a-droplet-based-microfluidic-approach-microsphere-pcr-amplification

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

microfluidic-acoustophoresis-for-flowthrough-separation-gram-negative

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and ...

pneumatically-driven-microfluidic-platform-for-micro-particle

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Inje University

Micro device Lab Co., Ltd

inje university

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL(-1), 80 pg mL(-1), and 800 fg mL(-1) when arraying the PSA antibody, H117 at the concentration 15 μg mL(-1), 35 μg mL(-1), and 154 μg mL(-1), respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL(-1) to 500 ng mL(-1). The microarray showed a LOD of 800 fg mL(-1) and a dynamic range of 800 fg mL(-1) to 80 ng mL(-1) in serum spiked samples.

Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody.

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

We fabricated droplet-based microfluidic platform for copolymerizable microspheres with acrydite modified DNA probe. The copolymerizable 3-D polyacrylamide microspheres were successfully produced from microcontinuous-flow synthesis with on-channel solidification. DNA copolymerization activity, surface presentation and thermostability were assessed by using fluorescent labeled complementary probe. The binding performance was only visible on the surface area of oligo-microspheres. We show that the resulting oligo-microspheres can be directly integrated into a streamlined microsphere-PCR protocol for amplifying ssDNA. Our microspheres could be utilized as a potential material for ssDNA analysis such as DNA microarray and automatic DNA SELEX process. 

On-Flow Synthesis of Co-Polymerizable Oligo-Microspheres and Application in ssDNA Amplification.

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 10(6) CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 10(4) CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 10(4) CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats.

Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate?specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol?gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.

A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen.

Spontaneous wrinkling of a polydimethylsiloxane (PDMS) surface was induced by repeated thermal shrinkage of liquid PDMS coated onto a cured PDMS layer. We investigated and evaluated the potential of the resulting surface as a cell culture substrate by monitoring the viability, spreading area, and proliferation rate of MG-63 cells cultured on native, wrinkled, and poly-L-lysine (PLL)-coated PDMS surfaces. Cells seeded on the wrinkled and PLL-coated PDMS surfaces spread and adhered better than those on native surfaces. The numbers of attached cells growing on wrinkled and PLL-coated PDMS surfaces were higher than those of cells on a native PDMS surface. The spreading area of cells on the wrinkled surface was similar to that of cells on the PLL-coated surface, and was much larger than that on native PDMS. The proliferation rate of cells on the wrinkled surface was more than double that of cells on native PDMS. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of integrin mRNA expression showed that cells on the wrinkled surface were more tightly attached due to higher expression of the protein than exhibited in cells on native PDMS. Thus, the novel findings of this study are that the induction of a wrinkled PDMS surface through a simple curing process produces a suitable cell culture substrate without need of surface modification, and that its effectiveness is comparable to that of a PLL-coated PDMS surface.

Inducement of a spontaneously wrinkled polydimethylsiloxane surface and its potential as a cell culture substrate.

Apple Scar Skin Viroid (ASSVd), a nonprotein coding, circular RNA pathogen is relatively difficult to detect by immunoassay. We report here a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to improve selectivity for diagnostic use in detecting ASSVd in plants. ASSVd RT-LAMP was accelerated using loop primers and was found to be highly sensitive with a detection limit of 10 copies of cDNA-ASSVd within 30 min. Real-time LAMP and melting curve analysis could differentiate between the true-positive LAMP amplicons and false-positive nonspecific primer amplification products. The optimized RT-LAMP was then followed by the addition of nonthiolated AuNP:poly-adenine (A10)-ASSVd LAMP barcodes, showing a high authentication capacity with colorimetric changes. This type of barcoding assay is a potential alternative for rapid and multiple viroid diagnosis, providing for visible sensing in the field that can be applied to viroid-free planting.

Poly-adenine-Coupled LAMP Barcoding to Detect Apple Scar Skin Viroid.

