules at high yield. In this study, a microfluidic droplet array chip was designed to execute the digital LAMP process. The novel device was capable of 1) creating emulsion droplets, 2) sorting them into a 30 × 8 droplet array, and 3) executing LAMP across the 240 trapped and separated droplets (with a volume of 0.22 nL) after only 40 min of reaction at 56 °C. Nucleic acids were accurately quantified across a dynamic range of 50 to 2.5 × 103 DNA copies per μL, and the limit of detection was a single DNA molecule. This is the first time that an arrayed emulsion droplet microfluidic device has been used for digital LAMP analysis. When compared to microwell digital nucleic acid amplification assays, this droplet array-based digital LAMP assay eliminates the constraint on the size of the digitized target, which was determined by the dimension of the microwells for its counterparts. Moreover, the capacity for hydrodynamic droplet trapping allows the chip to operate in a one-droplet-to-one-trap manner. This microfluidic chip may therefore become a promising device for digital LAMP-based diagnostics in the near future.

Detecting miRNA biomarkers from extracellular vesicles for cardiovascular disease with a microfluidic system

Abstract: According to World Health Organization reports, cardiovascular diseases (CVDs) are amongst the major causes of death globally and are responsible for over 18 million deaths every year. Traditional detection methods for CVDs include cardiac computerized tomography scans, electrocardiography, and myocardial perfusion imaging scans. Although diagnosis of CVDs through such bio-imaging techniques is common, these methods are relatively costly and cannot detect CVDs in their earliest stages. In contrast, the levels of certain micro RNA (miRNA) biomarkers extracted from extracellular vesicles (EVs) in the bloodstream have been recognized as promising indicators for early CVD detection. However, detection and quantification of miRNA using existing methods are relatively labor-intensive and time-consuming. In this study, a new integrated microfluidic system equipped with highly sensitive field-effect transistors (FETs) was capable of performing EV extraction, EV lysis, target miRNA isolation and miRNA detection within 5 h. The limit of detection was within the physiological range (femtomolar) for two targeted miRNAs, miR-21 and miR-126, meaning that this integrated microfluidic system has the potential to be used as a tool for early detection of CVDs.

Comparison of molecular diagnosis with serum markers and synovial fluid analysis in patients with prosthetic joint infection.

Abstract: Aims The aim of this study was to compare the results of 16S/28S rRNA sequencing with the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and synovial fluid analysis in the di...

Enumeration of circulating tumor cells and investigation of cellular responses using aptamer-immobilized AlGaN/GaN high electron mobility transistor sensor array

Abstract: This study reports the fabrication and characterization of electrical double layer gated AlGaN/GaN High electron mobility (HEMT) biosensor array to capture, detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC). GaN HEMT chips were assembled into a sensor array on a thermo-curable polymer substrate in a simple and robust packaging process. The array had multiple aptamer immobilized sensing sites and was highly sensitive and selective with up to single cell resolution. The present method can detect cells in their native electrolyte composition, in a very short time with extremely low cost compared to the conventional circulating tumor cell assays. The effect of cellular binding in different test environments was further studied and reported. The electrical response of the sensor was discovered to be relevant to the transmembrane potential of the cell. This whole cell sensor array has the potential to be used as a point-of-care diagnostic tool in varied applications such as detection of rare cells, pathogens and studying the dynamics of cellular interactions.

A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers.

Abstract: Bacteria such as Acinetobacter baumannii (AB) can cause serious infections, resulting in high mortality if not diagnosed early and treated properly; there is consequently a need for rapid and accurate detection of this bacterial species. Therefore, we developed a new, nitrocellulose-based microfluidic system featuring AB-specific aptamers capable of automating the bacterial detection process via the activity of microfluidic devices composed of magnetic-composite membranes. Electromagnets were used to actuate these microfluidic devices such that the entire diagnostic process could be conducted in the integrated microfluidic system within 40 minutes with a limit of detection as low as 450 CFU per reaction for AB. Aptamers were used to capture AB in complex samples on nitrocellulose membranes, and a simple colorimetric assay was used to estimate bacterial loads. Given the ease of use, portability, and sensitivity of this aptamer-based microfluidic system, it may hold great promise for point-of-care diagnostics.

