Manabu Tokeshi - Profile on Academia.edu (original) (raw)

Papers by Manabu Tokeshi

Paper-Based Analytical Device for the On-Site Detection of Nerve Agents

ACS Applied Bio Materials, 2021

RSC Advances, 2021

We developed a three-dimensional and symmetrically assembled microfluidic device named the 3D-iLi... more We developed a three-dimensional and symmetrically assembled microfluidic device named the 3D-iLiNP device. The 3D-iLiNP device allowed the precise size control of sub-100 nm sized lipid nanoparticles by the homogeneous and slow ethanol dilution.

ACS Omega, 2021

Analytical methods with fluorescence detection are in widespread use for detecting low abundance ... more Analytical methods with fluorescence detection are in widespread use for detecting low abundance analytes. Here, we report a simple method for fluorescence signal amplification utilizing a structure of an azide-unit pendant water-soluble photopolymer (AWP) in a microchannel. The AWP is a poly(vinyl alcohol)-based photocross-linkable polymer, which is often used in biosensors. We determined that the wall-like structure of the AWP (AWP-wall) constructed in a microchannel functioned as an amplifier of a fluorescence signal. When a solution of fluorescent molecules was introduced into the microchannel having the AWP-wall, the fluorescent molecules accumulated inside the AWP-wall by diffusion. Consequently, the fluorescence intensity inside the AWP-wall increased locally. Among the fluorescent molecules considered in this paper, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) (DDAO) showed the highest efficiency of fluorescence signal amplification. We prepared a calibration curve for DDAO using the fluorescence intensity inside the AWP-wall, and the sensitivity was 5-fold that for the microchannel without the AWP-wall. This method realizes the improved sensitivity of fluorescence detection easily because the fluorescence signal was amplified only by injecting the solution into the microchannel having the AWP-wall. Furthermore, since this method is not limited to only the use of microchannel, we expect it to be applicable in various fields.

This paper described a simple preparation method for small-size and monodispersed lipid nanoparti... more This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm.

Fluorescent polarization immunoassay (FPIA) is a singlestep immunoassay method that is applicable... more Fluorescent polarization immunoassay (FPIA) is a singlestep immunoassay method that is applicable to point-of-care testing; however, its applicability to large biomolecules has been restricted because ordinary FPIA is a competitive assay. Here, we report a noncompetitive FPIA using the variable domain from the heavy chain of a camelid antibody (VHH antibody). FPIA with VHH was successfully used to quantitate rabbit immunoglobulin G (IgG) and demonstrated a wider response range than that observed with antibody-binding (Fab) fragment. Then, using a portable FPIA instrument, a VHH-based immunoassay of human IgG in a human serum certified reference material was demonstrated.

Preparation of high-quality protein crystals is a major challenge in protein crystallography. Nat... more Preparation of high-quality protein crystals is a major challenge in protein crystallography. Natural convection is considered to be an uncontrollable factor of the crystallization process at the ground level as it disturbs the concentration gradient around the growing crystal, resulting in lower-quality crystals. A microfluidic environment expects an imitated microgravity environment because of the small Gr number. However, the mechanism of protein crystal growth in the microfluidic device was not elucidated due to limitations in measuring the crystal growth process within the device. Here, we demonstrate the real-time measurement of protein crystal growth rates within the microfluidic devices by laser confocal microscopy with differential interference contrast microscopy (LCM-DIM) at the nanometer scale. We confirmed the normal growth rates in the 20 and 30 μm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 μm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.

Analytical Sciences, 2018

We report on the effects of fabrication methods, photolithography, wax printing, screen printing,... more We report on the effects of fabrication methods, photolithography, wax printing, screen printing, and craft cutting, on selected properties of microfluidic paper-based analytical devices (μPADs): cost, fabrication precision, wicking rate, and analytical accuracy. Photolithography requires numerous fabrication steps, and an oxygen plasma treatment is necessary when using an aqueous solution. Although the boundary between the hydrophobic and hydrophilic areas in the μPAD is sharpest, the obtained K-scale intensity in measuring of protein concentrations is lower than those of the devices by other methods. Wax printing offers the simplest and fastest fabrication, although solution leakage measures should be taken to improve the wicking rate and to prevent cross-contamination. Screen printing also offers easy fabrication. The screenprinted μPAD has a good wicking performance and shows a high detection intensity. Craft cutting allows automated fabrication of many μPADs at once. The craft cut μPAD has the fastest wicking rate among the four μPADs due to bare cellulose fibers. We consider that the detection intensity of this μPAD can be raised by optimizing the evaporation rate.

