論文

Purification of basic drugs in reversed-phase mode is often difficult, mainly due to adsorption of positively charged compounds to the silica gel-based stationary phase. Since this adsorption can be suppressed under alkaline condition, columns with alkali-resistance are required. In addition, compounds with acid-sensitive structures are sometimes degraded during separation on silica gel-based columns which exhibit acidity due to their surface structure. We prepared an alkali-resistant reversed-phase packing material, Eggshell-PMAcO based on eggshells modified with an amphiphilic copolymer, poly(maleic acid-alt-1-octadecene) (PMAcO). The height equivalent to a theoretical plate (HETP) of the Eggshell-PMAcO column was improved by surface treatment with ammonium acetate buffer (900 mM, pH = 3.7), which is an inexpensive reagent, and the retention behavior for hydrophobic compounds was compared to a typical ODS column based on silica gel, resulting in sufficient selectivity of the eggshell-based column for hydrophobic compounds, as indicated by the ratio of retention factors of pentylbenzene and butylbenzene (Eggshell-PMAcO column: 1.55, ODS column: 1.65). Column temperature-dependent retention behavior of naphthalene was investigated in the temperature range from 25 °C to 45 °C, followed by the calculation of thermodynamic parameters. There was little difference in the standard molar enthalpy (Eggshell-PMAcO: −19.6 kJ/mol, ODS: −21.7 kJ/mol). The absolute value of the standard free Gibbs energy for the Eggshell-PMAcO column was much smaller than that of the ODS column (Eggshell-PMAcO: −0.284 kJ/mol, ODS: −13.0 kJ/mol), indicating that the Eggshell-PMAcO column had a weaker retention strength for naphthalene than the ODS column mainly due to the large difference in the standard molar entropy (Eggshell-PMAcO: −64.9 J/mol K, ODS column: −29.2 J/mol K). The retention capacities for imipramine under neutral (water/methanol) and alkaline (0.1% triethylamine water/methanol) conditions were 0.2 mg and 5 mg, respectively, based on injection mass-dependent HETP, retention factor and symmetry factor. Finally, the prepared column was applied to the purification of a building block for nucleic acid drugs. This study demonstrated that reversed-phase columns, which can be fabricated from eggshells and an amphiphilic copolymer in an inexpensive and eco-friendly way, have the ability to purify basic compounds and acid-sensitive compounds.

DNA analysis in water samples is attracting attention in various fields. However, conventional methods for DNA analysis require a work-intensive and time-consuming sample pre-treatment. In this study, a simplified pre-treatment method for analyzing DNA in water samples was evaluated. The process consists of filtration, DNA extraction, and amplification, which can be achieved within a short time. In the filtration process, two types of filters, firstly a tissue paper (Kimwipe) and then a glass filter (GF/F), were used in sequence. The first large pore size filter enabled a reduction in filtration time by removing large particulate matter impurities present in river water matrix. Cells spiked into 1 L of river water were recovered at more than 90% within approximately 5 min filtration time. Also, DNA was extracted from the captured cells directly on the surface of the filter in only 5 min. Thus, DNA collection and extraction from a water sample can be completed within about 10 min. Furthermore, PCR amplification was performed directly from DNA-attached filter sections, which greatly reduced the number of required pre-treatment steps. Finally, we succeeded in establishing a simple and fast on-site pre-treatment system by using a hand-driven syringe filtration method. This pre-treatment system is expected to offer the possibility for the future establishment of a rapid and easy DNA analysis method applicable to various types of water samples. Graphical abstract: (Figure presented.)

Despite the promising features of the CRISPR/Cas system for application to point-of-care nucleic acid tests, there are only a few reports on its integration into paper-based analytical devices (PADs) for the purpose of assay simplification. In most cases, paper platforms have only been used for the final signal readout in an assay otherwise performed in a test tube. Therefore, there is very limited information on the suitability of the CRISPR/Cas system for on-device reagent storage. To fill this gap, the current work primarily investigated the influence of various factors, including the type of paper, reagent drying method, effect of stabilizers, and storage condition on the storage stability of reagents necessary for CRISPR-based assays on paper substrates, by comparing the fluorescence signal emitted by the trans-cleavage of the dsDNA-activated Cas12a complex. The results obtained in the form of fluorescence signals emitted after trans-cleavage of a ssDNA probe through a dsDNA-activated Cas12a complex on paper substrates showed that CRISPR-related reagents spontaneously dried at room temperature on BSA blocked paper retained over 70% of their initial activity when stored at −20 °C for 28 days, independent of the type of paper substrates, which was improved by the addition of sucrose as a stabilizer. In addition, reagents dried on paper substrates under the optimized conditions exhibited stronger heat tolerance at temperatures above 65 °C compared to their corresponding solutions. This work is expected to contribute to the future development of fully integrated PADs relying on CRISPR/Cas systems for point-of-care applications requiring no additional reagent handling.

