Publications by authors named "Eiichi Tamiya"

128 Publications

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

Feasibility of a Novel Mobile C-Reactive Protein-Testing Device Using Gold-Linked Electrochemical Immunoassay: Clinical Performance Study.

JMIR Mhealth Uhealth 2020 09 7;8(9):e18782. Epub 2020 Sep 7.

Division of Endocrinology and Hypertension, Department of Cardiovascular and Internal Medicine, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan.

Background: Home-based care is one of the most promising solutions to provide sufficient medical care for several older patients in Japan. However, because of insufficient diagnostic devices, it is sometimes difficult to detect early signs of the occurrence or worsening of diseases, such as infections under home-based care settings. C-reactive protein (CRP) is highly sensitive to diagnosing infections, and its elevation can help diagnose acute infection in older patients. Therefore, a CRP-measuring device that can be used in such a specific occasion is needed for home-based care. However, aspects such as its size, weight, and procedure are still challenging with respect to the practical use of mobile devices that quantitatively measure CRP levels easily and quickly under home-based care settings.

Objective: We developed a new mobile, rapid CRP measurement device using a gold-linked electrochemical immunoassay (GLEIA) system. The aim of this study was to evaluate the feasibility of this mobile CRP-testing device.

Methods: First, we assessed the performance of bare GLEIA-based electrode chips as the foundation of the device. After embedding the bare GLEIA-based electrode chips in a special plastic case and developing the mobile CRP-testing device, we further tested the device prototype using clinical blood samples. Finally, we evaluated the intra-assay variability for precision in the same condition and inter-assay variability for reproducibility in different conditions.

Results: Blood samples for analysis were obtained by direct vein puncture from outpatients (N=85; females: 57/85; males: 28/85; age: 19-88 years) at Kanazawa University Hospital in Japan. For performance evaluation of bare GLEIA-based electrode chips, we used 85 clinical blood samples. There was a significant positive correlation between the electrode-predicted CRP levels and the reference CRP concentrations (R=0.947; P<.001). The assembled device was mobile (size 45×90×2.4 mm; weight 10 g) and disposable. The minimum volume of the sample needed for measuring CRP was 1.4 µL. The estimated preanalytical time was approximately 7 minutes and 40 seconds, and analysis time was approximately 1 minute and 10 seconds. Subsequently, for performance evaluation of the mobile CRP-testing device using GLEIA-based electrode chips, we used 26 clinical blood samples and found a significant positive correlation between the mobile device-predicted CRP levels and the reference CRP concentrations (R=0.866, P<.001). The intra-assay variabilities were 34.2%, 40.8%, and 24.5% for low, medium, and high CRP concentrations, respectively. The inter-assay variabilities were 46.5%, 38.3%, and 64.1% for low, medium, and high CRP concentrations, respectively.

Conclusions: Our findings suggest that this new mobile CRP-testing device might be suitable for use in home-based care settings.
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http://dx.doi.org/10.2196/18782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506539PMC
September 2020

Towards On-site Determination of Secretory IgA in Artificial Saliva with Gold-Linked Electrochemical Immunoassay (GLEIA) Using Portable Potentiostat and Disposable Printed Electrode.

Appl Biochem Biotechnol 2021 May 13;193(5):1311-1320. Epub 2020 Jun 13.

AIST-Osaka University Advanced Photonics and Biosensing Open Innovation Laboratory, AIST, Suita, Japan.

Mental stress is closely connected with our physical and mental wellness. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. In this paper, we detect the secretory immunoglobulin A (sIgA), which is the candidate of salivary stress markers, with original electrochemical immunoassay: gold-linked electrochemical immunoassay (GLEIA). This biosensor is based on a sandwich-type immunosensor and adopts the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires a low sample volume (10 μL). In addition, the GLEIA show high sensitivity and selectivity. We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Furthermore, we obtained acceptable selectivity in the various solution with salivary proteins such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme, and mucin. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress markers in saliva.
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http://dx.doi.org/10.1007/s12010-020-03332-8DOI Listing
May 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

Chemically Regulated ROS Generation from Gold Nanoparticles for Enzyme-Free Electrochemiluminescent Immunosensing.

Anal Chem 2018 05 17;90(9):5773-5780. Epub 2018 Apr 17.