Here, we report a simple and effective method for capturing and displacement of gram-negative bacteria using aptamer-modified microbeads and acoustophoresis. As acoustophoresis allows for simultaneous washing and size-dependent separation in continuous flow mode, we efficiently obtained gram-negative bacteria that showed high affinity without any additional washing steps. The proposed device has a simple and efficient channel design, utilizing a long, square-shaped microchannel that shows excellent separation performance in terms of the purity, recovery, and concentration factor. Microbeads (10 µm) coated with the GN6 aptamer can specifically bind gram-negative bacteria. After incubation of bacteria culture sample with aptamer affinity bead, gram-negative bacteria-bound microbeads, and other unbound/contaminants can be separated by size with high purity and recovery. The device demonstrated excellent separation performance, with high recovery (up to 98%), high purity (up to 99%), and a high-volume rate (500 µL/min). The acoustophoretic separation performances were conducted using 5 Gram-negative bacteria and 5 Gram-positive bacteria. Thanks to GN6 aptamer's binding affinity, aptamer affinity bead also showed binding affinity to multiple strains of gram-negative bacteria, but not to gram-positive bacteria. GN6 coated bead can capture Gram-negative bacteria but not Gram-positive bacteria. This study may present a different perspective in the field of early diagnosis in bacterial infectious diseases. In addition to detecting living bacteria or bacteria-derived biomarkers, this protocol can be extended to monitoring the contamination of water resources and may aid quick responses to bioterrorism and pathogenic bacterial infections.

Aptamer Affinity-Bead Mediated Capture and Displacement of Gram-Negative Bacteria Using Acoustophoresis.

We developed a microfluidic platform employing (normally open) pneumatic valves for particle concentration. The device features a three-dimensional network with a curved fluidic channel and three pneumatic valves (a sieve valve (Vs) that concentrates particles and two ON/OFF rubber-seal pneumatic valves that block the working fluid). Double-sided replication employing polydimethylsiloxane (PDMS) was used to fabricate the network, channel, and chamber. Particles were blocked by deformation of the Vs diaphragm, and then accumulated in the curved microfluidic channel. The working fluid was discharged via operation of the two ON/OFF valves. After concentration, particles were released to an outlet port. The Vs pressure required to block solid particles varying in diameter was determined based on the height of the curved microchannel and a finite element method (FEM) simulation of Vs diaphragm displacement. Our method was verified according to the temporal response of the fluid flow rate controlled by the pneumatic valves. Furthermore, all particles with various diameters were successfully blocked, accumulated, and released. The operating pressure, time required for concentration, and concentration ratio were dependent on the particle diameter. The estimated concentration percentage of 24.9 µm diameter polystyrene particles was about 3.82% for 20 min of operation.

Fabrication of a Pneumatic Microparticle Concentrator.

Flow cytometry has become an indispensable tool for counting, analyzing, and sorting large cell populations in biological research and medical practice. Unfortunately, it has limitations in the analysis of non-spherically shaped cells due to the variation of their alignment with respect to the flow direction and, hence, the optical interrogation axis, resulting in unreliable cell analysis. Here, we present a simple on-chip acoustofluidic method to fix the orientation of ellipsoidal cells and focus them into a single, aligned stream. Specifically, by generating acoustic standing waves inside a 100 ⋅ 100 µm square-shaped microchannel, we successfully aligned and focused up to 97.7% of a population of Euglena gracilis (an ellipsoidal shaped microalgal species) cells in the center of the microchannel with high precision at a volume rate of 25 to 200 µL min. Uniform positioning of ellipsoidal cells is essential for making flow cytometry applicable to the investigation of a greater variety of cell populations and is expected to be beneficial for ecological studies and aquaculture.

Ok Chan Jeong

Department of Biomedical Engineering,
Institute of Digital Anti-Aging Healthcare,
Department of Digital Anti-Aging Health Care

Inje University

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

Ok Chan Jeong

Department of Biomedical Engineering,Institute of Digital Anti-Aging Healthcare,Department of Digital Anti-Aging Health Care

Inje University

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

Department of Biomedical Engineering,
Institute of Digital Anti-Aging Healthcare,
Department of Digital Anti-Aging Health Care