Direct detection of DNA using electrical double layer gated high electron mobility transistor in high ionic strength solution with high sensitivity and specificity

Abstract: In this research, we have realized an electrical double layer (EDL) gated high electron mobility transistor (HEMT) as DNA sensor. The sensing area on the gate electrode which is separated from the transistor channel is immobilized with probe DNA to capture target DNA from physiological salt environment. The detection limit of the sensor can be as low as 1 fM with very high sensitivity. The specificity of the DNA sensor is also demonstrated by controlling the hybridization temperature. By choosing the hybridization temperature slightly lower than the melting temperature of the well-matched sequence, the binding ratio can be controlled between the fully-matched and mismatched one. The sensor has demonstrated specificity, with the ability to achieve single base mismatch resolution. The sensor has the potential for rapid DNA sensing applications in cells, biomarkers and viruses.

Dynamic monitoring of transmembrane potential changes: a study of ion channels using an electrical double layer-gated FET biosensor.

Abstract: In this research, we have designed, fabricated and characterized an electrical double layer (EDL)-gated AlGaN/GaN high electron mobility transistor (HEMT) biosensor array to study the transmembrane potential changes of cells. The sensor array platform is designed to detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC) and investigate cellular bioelectric signals. Using the EDL FET biosensor platform, cellular responses can be studied in physiological salt concentrations, thereby eliminating complex automation. Upon investigation, we discovered that our sensor response follows the transmembrane potential changes of captured cells. Our whole cell sensor platform can be used to monitor the dynamic changes in the membrane potential of cells. The effects of continuously changing electrolyte ion concentrations and ion channel blocking using cadmium are investigated. This methodology has the potential to be used as an electrophysiological probe for studying ion channel gating and the interaction of biomolecules in cells. The sensor can also be a point-of-care diagnostic tool for rapid screening of diseases.

Automatic cell fusion via optically-induced dielectrophoresis and optically-induced locally-enhanced electric field on a microfluidic chip.

Abstract: Cell fusion technology has been exploited in a wide variety of biomedical applications, and physical, chemical, and biological approaches can all be used to fuse two different types of cells; however, no current technique is adept at inducing both cell pairing and fusion at high efficiencies and yields. Hence, we developed a new method featuring the use of optically induced dielectrophoresis (ODEP) in conjunction with an optically induced, locally enhanced electric field for accurate and automatic cell pairing and fusion on a microfluidic device. After pairing cells via ODEP, a locally enhanced electric field generated by “virtual electrodes” by projecting light patterns was enacted to induce a proper transmembrane potential at the cell contact area such that cell fusion could be triggered by white light exposure. As a fusion yield of 9.67% was achieved between Pan1 and A549 cells, we believe that this may be a promising technique for automatically fusing different cell types.

Microfluidic platforms for rapid screening of cancer affinity reagents by using tissue samples

Abstract: Cancer is the most serious disease worldwide, and ovarian cancer (OvCa) is the second most common type of gynecological cancer. There is consequently an urgent need for early-stage detection of OvCa, which requires affinity reagent biomarkers for OvCa. Systematic evolution of ligands by exponential enrichment (SELEX) and phage display technology are two powerful technologies for identifying affinity reagent biomarkers. However, the benchtop protocols for both screening technologies are relatively lengthy and require well-trained personnel. We therefore developed a novel, integrated microfluidic system capable of automating SELEX and phage display technology. Instead of using cancer cell lines, it is the first work which used tissue slides as screening targets, which possess more complicated and uncovered information for affinity reagents to recognize. This allowed for the identification of aptamer (nucleic acid) and peptide probes specific to OvCa cells and tissues. Furthermore, this developed system could be readily modified to uncover affinity reagents for diagnostics or even target therapy of other cancer cell types in the future.

Visible light induced electropolymerization of suspended hydrogel bioscaffolds in a microfluidic chip.