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

In the present work, we report on an enzyme-free electrochemiluminescent (ECL) immunosensing scheme utilizing the catalytic generation of reactive oxygen species (ROS) from gold nanoparticles (AuNPs) (diameter ≥5 nm) dispersed in aqueous solutions of trishydroxymethylaminomethane (Tris). First, to examine this catalytic pathway in detail, the effects of various factors such as the AuNP size and concentration, dispersant type and concentration, and dissolved oxygen were investigated using the electrochemiluminescence (ECL) of luminol. It was found that the catalytic generation of ROS from AuNPs can be regulated chemically by altering conditions such as the type, concentration, and pH of the solution that the AuNPs are dispersed in. Under the best conditions studied in this work, the AuNPs displayed high catalytic activity toward ROS generation, with an estimated apparent turnover number per AuNP of 0.1 s, comparable to those of several common peroxide-producing enzymes. Following these studies, this phenomenon was applied to develop a one-step enzyme-free ECL immunosensor based on sandwiching the target analyte using antibody-conjugated magnetic beads (MB) and AuNPs. Using IgA as a model analyte, the developed immunosensor was able to detect the target in the range of 1 ng/mL to 10 μg/mL, with the lower detection limit being comparable to those of commercial assays for the same target. Altering the antibodies used to modify the MB and AuNPs could further improve the detection limit as well as expand the applicability of this immunoassay to the detection of other analytes.
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http://dx.doi.org/10.1021/acs.analchem.8b00118DOI Listing
May 2018

Trends in Paper-based Electrochemical Biosensors: From Design to Application.

Anal Sci 2018 ;34(1):7-18

Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam.

Electrochemical bio-sensing using paper-based detection systems is the main focus of this review. The different existing designs of 2-dimensional and 3-dimensional sensors, and fabrication techniques are discussed. This review highlights the effect of adopting different sensor designs, distinct fabrication techniques, as well as different modification methods, in order to produce reliable and reproducible reading. The use of various nanomaterials have been demonstrated in order to modify the surface of electrodes during fabrication to further enhance the signal for subsequent analysis. The reviewed sensors were classified into categories based on their applications, such as diagnostics, environmental and food testing. One of the major advantages of using paper-based electrochemical sensors is the potential for miniaturization, which only requires relatively small amount of samples, and the low cost for the purpose of mass production. Additionally, most of the devices reviewed were made to be portable, making them well-suited for on-site detection. Finally, paper-based detection is an ideal platform to fabricate cost-effective, user-friendly and sensitive electrochemical biosensors, with large capacity for customization depending on functional needs.
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http://dx.doi.org/10.2116/analsci.34.7DOI Listing
August 2018

Carbon-Based Nanomaterials in Biomass-Based Fuel-Fed Fuel Cells.

Sensors (Basel) 2017 Nov 10;17(11). Epub 2017 Nov 10.

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

Environmental and sustainable economical concerns are generating a growing interest in biofuels predominantly produced from biomass. It would be ideal if an energy conversion device could directly extract energy from a sustainable energy resource such as biomass. Unfortunately, up to now, such a direct conversion device produces insufficient power to meet the demand of practical applications. To realize the future of biofuel-fed fuel cells as a green energy conversion device, efforts have been devoted to the development of carbon-based nanomaterials with tunable electronic and surface characteristics to act as efficient metal-free electrocatalysts and/or as supporting matrix for metal-based electrocatalysts. We present here a mini review on the recent advances in carbon-based catalysts for each type of biofuel-fed/biofuel cells that directly/indirectly extract energy from biomass resources, and discuss the challenges and perspectives in this developing field.
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http://dx.doi.org/10.3390/s17112587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713132PMC
November 2017

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

Sensitive Detection of Glycated Albumin in Human Serum Albumin Using Electrochemiluminescence.

Anal Chem 2017 06 16;89(11):5909-5915. Epub 2017 May 16.