© 2020 CBMS-0001 Microfluidic paper-based analytical devices (µPADs) that display semi-quantitative analysis results by reading a QR code have been developed. The only user operation required is to apply the sample and to read the displayed QR code with a mobile device to obtain the result. Copper ions were detected as a model target and the copper ion concentration-dependent display of QR codes was achieved. This simple assay method can be applied to other targets and has great potential for point-of-care testing (POCT) applications.

© 2020 CBMS-0001 This work reports an intuitive signaling approach enabling naked-eye semi-quantitative result readout in the form of a “traffic-light” for microfluidic paper-based analytical devices (µPADs). The detection of hydrogen peroxide has been demonstrated with the aim of quantifying metabolites relevant as biomarkers of various lifestyle-related diseases. User operation is limited to simple sample application, followed by observing the traffic-light signal after approximately 10 min. Due to its intuitiveness with very low probability of result misinterpretation, this system is expected to offer great potential for home-based self-diagnostic point-of-care testing (POCT) applications.

© 2020 CBMS-0001 This report suggests utilizing “cellophane” as a new substrate material for point-of-care testing (POCT) devices. Cellophane-based microfluidic devices were fabricated by a print, cut and lamination (PCL) methodology and sample liquid was transported into four assay areas without external pumping. Furthermore, making use of the transparency of cellophane, background-tinted devices were fabricated. Based on a blue background-tinted cellophane device, the dynamic range of a hue-based H2O2 assay was 46-fold increased compared to performing the same assay on a white background device. Finally, turbidity testing for albumin was implemented on black background-tinted devices with a visual detection limit of 15 mg/dL.

© 2019 CBMS-0001. We present two diagnostic platforms, a microfluidic thread-based and a conventional immunochromatographic strip approach, for single-step lateral flow immunoassays (LFIAs) with bioluminescence (BL) signaling requiring only sample application and detection of emission color changes by a digital camera. Both rely on bioluminescence resonance energy transfer (BRET)-switching proteins. The detection of three clinically relevant targets is demonstrated: Cetuximab as example of an antibody-based anticancer drug requiring monitoring of blood drug levels in patients, and HIV1-p17 antibodies and antigens as examples of infectious disease markers.

© 2019 CBMS-0001. A disposable and simple PPAD (microfluidic paper-based analytical device) for the quantitative colorimetric detection of salivary cation species, relying on ISOs (ion-selective optodes) is introduced. This work demonstrates the applicability of ISO-integrated PPADs to real saliva samples. Calibration enabling accurate salivary cation quantification was achieved by using artificial saliva. Multiple sensing areas on a single device can detect three different types of ions within 3 min by simple application of 20 PL sample. Three kinds of ISOs were optimized for salivary Na+, K+ and Ca2+ quantification. For proof-of-concept, salivary Na+ concentration was accurately quantified by the developed PPAD.

© 2019 CBMS-0001. This work reports a basic study to simplify urinalysis, using distance-based microfluidic paper-based analytical devices (D-µPADs) for multiplexed detection. Intuitive detection of albumin (Alb), glucose (Glu) and hemoglobin (Hb) in artificial urine samples is demonstrated by a colorimetric distance-based readout method. The response ranges for the three analytes (15-1000 mg/dL, 25-2000 mg/dL, 0.25-1.0 mg/dL), fully cover the physiologically relevant domain. User operation is limited to sample application and readout of the color-changed length corresponding to the analyte concentration.

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Competitive lateral flow immunochromatographic assays (LFIAs) enabling naked-eye semi-quantitative result readout in the form of “text” have been developed. User operation is limited to sample application, followed by direct reading of assay results written in text representing the target concentration. LFIA strips enabled the detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) at concentrations down to 5 ng/mL, which is comparable to existing immunochromatography using external readout equipment. The system is readily adaptable to other analytical targets and allows for high flexibility in text design and detectable analyte concentrations. The simple assay handling and intuitive data interpretation offer great potential for point-of-care testing applications.

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Bioluminescent protein integrating microfluidic thread-based analytical devices (µTADs) for point-of-care testing (POCT) of antibodies in whole blood have been developed. Devices consist of a blood separation membrane and a plastic film layer with sewn-in cotton thread, in which proteins and luciferin for signal generation are pre-deposited. Colorimetry-based response curves for three model antibodies spiked into porcine whole blood have been obtained. Stable signals from 6-18 min after sample application and no cross-talk when assaying multiple different antibodies are observed. The µTADs offer quantitative simultaneous detection of multiple antibodies in a small volume of whole blood (8 µL).

Bio)chemical sensors based on paper substrates show attractive features such as being low cost, portable and disposable. Therefore, the technology is currently used in urinary dipsticks and immunochromatographic test strip. In recent years, many research groups report studies on microfluidic paper-based analytical devices (μPADs). In spite of the simplicity of paper-based platforms, μPADs enable performing complicated analytical procedures, multiplex target analysis and equipment-free detection. Due to these useful functions, μPADs are paid attention for point-of-care testing (POCT) and for field-deployable analysis. Herein, we introduce the fabrication methodology and clinical applications of μPADs developed by our research group.