Abstract: The development of microengineered hydrogels co-cultured with cells in vitro could advance in vivo bio-systems in both structural complexity and functional hierarchy, which holds great promise for applications in regenerative tissues or organs, drug discovery and screening, and bio-sensors or bio-actuators. Traditional hydrogel microfabrication technologies such as ultraviolet (UV) laser or multiphoton laser stereolithography and three-dimensional (3D) printing systems have advanced the development of 3D hydrogel micro-structures but need either expensive and complex equipment, or harsh material selection with limited photoinitiators. Herein, we propose a simple and flexible hydrogel microfabrication method based on a ubiquitous visible-light projection system combined with a custom-designed photosensitive microfluidic chip, to rapidly (typically several to tens of seconds) fabricate various two-dimensional (2D) hydrogel patterns and 3D hydrogel constructs. A theoretical layer-by-layer model that involves continuous polymerizing-delaminating-polymerizing cycles is presented to explain the polymerization and structural formation mechanism of hydrogels. A large area of hydrogel patterns was efficiently fabricated without the usage of costly laser systems or photoinitiators, i.e., a stereoscopic mesh-like hydrogel network with intersecting hydrogel micro-belts was fabricated via a series of dynamic-changing digital light projections. The pores and gaps of the hydrogel network are tunable, which facilitates the supply of nutrients and discharge of waste in the construction of 3D thick bio-models. Cell co-culture experiments showed the effective regulation of cell spreading by hydrogel scaffolds fabricated by the new method presented here. This visible light enabled hydrogel microfabrication method may provide new prospects for designing cell-based units for advanced biomedical studies, e.g., for 3D bio-models or bio-actuators in the future.

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Abstract: Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.

An automatic integrated microfluidic system for allergy microarray chips

Abstract: Billions of people suffer from allergies, though in many cases, the source allergen is unknown. If one knows which allergens to avoid, this would result in an improved quality of life. Since a rapid, high-throughput, automatic allergen detection method is of great need, an integrated system combining microfluidic techniques and microarray chips has been developed herein to automate the allergen detection process. The developed microfluidic system could automatically carry out the entire procedure such as reagent incubation, hybridization, transport, and washing without any intermediate step. The microarray chip could be easily detached from the microfluidic chip afterwards, enabling it to be read under a fluorescence scanner. The experimental results indicated that the developed microfluidic system can automatically perform all the incubation processes, including hybridization, reagent transportation, and washing. It is worth noting that active mixing has been applied in the present study which is different from our previous study using micro-channels for passive incubation. Comparable results to a conventional benchtop approach were obtained in ∼30% less time with ∼25% less samples/reagents. Similar results were also demonstrated while detecting immunoglobulin E samples. The developed system could therefore provide a rapid, reliable, and automated approach for detecting allergen-specific antibodies in human serum.

Rapid Assembly of Carbon Nanoparticles Into Electrical Elements by Optically-Induced Electroosmotic Flow

Abstract: We report the rapid assembly of carbon nanoparticles (CNPs) into functional electrical elements utilizing optically induced electroosmotic flow (OIEF) in a specialized microfluidics chip Numerical simulations of various optically induced electrokinetics forces exerted on CNPs were initially performed to ascertain the viable forces that could overcome the Brownian motion and produce sufficient force to manipulate and assemble the CNPs The results confirmed the theoretical prediction that only the force induced by OIEF could manipulate the CNPs Subsequently, a series of experiments for assembling CNPs were conducted This was followed by electrical characterization of the assembled three-dimensional CNP microstructures The results proved that OIEF could effectively and rapidly assemble CNPs in about 45 s We used measurements of the current–voltage relationship to validate that the CNP-based microstructures are resistive elements, and their resistance could be controlled by the width and length of the microstructures We believe that, by using the OIEF technique, different nanoparticles of varying electrical properties could be assembled rapidly in a specialized microfluidics chip to create micro-electrical elements in the future, including integrating different nanoparticle elements into functional electrical devices

Automatic microfluidic system for antibiotic susceptibility testing and method of operating thereof

Abstract: An automatic microfluidic system for antibiotic susceptibility testing of the present disclosure at least includes a microfluidic chip. The microfluidic chip includes a fluid storage unit, a reaction unit, a pneumatic micro-pumping unit and a plurality of valve units. The fluid storage unit is provided for storing a bacterial suspension, a broth and an antibiotic solution. The reaction unit includes a first reaction chamber and at least two second reaction chambers. The pneumatic micro-pumping unit is adjacently disposed to the fluid storage unit and the reaction unit for selectively, repeatedly and quantitatively transporting the broth, the bacterial suspension and the antibiotic solution to the reaction unit to form a first mixing solution and at least two second mixing solutions. The valve units include a plurality of pneumatic micro-valves and a plurality of valve control air holes for controlling the opening and closing of the pneumatic micro-valves.