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

Monitoring of blood glucose content is vital for diabetes patients. The conventional widely used method involves an invasive procedure for blood sampling. In addition, blood glucose measured by this way is affected by immediate food consumption and it does not show accurate baseline blood glucose measurement. Thus, monitoring blood glucose by a noninvasive method that accurately reflects baseline blood glucose content is important. Glycated albumin (GA), a biomarker for diabetes indicating the average blood glucose over 2 weeks, can be used for semilong-term blood glucose monitoring. Detection of GA in saliva is a noninvasive method that alleviates the use of needles for diabetic patients; however, its content in saliva is in the nanomolar range. Therefore, the GA enzymatic detection method was combined with the ECL method for a highly sensitive detection of GA in human serum albumin and in the saliva sample. Here, the standard curve was constructed using model substrate, FZK, between 0.1 and 2 μM, and GA in human serum albumin was measured in this range. Also, we successfully demonstrated the detection limit of 0.1 μM GA in human serum albumin sample using ECL, which has seen improvement of about 70 times more than the colorimetric methods. The detection of GA in real saliva sample suggested that sample dilution of 5 times may be necessary to suppress the ECL quenching effect by impurities.
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http://dx.doi.org/10.1021/acs.analchem.7b00280DOI Listing
June 2017

Synthesis of Recombinant Mouse Crystallin Proteins and in Vitro Measurement of Their Refractivity.

ACS Biomater Sci Eng 2017 Apr 6;3(4):502-508. Epub 2017 Mar 6.

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

The eye lens is an organ that focuses light onto the retina and is reported to have a high refractive index in vertebrates. An analysis of refractivity was conducted using recombinant mouse Crystallin proteins produced in () compared with bovine serum albumin (BSA) and other commercially available proteins. Not only did we measure the refractivity but for one of the crystallins, Cryba1, we also confirmed that it responds uniquely to its environmental conditions. The crystallin showed high refractivity, as expected, and we confirmed that the electrical charge of the Cryba1 molecule influences its refractivity.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00605DOI Listing
April 2017

DEP-On-Go for Simultaneous Sensing of Multiple Heavy Metals Pollutants in Environmental Samples.

Sensors (Basel) 2016 Dec 27;17(1). Epub 2016 Dec 27.

Biyani BioSolutions Pvt. Ltd., Biyani Research Group, R-4, Sector 3, Vidhyadhar Nagar, Jaipur 302023, India.

We describe a simple and affordable "Disposable electrode printed (DEP)-On-Go" sensing platform for the rapid on-site monitoring of trace heavy metal pollutants in environmental samples for early warning by developing a mobile electrochemical device composed of palm-sized potentiostat and disposable unmodified screen-printed electrode chips. We present the analytical performance of our device for the sensitive detection of major heavy metal ions, namely, mercury, cadmium, lead, arsenic, zinc, and copper with detection limits of 1.5, 2.6, 4.0, 5.0, 14.4, and, 15.5 μg·L, respectively. Importantly, the utility of this device is extended to detect multiple heavy metals simultaneously with well-defined voltammograms and similar sensitivity. Finally, "DEP-On-Go" was successfully applied to detect heavy metals in real environmental samples from groundwater, tap water, house dust, soil, and industry-processed rice and noodle foods. We evaluated the efficiency of this system with a linear correlation through inductively coupled plasma mass spectrometry, and the results suggested that this system can be reliable for on-site screening purposes. On-field applications using real samples of groundwater for drinking in the northern parts of India support the easy-to-detect, low-cost (<1 USD), rapid (within 5 min), and reliable detection limit (ppb levels) performance of our device for the on-site detection and monitoring of multiple heavy metals in resource-limited settings.
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http://dx.doi.org/10.3390/s17010045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298618PMC
December 2016

Printable Electrochemical Biosensors: A Focus on Screen-Printed Electrodes and Their Application.

Sensors (Basel) 2016 Oct 21;16(10). Epub 2016 Oct 21.

Department of Applied Physics, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.

In this review we present electrochemical biosensor developments, focusing on screen-printed electrodes (SPEs) and their applications. In particular, we discuss how SPEs enable simple integration, and the portability needed for on-field applications. First, we briefly discuss the general concept of biosensors and quickly move on to electrochemical biosensors. Drawing from research undertaken in this area, we cover the development of electrochemical DNA biosensors in great detail. Through specific examples, we describe the fabrication and surface modification of printed electrodes for sensitive and selective detection of targeted DNA sequences, as well as integration with reverse transcription-polymerase chain reaction (RT-PCR). For a more rounded approach, we also touch on electrochemical immunosensors and enzyme-based biosensors. Last, we present some electrochemical devices specifically developed for use with SPEs, including USB-powered compact mini potentiostat. The coupling demonstrates the practical use of printable electrode technologies for application at point-of-use. Although tremendous advances have indeed been made in this area, a few challenges remain. One of the main challenges is application of these technologies for on-field analysis, which involves complicated sample matrices.
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http://dx.doi.org/10.3390/s16101761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087545PMC
October 2016

Versatile Micropatterning of Plasmonic Nanostructures by Visible Light Induced Electroless Silver Plating on Gold Nanoseeds.