Analytical Sciences, 2017

Recently, we developed a label-free detection method based on optical diffraction, and implemente... more Recently, we developed a label-free detection method based on optical diffraction, and implemented it in on our fabricated micro-and nanofluidic device. This detection method is simple and useful for detecting biomolecules, but the device fabrication consists of complicated processes. In this paper, we propose a simple method for fabricating the micro-and nanofluidic device; the fabrication combines laser interference lithography with conventional photolithography. The performance of a device fabricated by the proposed method is comparable to the performance of the device in our previous study.

ACS Omega, 2019

Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute ... more Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23−100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.

Analytical Sciences, 2019

In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer ... more In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer having a dithiol-based anchor, which exhibited higher stability on a gold electrode than a monothiol-based aptamer because of its two anchors. The sensor was also based on a signal-on scheme that produces a signal current resulting from structure-switching of the aptamer upon interaction with OTA. For simple fabrication of this sensor, the non-covalent interaction of methylene blue with the aptamer was also employed as an electrochemical indicator. In this study, the performance of the sensor, including the dissociation constant of the aptamer-OTA complex, was characterized. The proposed sensor exhibited high reproducibility and enough sensitivity to detect the minimum amount of OTA required for the analysis of real food samples with a limit of detection of 113 pM.

ACS Omega, 2018

The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) car... more The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device; these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.

Review of Scientific Instruments, 2018

Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementabl... more Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementable in molecular interaction analysis. Previously we have developed a unique FP measurement system using a liquid crystal (LC) layer and an image sensor. The system is based on a principle of synchronized detection between the switching rate of the LC layer and the sampling rate of the CCD. The FP system realized simultaneous multiple sample detection; however, the measurement precision was lower than that of the conventional FP apparatus. The main drawbacks were low light transmittance of the LC layer and insufficient synchronization between the LC layer and CCD. In this paper, we developed a new FP analyzer based on LC-CCD synchronization detection. By using a newly designed LC with high transmittance and improving synchronization, the performance of the system has been dramatically improved. Additionally, we reduced the cost by using an inexpensive CCD and an LED as the excitation source. Simultaneous FP immunoassay of multiple samples of prostaglandin E2 was performed. The error rate of the FP system is reduced from 16.9% to 3.9%, as comparable to the commercial conventional FP system.

ACS Omega, 2018

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a poten... more Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a potential carcinogen and responsible for many diseases affecting humans. Consequently, a sensitive, portable device for the detection of OTA is highly desirable. In this study, a miniaturized electrochemical aptamer-based sensor was developed for the label-free, sensitive detection of OTA. For the construction of the sensor, a gold thin-film three-electrode system was fabricated using standard microfabrication techniques on a polystyrene substrate (25 mm × 25 mm). Subsequently, the thiol-modified linker, 6-mercaptohexanol, DNA aptamer, and methylene blue (MB) were sequentially applied to the working electrode to construct a sensing layer. MB served as a redox indicator that interacted with the aptamer via the guanine bases and phosphate backbone to form complexes. The addition of OTA to the sensor induced the folding of the aptamer, which was accompanied by the release of the aptamer−MB−OTA complex from the sensor. Thus, the amount of MB decreased with increasing concentration of OTA. Differential pulse voltammetry was used for monitoring the highly sensitive detection. The standard curve for OTA exhibited a wide linearity ranging from 0.1 to 300 ng mL −1 with a detection limit of 78.3 pg mL −1 (S/N = 3). The selectivity test confirmed that the aptamer had high affinity only for the target. The OTA recoveries with the proposed sensor in commercial samples of coffee and beer were 86.4−107%.

Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed,... more Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed, aptamers have made significant contributions as bio-recognition sensors. Microdevice systems allow for low reagent consumption, high-throughput of samples, and disposability. Due to these advantages, there has been an increasing demand to develop microfluidic-based aptasensors for analytical technique applications. This review introduces the principal concepts of aptasensors and then presents some advanced applications of microdevice-based aptasensors on several platforms. Highly sensitive detection techniques such as electrochemical and optical detection have been integrated into lab-on-a-chip devices and researchers have moved towards the goal of establishing point-of-care diagnoses for target analyses.

PloS one, 2017

Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines.... more Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP...

The Analyst, Jan 28, 2016

The development of a competitive immunoassay system for colorimetric detection on microfluidic pa... more The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (μPADs) is reported. The μPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. μPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody ...

Analytical Sciences, 2016

We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an importan... more We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an important marker for chronic kidney disease in cats, using immuno-pillar chips. The required amount of reagent solution is 200 times smaller than that for the conventional ELISA in the 96-well microplate (0.5 μL versus 100 μL). In addition, the total assay time in the proposed method is more than 12 times shorter than in the conventional method (20 min versus 240 min). The limit of detection in the new method of 3 ng mL-1 is comparable to that of the conventional method (1 ng mL-1) and it is in the clinically relevant range.

Analytical and bioanalytical chemistry, 2016

A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based o... more A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow μPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model μPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 μg mL(-1). Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and...

Analytical Sciences, 2016

We report on the colorimetric oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen perox... more We report on the colorimetric oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide using horseradish peroxidase on photolithography-fabricated (P-PAD) and wax-printed (W-PAD) paper-based analytical devices. Fabricating PADs via photolithography exposes the hydrophilic areas to polymers (photoresists) and solvents, not only reducing the hydrophilicity, but also affecting the TMB-H2O2 assay system with an unavoidable incomplete elimination of photoresist during fabrication. Detection signals are then observed in the presence of photoresist residues on the P-PAD, even at a blank HRP concentration.

Paper-Based Analytical Device for the On-Site Detection of Nerve Agents

ACS Applied Bio Materials, 2021

RSC Advances, 2021

We developed a three-dimensional and symmetrically assembled microfluidic device named the 3D-iLi... more We developed a three-dimensional and symmetrically assembled microfluidic device named the 3D-iLiNP device. The 3D-iLiNP device allowed the precise size control of sub-100 nm sized lipid nanoparticles by the homogeneous and slow ethanol dilution.

ACS Omega, 2021

Analytical methods with fluorescence detection are in widespread use for detecting low abundance ... more Analytical methods with fluorescence detection are in widespread use for detecting low abundance analytes. Here, we report a simple method for fluorescence signal amplification utilizing a structure of an azide-unit pendant water-soluble photopolymer (AWP) in a microchannel. The AWP is a poly(vinyl alcohol)-based photocross-linkable polymer, which is often used in biosensors. We determined that the wall-like structure of the AWP (AWP-wall) constructed in a microchannel functioned as an amplifier of a fluorescence signal. When a solution of fluorescent molecules was introduced into the microchannel having the AWP-wall, the fluorescent molecules accumulated inside the AWP-wall by diffusion. Consequently, the fluorescence intensity inside the AWP-wall increased locally. Among the fluorescent molecules considered in this paper, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) (DDAO) showed the highest efficiency of fluorescence signal amplification. We prepared a calibration curve for DDAO using the fluorescence intensity inside the AWP-wall, and the sensitivity was 5-fold that for the microchannel without the AWP-wall. This method realizes the improved sensitivity of fluorescence detection easily because the fluorescence signal was amplified only by injecting the solution into the microchannel having the AWP-wall. Furthermore, since this method is not limited to only the use of microchannel, we expect it to be applicable in various fields.

This paper described a simple preparation method for small-size and monodispersed lipid nanoparti... more This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm.