Integrated microfluidic system with field effect transistor for automatic detection of multiple cardiovascular biomarkers

Abstract: This study presents an integrated microfluidic platform equipped with a field-effect-transistor sensor array which is capable of performing analysis beyond Debye length and detecting protein biomarkers associated with cardiovascular diseases from clinical samples within a short period of time. This work also demonstrates a prototype for future point-of-care device on which antibodies could be replaced by highly specific aptamers. In order to demonstrate the capability of the developed device, four cardiovascular biomarkers (CRP, NT-proBNP, troponin I, fibrinogen) were chosen and their physiological as well as risk concentrations were analyzed simultaneously in a single chip.

Aptamer-functionalized AlGaN/GaN High-electron-mobility Transistor for Rapid Diagnosis of Fibrinogen in Human Plasma

Abstract: 1Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C. 2Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C. 3Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C. 4Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan City 701, Taiwan, R.O.C.

An integrated microfluidic system for identification of live mycobacterium tuberculosis by real-time polymerase chain reaction

Abstract: Mycobacterium tuberculosis (TB) infection poses a huge challenge worldwide. It is challenging for rapid identification of TB due to its low growth rate, and therefore it could be widely spread. For this reason, a novel combination of propidium monoazide (PMA) and real-time polymerase chain reaction (RT-PCR) was reported in this work for fast detection of live TB. Experimental results showed that the PMA-RT-PCR protocol can detect live TB with a detection limit as low as 14 colony formation units (CFU)/reaction by measuring the threshold cycle number (Ct value) in a calibration curve. Furthermore, PMA could be used successfully to distinguish dead TB such that no PCR products could be generated. More importantly, all the procedure could be finished within 2 hours. This is the first time that an integrated microfluidic system was demonstrated to use molecular diagnostics for fast detection of TB and could be potentially used for prognosis monitoring of TB treatment.

Thermometry of photosensitive and optically induced electrokinetics chips

Abstract: Optically induced electrokinetics (OEK)-based technologies, which integrate the high-resolution dynamic addressability of optical tweezers and the high-throughput capability of electrokinetic forces, have been widely used to manipulate, assemble, and separate biological and non-biological entities in parallel on scales ranging from micrometers to nanometers. However, simultaneously introducing optical and electrical energy into an OEK chip may induce a problematic temperature increase, which poses the potential risk of exceeding physiological conditions and thus inducing variations in cell behavior or activity or even irreversible cell damage during bio-manipulation. Here, we systematically measure the temperature distribution and changes in an OEK chip arising from the projected images and applied alternating current (AC) voltage using an infrared camera. We have found that the average temperature of a projected area is influenced by the light color, total illumination area, ratio of lighted regions to the total controlled areas, and amplitude of the AC voltage. As an example, optically induced thermocapillary flow is triggered by the light image-induced temperature gradient on a photosensitive substrate to realize fluidic hydrogel patterning. Our studies show that the projected light pattern needs to be properly designed to satisfy specific application requirements, especially for applications related to cell manipulation and assembly.

Non-UV Patterning of Gelatin Methacryloyl Hydrogel by Optically Induced Electropolymerization

Abstract: The microenvironment for culturing of cells is important in tissue engineering and biomedical applications. Owing to their excellent biocompatibility, hydrogels are widely used to create microenvironments. One of the most useful hydrogels, gelatin methacryloyl (GeIMA), can be cured by ultraviolet (UV) light to form a polymer. However, the use of a photoinitiator in this process results in cellular toxicity. In this study, we developed a novel method to polymerize GelMA hydrogel into desired patterns based on the principle of optically induced electropolymerization. For this technique, the polymer films were electrodeposited by optical virtual electrodes at the surface of a photoconductive substrate, instead of real conductive metallic electrodes. The shapes of the virtual electrodes depend on digitally projected images. The thickness of the deposited films, ranging from nanometers to micrometers, is controlled by the duration of the applied AC voltage. In this paper, we discuss the parameters used during the optically induced electropolymerization process to realize several microstructures of GelMA hydrogel with different shapes and sizes.