ACS Appl Mater Interfaces 2016 Sep 1;8(36):23932-40. Epub 2016 Sep 1.

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

A versatile fabrication technique for plasmonic silver (Ag) nanostructures that uses visible light exposure for micropatterning and plasmon resonance tuning is presented. The surface of a glass substrate modified with gold (Au) nanoseeds by a thermal dewetting process was used as a Ag plating platform. When a solution containing silver nitrate and sodium citrate was dropped on the Au nanoseeds under visible light exposure, the plasmon-mediated reduction of Ag ions was induced on the Au nanoseeds to form Ag nanostructures. The plasmon resonance spectra of Ag nanostructures were examined by an absorption spectral measurement and a finite-difference time-domain (FDTD) simulation. Some examples of Ag nanostructure patterning were demonstrated by means of light exposure through a photomask, direct writing with a focused laser beam, and the interference between two laser beams. Surface enhanced Raman spectroscopy (SERS) of 4-aminothiophenol (4-ATP) was conducted with fabricated Ag nanostructures.
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http://dx.doi.org/10.1021/acsami.6b07661DOI Listing
September 2016

Editorial: Translating the advances of biosensors from bench to bedside.

Biotechnol J 2016 Jun;11(6):727-8

Biosensors have been found with numerous applications in many areas including genetic analysis, detection of infectious diseases, environmental monitoring and forensic analysis. We have witnessed rapid advances in this field, especially with the emergence of nanotechnology in the past decade.
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http://dx.doi.org/10.1002/biot.201676010DOI Listing
June 2016

Impact of New Quick Gold Nanoparticle-Based Cortisol Assay During Adrenal Vein Sampling for Primary Aldosteronism.

J Clin Endocrinol Metab 2016 06 24;101(6):2554-61. Epub 2016 Mar 24.

Program Management Office for Paradigms-Establishing Centers for Fostering Medical Researchers of the Future (T.Y., M.K.), Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan; Division of Endocrinology and Hypertension (T.Y., S.K., M.K., M.U., R.O., Yoshim. Takeda, Yoshiyu Takeda), Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan; Departments of Hygiene (M.D.) and Radiology (J.S., T.M., W.K., T.G.), Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8640, Japan; Department of Advanced Medical Imaging (O.M.), Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa 920-8640, Japan; Department of Materials Science (K.I., Y.Taka.), School of Material Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan; Department of Applied Physics (E.T.), Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan; Hoju Memorial Hospital (M.O., M.N.), Nomi, Ishikawa 923-1226, Japan; Takaoka Municipal Hospital (S.M., N.T.), Takaoka, Toyama 933-8550, Japan; Sanda Municipal Hospital (Y.M.), Sanda, Hyogo 669-1321, Japan; Akashi Medical Center (K.K.), Akashi, Hyogo 674-0063, Japan; Ishikawa Prefectural Hospital (S.F.), Kanazawa, Ishikawa 920-8530, Japan; Department of Endocrinology (T.Se., T.Sa.), Saisekai Ishikawa Hospital, Kanazawa, Ishikawa 920-0353, Japan; Division of Cardiology (K.H., M.Y.), Department of Internal Medicine, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8640, Japan; and Innovative Clinical Research Center (Yoshiyu Takeda), Kanazawa University Hospital, Kanazawa, Ishikawa, 920-8640, Japan.

Context: Adrenal vein sampling (AVS) is essential for identifying a surgically curable form of primary aldosteronism (PA), but accurate placement of the sampling catheter is technically challenging. Intraprocedural cortisol measurement can confirm the catheter's position, thereby increasing the AVS success rate.

Objective And Methods: We developed a quick cortisol assay (QCA) that uses immunochromatography and gold nanoparticles and can be performed either semiquantitatively or quantitatively. The assay was evaluated in two studies. In a single-center study, PA patients were assigned to undergo AVS incorporating the semiquantitative QCA (n = 30), the quantitative QCA (n = 30), or without the QCA (n = 30), and the rates of successful AVS were determined. In a prospective multicenter randomized, controlled study, the success rates of AVS performed with (n = 148) or without (n = 145) the semiquantitative QCA were determined.

