Publications by authors named "Wilfred Espulgar"

10 Publications

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A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis.

Sci Rep 2021 Jun 21;11(1):12995. Epub 2021 Jun 21.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve's optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip's throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.
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http://dx.doi.org/10.1038/s41598-021-92472-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217553PMC
June 2021

Deskilled and Rapid Drug-Resistant Gene Detection by Centrifugal Force-Assisted Thermal Convection PCR Device.

Sensors (Basel) 2021 Feb 9;21(4). Epub 2021 Feb 9.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Here we report the improved Cyclo olefin polymer (COP) microfluidic chip and polymerase chain reaction (PCR) amplification system for point-of-care testing (POCT) in rapid detection of Carbapenem-resistant Enterobacteriaceae (CRE). The PCR solution and thermal cycling is controlled by the relative gravitational acceleration (7G) only and is expected to pose minimal problem in operation by non-expert users. Detection is based on identifying the presence of carbapenemase encoding gene through the corresponding fluorescence signal after amplification. For preliminary tests, the device has been demonstrated to detect from patients stool samples. From the prepared samples, 96.4 fg/µL was detected with good certainty within 15 min (~106 thermocycles,) which is significantly faster than the conventional culture plate method. Moreover, the device is expected to detect other target genes in parallel as determination of the presence of and from control samples has also been demonstrated. With the rising threat of drug-resistant bacteria in global healthcare, this technology can greatly aid the health sector by enabling the appropriate use of antibiotics, accelerating the treatment of carriers, and suppressing the spread.
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http://dx.doi.org/10.3390/s21041225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916093PMC
February 2021

study of monocytic THP-1 leukemia cell membrane elasticity with a single-cell microfluidic-assisted optical trapping system.

Biomed Opt Express 2020 Oct 1;11(10):6027-6037. Epub 2020 Oct 1.

OPTICS Research Unit, CENSER, De La Salle University (DLSU), Manila, Philippines.

We studied the elastic profile of monocytic THP-1 leukemia cells using a microfluidic-assisted optical trap. A 2-m fused silica bead was optically trapped to mechanically dent an immobilized single THP-1 monocyte sieved on a 15-µm microfluidic capture chamber. Cells treated with Zeocin and untreated cells underwent RT-qPCR analysis to determine cell apoptosis through gene expression in relation to each cell's deformation profile. Results showed that untreated cells with 43.05 ± 6.68 Pa are more elastic compared to the treated cells with 15.81 ± 2.94 Pa. THP-1 monocyte's elastic modulus is indicative of cell apoptosis shown by upregulated genes after Zeocin treatment. This study clearly showed that the developed technique can be used to distinguish between cells undergoing apoptosis and cells not undergoing apoptosis and which may apply to the study of other cells and other cell states as well.
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http://dx.doi.org/10.1364/BOE.402526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587289PMC
October 2020

The future of microfluidics in immune checkpoint blockade.

Cancer Gene Ther 2021 Sep 27;28(9):895-910. Epub 2020 Oct 27.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.

Recent advances in microfluidic techniques have enabled researchers to study sensitivities to immune checkpoint therapy, to determine patients' response to particular antibody treatment. Utilization of this technology is helpful in antibody discovery and in the design of personalized medicine. A variety of microfluidic approaches can provide several functions in processes such as immunologic, genomic, and/or transcriptomic analysis with the aim of improving the efficacy and coverage of immunotherapy, particularly immune checkpoint blockade (ICB). To achieve this requires researchers to overcome the challenges in the current state of the technology. This review looks into the advancements in microfluidic technologies applied to researches on immune checkpoint blockade treatment and its potential shift from proof-of-principle stage to clinical application.
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http://dx.doi.org/10.1038/s41417-020-00248-7DOI Listing
September 2021

Real-Time Monitoring and Detection of Single-Cell Level Cytokine Secretion Using LSPR Technology.

Micromachines (Basel) 2020 Jan 19;11(1). Epub 2020 Jan 19.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Cytokine secretion researches have been a main focus of studies among the scientists in the recent decades for its outstanding contribution to clinical diagnostics. Localized surface plasmon resonance (LSPR) technology is one of the conventional methods utilized to analyze these issues, as it could provide fast, label-free and real-time monitoring of biomolecule binding events. However, numerous LSPR-based biosensors in the past are usually utilized to monitor the average performance of cell groups rather than single cells. Meanwhile, the complicated sensor structures will lead to the fabrication and economic budget problems. Thus, in this paper, we report a simple synergistic integration of the cell trapping of microwell chip and gold-capped nanopillar-structured cyclo-olefin-polymer (COP) film for single cell level Interleukin 6 (IL-6) detection. Here, in-situ cytokine secreted from the trapped cell can be directly observed and analyzed through the peak red-shift in the transmittance spectrum. The fabricated device also shows the potential to conduct the real-time monitoring which would greatly help us identify the viability and biological variation of the tested single cell.
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http://dx.doi.org/10.3390/mi11010107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019717PMC
January 2020

Single Cell Analysis of Neutrophils NETs by Microscopic LSPR Imaging System.