Automatic and rapid antimicrobial susceptibility test on an integrated microfluidic device

Abstract: This study presented an integrated microfluidic device which can perform rapid antimicrobial susceptibility tests (AST) of antibiotics combination against clinical bacteria. A new approach to measure either drug interactions of three antibiotics or minimum inhibitory concentration (MIC) of antibiotics by performing bacteria dispensation and antibiotics dilution automatically was proposed and it substantially reduced detection time (from 24 hr to 4 hr) and bacteria consumption (from 100 μL to 3 μL) when compared to the broth microdilution method. A new membrane-type micromixer composed of two circular micropumps was integrated in order to achieve on-chip pumping/mixing with high accuracy and efficiency. Experimental results revealed that the proposed device can produce accurate liquid transportation and chaotic mixing and visually interpret AST results including individual MIC determination and drug interactions. Synergistic and antagonistic effects of pairwise and triple interactions among three common antibiotics (vancomycin, gentamicin and ceftazidime) with ten different combinations were demonstrated on one clinical isolate of vancomycin-intermediate staphylococcus aureus. It is the first time that a microfluidic device was demonstrated for drug screening to inhibit bacteria growth with three antibiotics by performing AST automatically. The developed device could be utilized as a robust tool to rapidly explore drug screening for drug cocktail therapy.

Detection of micro ribonucleic acids from extracted extracellular vesicles for cardiovascular diseases by using an integrated microfluidic system

Abstract: Recently, micro ribonucleic acids (miRNAs) extracted from extracellular vesicles (EVs) have been recognized as promising biomarkers for early detection of cardiovascular diseases (CVDs). However, the detection and quantification of miRNAs by using the existing methods are relatively labor-intensive and time-consuming. In this work, an integrated microfluidic system equipped with highly sensitive field-effect transistors which is capable of performing EVs extraction, EVs lysis, target miRNAs extraction and miRNAs detection was reported. The entire detection process could be automated within three hours on a single chip and the detection limit of miRNAs was observed to be in a femtomolar range, which meets the requirement of physiological concentrations. This integrated microfluidic system has great potential to be a tool for early detection of CVDs.

An Integrated Microfluidic System for Bacteria Identification from Human Joint Fluids

Abstract: An integrated microfluidic system was developed to identify bacteria in human joint fluid (HJF) in this study. The entire process including bacteria isolation in HJF, bacteria lysis, polymerase chain reaction (PCR) and optical detection could be automated on a single chip. An universal 16S rRNA primer set and four designed primers to identify Staphylococcus aureus (SA), methicillin-resistance Staphylocuccus aureus (MRSA), Escherichia coli (E. coli), and Acinetobacter baumannii $(\pmb{A}.\pmb{B}.)$ were used for bacteria identification. In addition, N-acetyl-L-cysteine for dissolving sticky mucus of HJF samples and vancomycin-coated magnetic beads were applied for bacteria isolation such that the detection limit could be significantly improved. The entire process could be automated by using a microfluidic system. Experimental results showed that the limit of detection (LOD) could be significantly improved to 100 colony formation units (CFU) per milliliter (or 20 CFU/reaction), which meets the demand on clinical diagnosis. Furthermore, the whole process could be performed within 90 minutes, which is much faster when compared to the traditional method (usually 3–7 days).

Aptamer specific to ovarian cancer and detection method for ovarian cancer

Abstract: An aptamer specific to ovarian cancer and a detection method for ovarian cancer are provided. The aptamer includes a following nucleotide sequence: 5′-ncaaannncnnnnanncnnnnnnnnnnngaannnannngg-3′, wherein n is a nucleotide independently selected from “a,” “t,” “c,” and “g.”

Microfluidic in-vitro screening chip system and method of using the same

Abstract: A microfluidic in-vitro screening chip system including a first micromixer chamber, a plurality of first storage chambers, a second micromixer chamber and a plurality of second storage chambers The first micromixer chamber includes a non-target disease tissue slice region The plurality of first storage chambers are connected to the first micromixer chamber, wherein at least one first storage chamber is used for storing a library The second micromixer chamber is connected to the first micromixer chamber, and the second micromixer chamber includes a target disease tissue slice region The plurality of second storage chambers are connected to the second micromixer chamber