Results: Cortisol concentrations were measured during AVS in 6 minutes or less in the radiology suite, without additional technical assistance, and significantly correlated with a conventional reference assay (R(2) = 0.994; P < .001). In the single-center study, the differences in the AVS success rates associated with semiquantitative and quantitative QCAs were not significant (both 93%); however, the success rates were significantly higher than the rate of successful AVS performed without using the QCA (63%; P < .001). The success rate of AVS performed in the multicenter study was 94% for the semiquantitative QCA, which was significantly higher than the rate for the patients without QCA (60%; P < .001).

Conclusions: Our novel QCA was rapidly and easily performed at the point of care and improved the rate of successful AVS.
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http://dx.doi.org/10.1210/jc.2016-1011DOI Listing
June 2016

PEP-on-DEP: A competitive peptide-based disposable electrochemical aptasensor for renin diagnostics.

Biosens Bioelectron 2016 Oct 24;84:120-5. Epub 2015 Dec 24.

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.

Antibody-based immunosensors are relatively less accessible to a wide variety of unreachable targets, such as low-molecular-weight biomarkers that represent a rich untapped source of disease-specific diagnostic information. Here, we present a peptide aptamer-based electrochemical sensor technology called 'PEP-on-DEP' to detect less accessible target molecules, such as renin, and to improve the quality of life. Peptide-based aptamers represent a relatively smart class of affinity binders and show great promise in biosensor development. Renin is involved in the regulation of arterial blood pressure and is an emerging biomarker protein for predicting cardiovascular risk and prognosis. To our knowledge, no studies have described aptamer molecules that can be used as new potent probes for renin. Here, we describe a portable electrochemical biosensor platform based on the newly identified peptide aptamer molecules for renin. We constructed a randomized octapeptide library pool with diversified sequences and selected renin specific peptide aptamers using cDNA display technology. We identified a few peptide aptamer sequences with a KD in the µM binding affinity range for renin. Next, we grafted the selected peptide aptamers onto gold nanoparticles and detected renin in a one-step competitive assay using our originally developed DEP (Disposable Electrochemical Printed) chip and a USB powered portable potentiostat system. We successfully detected renin in as little as 300ngmL(-1) using the PEP-on-DEP method. Thus, the generation and characterization of novel probes for unreachable target molecules by merging a newly identified peptide aptamer with electrochemical transduction allowed for the development of a more practical biosensor that, in principle, can be adapted to develop a portable, low-cost and mass-producible biosensor for point-of-care applications.
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http://dx.doi.org/10.1016/j.bios.2015.12.078DOI Listing
October 2016

Modified screen printed electrode for development of a highly sensitive label-free impedimetric immunosensor to detect amyloid beta peptides.

Anal Chim Acta 2015 Sep 28;892:69-76. Epub 2015 Aug 28.

School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan; Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1-21-24, Kagoshima City, Kagoshima, 890-0065, Japan. Electronic address:

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting approximately 26 million people world-wide, and the number is increasing as life expectancy increases. Since the only reliable diagnosis for the pathology is histochemical post-mortem examination, there is a rather urgent need for reliable, sensitive and quick detection techniques. Amyloid beta, being one of the established and widely accepted biomarkers of AD is a target biomolecule. Herein, we present fabrication of a labelless impedimetric amyloid beta immunosensor on carbon DEP (disposable electrochemical printed) chip. Three types of amyloid β impedimetric immunosensors were fabricated in a systematic step-wise manner in order to understand the effects that each surface modification chemistry had on detection sensitivity. We found that compared to a bare electrode, surface modification through formation of SAM of AuNPs increased sensitivity by approximately three orders of magnitude (LoD from 2.04 μM to 2.65 nM). A further modification using protein G, which helps orientate antibodies to an optimum position for interaction with antigen, lowered the LoD further to 0.57 nM. We have demonstrated that the presence of one of the most abundance proteins in biological fluids, bovine serum albumin (BSA), did not interfere with the sensitivity of the sensor. Since the DEP chips are disposable and the detection platform label-free, the developed sensor is relatively fast and cheap. These methods could easily be applied for detection of other antigens, with selection of the detection platform based on the desired for sensitivity.
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http://dx.doi.org/10.1016/j.aca.2015.08.036DOI Listing
September 2015

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

Time-lapse Raman imaging of osteoblast differentiation.