Micromachines (Basel) 2019 Dec 31;11(1). Epub 2019 Dec 31.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.

A simple microengraving cell monitoring method for neutrophil extracellular traps (NETs) released from single neutrophils has been realized using a polydimethylsiloxane (PDMS) microwell array (MWA) sheet on a plasmon chip platform. An imbalance between NETs formation and the succeeding degradation (NETosis) are considered associated with autoimmune disease and its pathogenesis. Thus, an alternative platform that can conduct monitoring of this activity on single cell level at minimum cost but with great sensitivity is greatly desired. The developed MWA plasmon chips allow single cell isolation of neutrophils from 150 µL suspension (6.0 × 10 cells/mL) with an efficiency of 36.3%; 105 microwells with single cell condition. To demonstrate the utility of the chip, trapped cells were incubated between 2 to 4 h after introducing with 100 nM phorbol 12-myristate 13-acetate (PMA) before measurement. Under observation using a hyperspectral imaging system that allows high-throughput screening, the neutrophils stimulated by PMA solution show a significant release of fibrils and NETs after 4 h, with observed maximum areas between 314-758 µm. An average absorption peak wavelength shows a redshift of Δλ = 1.5 nm as neutrophils release NETs.
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http://dx.doi.org/10.3390/mi11010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019790PMC
December 2019

A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling.

Theranostics 2020 1;10(1):123-132. Epub 2020 Jan 1.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, JAPAN.

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. : A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. : GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. : As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
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http://dx.doi.org/10.7150/thno.37728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929635PMC
April 2021

Utility of Centrifugation-Controlled Convective (C3) Flow for Rapid On-chip ELISA.

Sci Rep 2019 12 27;9(1):20150. Epub 2019 Dec 27.

Department of Applied Physics, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Osaka, Japan.

Miniaturizing the enzyme-linked immunosorbent assay (ELISA) protocols in microfluidics is sought after by researchers for a rapid, high throughput screening, on-site diagnosis, and ease in operation for detection and quantification of biomarkers. Herein, we report the use of the centrifugation-controlled convective (C3) flow as an alternative method in fluid flow control in a ring-structured channel for enhanced on-chip ELISA. A system that consists of a rotating heater stage and a microfluidic disk chip has been developed and demonstrated to detect IgA. The ring-structured channel was partially filled with microbeads (250 µm in diameter) carrying the capture antibodies and the analyte solution was driven by thermal convection flow (50 µL/min) to promote the reaction. The remaining part of the circular channel without microbeads served as the observation area to measure the absorbance value of the labeled protein. Currently, the system is capable of conducting four reactions in parallel and can be performed within 30 min at 300 G. A detection limit of 6.16 ng/mL using 24 µL of target sample (IgA) was observed. By simply changing the capture antibodies, the system is expected to be versatile for other immunoassays.
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http://dx.doi.org/10.1038/s41598-019-56772-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934823PMC
December 2019

Centrifugation-Controlled Thermal Convection and Its Application to Rapid Microfluidic Polymerase Chain Reaction Devices.

Anal Chem 2017 Dec 17;89(23):12797-12804. Epub 2017 Nov 17.

Department of Applied Physics, Graduate School of Engineering, Osaka University , 2-1, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.

Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (β-actin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications.
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http://dx.doi.org/10.1021/acs.analchem.7b03107DOI Listing
December 2017

Optical microscopy imaging for the diagnosis of the pharmacological reaction of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs).

Analyst 2015 Oct 26;140(19):6500-7. Epub 2015 Aug 26.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan.

Quantitative diagnosis of pharmacological chronotropic reactions on mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) was successfully performed by utilizing derivative imaging analysis of videos recorded with a microscope camera at 30 Hz frame rate and 680 × 510 pixel resolution. The imaging analysis algorithm, developed in our lab, generated the contractile profile of the cells which was exploited for drug effect profiling. Six drugs such as isoproterenol (0.01-1 μM), quinidine (2-200 μM), propranolol (0.03-30 μM), verapamil (0.01-1 μM), sotalol (1-100 μM), and acetylsalicylic acid (0.1-10 μM) were administered and the quantitative medication effect was determined. Among the negative chronotropic agents administered, verapamil was found to be the most potent while sotalol was found to be the least potent at the micromolar level. Simultaneous measurement of the field potential and contractile motion in the verapamil effect test showed a coherent result. Moreover, this approach can provide insights into the contraction-relaxation conditions which are not available in the common electrophysiological approach. With these findings, it is expected that this study can aid in providing a simple and reliable in vitro mESC-CM-based screening platform for cardiovascular effect profiling of candidate drugs.
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http://dx.doi.org/10.1039/c5an01144bDOI Listing
October 2015
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