Fluorescent polarization immunoassay (FPIA) is a singlestep immunoassay method that is applicable... more Fluorescent polarization immunoassay (FPIA) is a singlestep immunoassay method that is applicable to point-of-care testing; however, its applicability to large biomolecules has been restricted because ordinary FPIA is a competitive assay. Here, we report a noncompetitive FPIA using the variable domain from the heavy chain of a camelid antibody (VHH antibody). FPIA with VHH was successfully used to quantitate rabbit immunoglobulin G (IgG) and demonstrated a wider response range than that observed with antibody-binding (Fab) fragment. Then, using a portable FPIA instrument, a VHH-based immunoassay of human IgG in a human serum certified reference material was demonstrated.

Preparation of high-quality protein crystals is a major challenge in protein crystallography. Nat... more Preparation of high-quality protein crystals is a major challenge in protein crystallography. Natural convection is considered to be an uncontrollable factor of the crystallization process at the ground level as it disturbs the concentration gradient around the growing crystal, resulting in lower-quality crystals. A microfluidic environment expects an imitated microgravity environment because of the small Gr number. However, the mechanism of protein crystal growth in the microfluidic device was not elucidated due to limitations in measuring the crystal growth process within the device. Here, we demonstrate the real-time measurement of protein crystal growth rates within the microfluidic devices by laser confocal microscopy with differential interference contrast microscopy (LCM-DIM) at the nanometer scale. We confirmed the normal growth rates in the 20 and 30 μm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 μm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.

Analytical Sciences, 2018

We report on the effects of fabrication methods, photolithography, wax printing, screen printing,... more We report on the effects of fabrication methods, photolithography, wax printing, screen printing, and craft cutting, on selected properties of microfluidic paper-based analytical devices (μPADs): cost, fabrication precision, wicking rate, and analytical accuracy. Photolithography requires numerous fabrication steps, and an oxygen plasma treatment is necessary when using an aqueous solution. Although the boundary between the hydrophobic and hydrophilic areas in the μPAD is sharpest, the obtained K-scale intensity in measuring of protein concentrations is lower than those of the devices by other methods. Wax printing offers the simplest and fastest fabrication, although solution leakage measures should be taken to improve the wicking rate and to prevent cross-contamination. Screen printing also offers easy fabrication. The screenprinted μPAD has a good wicking performance and shows a high detection intensity. Craft cutting allows automated fabrication of many μPADs at once. The craft cut μPAD has the fastest wicking rate among the four μPADs due to bare cellulose fibers. We consider that the detection intensity of this μPAD can be raised by optimizing the evaporation rate.

Analytical Sciences, 2017

Recently, we developed a label-free detection method based on optical diffraction, and implemente... more Recently, we developed a label-free detection method based on optical diffraction, and implemented it in on our fabricated micro-and nanofluidic device. This detection method is simple and useful for detecting biomolecules, but the device fabrication consists of complicated processes. In this paper, we propose a simple method for fabricating the micro-and nanofluidic device; the fabrication combines laser interference lithography with conventional photolithography. The performance of a device fabricated by the proposed method is comparable to the performance of the device in our previous study.

ACS Omega, 2019

Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute ... more Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23−100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.

Analytical Sciences, 2019

In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer ... more In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer having a dithiol-based anchor, which exhibited higher stability on a gold electrode than a monothiol-based aptamer because of its two anchors. The sensor was also based on a signal-on scheme that produces a signal current resulting from structure-switching of the aptamer upon interaction with OTA. For simple fabrication of this sensor, the non-covalent interaction of methylene blue with the aptamer was also employed as an electrochemical indicator. In this study, the performance of the sensor, including the dissociation constant of the aptamer-OTA complex, was characterized. The proposed sensor exhibited high reproducibility and enough sensitivity to detect the minimum amount of OTA required for the analysis of real food samples with a limit of detection of 113 pM.

ACS Omega, 2018

The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) car... more The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device; these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.