Sci Rep 2015 Jul 27;5:12529. Epub 2015 Jul 27.

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

Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.
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http://dx.doi.org/10.1038/srep12529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515588PMC
July 2015

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

Biosens Bioelectron 2015 Dec 9;74:725-30. Epub 2015 Jul 9.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 575-0871, Japan. Electronic address:

Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing.
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http://dx.doi.org/10.1016/j.bios.2015.07.009DOI Listing
December 2015

Toward the development of smart and low cost point-of-care biosensors based on screen printed electrodes.

Crit Rev Biotechnol 2016 12;36(3):495-505. Epub 2015 Jan 12.

e Nanobioengineering Laboratory, Department of Applied Physics , Graduate School of Engineering, Osaka University , Osaka , Japan.

Screen printing technology provides a cheap and easy means to fabricate disposable electrochemical devices in bulk quantities which are used for rapid, low-cost, on-site, real-time and recurrent industrial, pharmaceutical or environmental analyses. Recent developments in micro-fabrication and nano-characterization made it possible to screen print reproducible feature on materials including plastics, ceramics and metals. The processed features forms screen-printed disposable biochip (SPDB) upon the application of suitable bio-chemical recognition receptors following appropriate methods. Adequacy of biological and non-biological materials is the key to successful biochip development. We can further improve recognition ability of SPDBs by adopting new screen printed electrode (SPE) configurations. This review covers screen-printing theory with special emphasis on the technical impacts of SPE architectures, surface treatments, operational stability and signal sensitivity. The application of SPE in different areas has also been summarized. The article aims to highlight the state-of-the-art of SPDB at the laboratory scale to enable us in envisaging the deployment of emerging SPDB technology on the commercial scale.
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http://dx.doi.org/10.3109/07388551.2014.992387DOI Listing
December 2016

Rapid and highly sensitive detection by a real-time polymerase chain reaction using a chip coated with its reagents.

Anal Sci 2014 ;30(5):569-74

Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST).

On-site detection by flow-through polymerase chain reaction (PCR) microfluidic systems for rapid and highly sensitive analysis, are significantly desired for bioanalytical and medical research. The conventional continuous-flow PCR chips realized rapid detection, but their sensitivity was very low (10(6) to 10(8) copies μL(-1)). We improved this drawback by coating the chip with a PCR reagents mixture, and succeed to obtain a rapid and highly sensitive detection by using a segment-flow PCR system. In the present work, we developed a portable segment-flow PCR system for practical use. PCR was performed for the uid A gene in E. coli. By real-time segment-flow PCR using coated chips, we realized rapid detection in 8 min and a high sensitivity of 4 cells μL(-1). The sensitivity by the segment-flow PCR chip was the same as that of a conventional thermal cycler. Moreover, the detection speed of our segment-flow PCR chip was 15-times as rapid as that of the conventional thermal cycler.
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http://dx.doi.org/10.2116/analsci.30.569DOI Listing
April 2015

Integrating reductive and synthetic approaches in biology using man-made cell-like compartments.

Sci Rep 2014 Apr 17;4:4722. Epub 2014 Apr 17.

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

We propose 'integrated synthetic genetics' as a novel methodology that integrates reductive and synthetic approaches used in life science research. Integrated synthetic genetics enables determinations of sets of genes required for the functioning of any biological subsystem. This method utilizes artificial cell-like compartments, including a randomly introduced whole gene library, strictly defined components for in vitro transcription and translation and a reporter that fluoresces 'only when a particular function of a target biological subsystem is active.' The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent artificial cells and multiplex next-generation sequencing of genes included in these cells. The importance of this methodology is that screening for the set of genes involved in a subsystem and reconstructing the entire subsystem can be done simultaneously. This methodology can be applied to any biological subsystem of any species and may remarkably accelerate life science research.
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http://dx.doi.org/10.1038/srep04722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989557PMC
April 2014

Localized surface plasmon resonance detection of biological toxins using cell surface oligosaccharides on glyco chips.

ACS Appl Mater Interfaces 2013 May 13;5(10):4173-80. Epub 2013 May 13.

Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Japan.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.
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http://dx.doi.org/10.1021/am4002937DOI Listing
May 2013
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