Review of Scientific Instruments, 2018

Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementabl... more Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementable in molecular interaction analysis. Previously we have developed a unique FP measurement system using a liquid crystal (LC) layer and an image sensor. The system is based on a principle of synchronized detection between the switching rate of the LC layer and the sampling rate of the CCD. The FP system realized simultaneous multiple sample detection; however, the measurement precision was lower than that of the conventional FP apparatus. The main drawbacks were low light transmittance of the LC layer and insufficient synchronization between the LC layer and CCD. In this paper, we developed a new FP analyzer based on LC-CCD synchronization detection. By using a newly designed LC with high transmittance and improving synchronization, the performance of the system has been dramatically improved. Additionally, we reduced the cost by using an inexpensive CCD and an LED as the excitation source. Simultaneous FP immunoassay of multiple samples of prostaglandin E2 was performed. The error rate of the FP system is reduced from 16.9% to 3.9%, as comparable to the commercial conventional FP system.

ACS Omega, 2018

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a poten... more Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a potential carcinogen and responsible for many diseases affecting humans. Consequently, a sensitive, portable device for the detection of OTA is highly desirable. In this study, a miniaturized electrochemical aptamer-based sensor was developed for the label-free, sensitive detection of OTA. For the construction of the sensor, a gold thin-film three-electrode system was fabricated using standard microfabrication techniques on a polystyrene substrate (25 mm × 25 mm). Subsequently, the thiol-modified linker, 6-mercaptohexanol, DNA aptamer, and methylene blue (MB) were sequentially applied to the working electrode to construct a sensing layer. MB served as a redox indicator that interacted with the aptamer via the guanine bases and phosphate backbone to form complexes. The addition of OTA to the sensor induced the folding of the aptamer, which was accompanied by the release of the aptamer−MB−OTA complex from the sensor. Thus, the amount of MB decreased with increasing concentration of OTA. Differential pulse voltammetry was used for monitoring the highly sensitive detection. The standard curve for OTA exhibited a wide linearity ranging from 0.1 to 300 ng mL −1 with a detection limit of 78.3 pg mL −1 (S/N = 3). The selectivity test confirmed that the aptamer had high affinity only for the target. The OTA recoveries with the proposed sensor in commercial samples of coffee and beer were 86.4−107%.

Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed,... more Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed, aptamers have made significant contributions as bio-recognition sensors. Microdevice systems allow for low reagent consumption, high-throughput of samples, and disposability. Due to these advantages, there has been an increasing demand to develop microfluidic-based aptasensors for analytical technique applications. This review introduces the principal concepts of aptasensors and then presents some advanced applications of microdevice-based aptasensors on several platforms. Highly sensitive detection techniques such as electrochemical and optical detection have been integrated into lab-on-a-chip devices and researchers have moved towards the goal of establishing point-of-care diagnoses for target analyses.

PloS one, 2017

Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines.... more Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP...

The Analyst, Jan 28, 2016

The development of a competitive immunoassay system for colorimetric detection on microfluidic pa... more The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (μPADs) is reported. The μPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. μPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody ...

Analytical Sciences, 2016

We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an importan... more We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an important marker for chronic kidney disease in cats, using immuno-pillar chips. The required amount of reagent solution is 200 times smaller than that for the conventional ELISA in the 96-well microplate (0.5 μL versus 100 μL). In addition, the total assay time in the proposed method is more than 12 times shorter than in the conventional method (20 min versus 240 min). The limit of detection in the new method of 3 ng mL-1 is comparable to that of the conventional method (1 ng mL-1) and it is in the clinically relevant range.

Analytical and bioanalytical chemistry, 2016

A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based o... more A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow μPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model μPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 μg mL(-1). Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and...

Analytical Sciences, 2016

We report on the colorimetric oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen perox... more We report on the colorimetric oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide using horseradish peroxidase on photolithography-fabricated (P-PAD) and wax-printed (W-PAD) paper-based analytical devices. Fabricating PADs via photolithography exposes the hydrophilic areas to polymers (photoresists) and solvents, not only reducing the hydrophilicity, but also affecting the TMB-H2O2 assay system with an unavoidable incomplete elimination of photoresist during fabrication. Detection signals are then observed in the presence of photoresist residues on the P-PAD, even at a blank HRP concentration.