論文 - チッテリオ，　ダニエル

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

Lomae A., Teekayupak K., Preechakasedkit P., Pasomsub E., Ozer T., Henry C.S., Citterio D., Vilaivan T., Chailapakul O., Ruecha N.

Talanta 279 2024年11月

ISSN  00399140

　概要を見る

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

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Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices

Tanifuji Y., Suzuki H., Tong G., Hiruta Y., Citterio D.

Analytical Methods 16 （ 25 ） 4143 - 4149 2024年05月

ISSN  17599660

　概要を見る

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.

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Rational design of pH-responsive near-infrared spirocyclic cyanines: the effects of substituents and the external environment

Sakama A., Seo H., Hara J., Shindo Y., Ikeda Y., Oka K., Citterio D., Hiruta Y.

Chemical Communications 60 （ 46 ） 5984 - 5987 2024年05月

ISSN  13597345

　概要を見る

pH-responsive spirocyclic cyanine dyes were designed and synthesized. The equilibrium constant for cyclization (pKcycl) could be rationally controlled by changing the nucleophilic moiety and the side chains. Encapsulation in polymeric micelles inhibited the H-aggregation of the dye, and the pKcycl could be shifted according to the amphiphilic polymer employed.

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Simplified capture, extraction, and amplification of cellular DNA from water samples

Kawaguchi M., Aoki H., Kamo H., Miura K., Hiruta Y., Simizu S., Citterio D.

Analytical Sciences （Analytical Sciences）  40 （ 3 ） 501 - 510 2024年03月

ISSN  09106340

　概要を見る

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.)

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Peroxidase-like Activity of Aptamer-Gold Nanoparticles for Selective and Sensitive Fluorescence Detection of Low-Density Lipoproteins

Prakobkij A., Saenmuangchin R., Chunta S., Amatatongchai M., Citterio D., Jarujamrus P.

ACS Applied Nano Materials （ACS Applied Nano Materials）  7 （ 11 ） 12356 - 12365 2024年

　概要を見る

Low-density lipoprotein cholesterol (LDL-C), commonly called “bad cholesterol”, is crucial to cardiovascular health. Increased LDL-C levels pose a substantial risk to human health. As a result, there is a demand for reliable, affordable, and highly sensitive analytical methods for LDL-C detection. Herein, a facile fluorometric aptasensor for LDL-C detection based on the aptamer-enhanced peroxidase-mimicking activity of gold nanoparticles (AuNPs) has been developed. AuNPs were functionalized with LDL-C-specific thiolated aptamer to enhance their intrinsic peroxidase-like activity, which could effectively catalyze the oxidation of o-phenylenediamine dihydrochloride (OPD) by H2O2 into the yellow-fluorescent product 2,3-diamino phenazine (DAP). The characterization studies confirmed that the presence of the aptamer enhances the affinity of AuNPs toward the OPD substrate, thereby leading to a marked increase in the peroxidase-mimicking activity. Moreover, after being functionalized with an aptamer, increased dispersibility and substrate affinity of AuNPs were achieved. Using LDL-C as a target analyte, under optimum conditions, a linear relationship between smartphone-recorded signal intensity and the logarithm of the analyte concentration was observed in the range of 0.05-1 mg dL-1 and the limit of detection was 0.0230 mg dL-1. The results agree with those obtained by a clinical laboratory method. This proposed method is readily deployable and a promising prototype for diverse diagnostic applications, particularly in biomarker detection within serum or plasma samples. It holds the potential to yield substantial advantages in point-of-care testing.

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Development of Phosphinate Ligand-Based Low-Affinity Ca²⁺ Fluorescent Probes and Application to Intracellular Ca²⁺ Imaging

Kumada R., Sakama A., Shindo Y., Kuronuma Y., Iwasawa N., Citterio D., Oka K., Hiruta Y.

Analytical Chemistry （Analytical Chemistry）  95 （ 45 ） 16683 - 16691 2023年11月

ISSN  00032700

　概要を見る

Divalent metal cations such as calcium ion (Ca2+) and magnesium ion (Mg2+) are indispensable to the regulation of various cellular activities. In this research, we developed the KLCA series utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a target binding site, which was reported recently as a highly free Mg2+-selective ligand. KLCA-301 with orange fluorescence based on a rhodamine fluorophore and KLCA-501 with near-infrared (NIR) fluorescence based on a Si-rhodamine fluorophore were synthesized, intended for application to multicolor imaging. The evaluation of the fluorescence response to Ca2+ and Mg2+ of the KLCA series indicated the applicability as low-affinity Ca2+ probes. While KLCA-301 mainly localized in the cytosol in cultured rat hippocampal neurons, KLCA-501 localized to the cytosol and granular organelles in neurons. Comparison of the fluorescence response of KLCA-301 and the high-affinity Ca2+ probe Fluo-4 upon stimulation by glutamate in stained neurons revealed that KLCA-301 could reflect the secondary large rise of intracellular Ca2+, which Fluo-4 could not detect. In addition, KLCA-501 showed a fluorescence response similar to the low-affinity Ca2+ probe Fluo-5N upon stimulation by glutamate in stained neurons, concluding that KLCA-301 and KLCA-501 could be used as low-affinity Ca2+ probes. The KLCA series offers new options for low-affinity Ca2+ probes. Moreover, KLCA-501 achieved simultaneous visualization of the change in Ca2+ and ATP concentrations and also in mitochondrial inner membrane potential in neurons. KLCA-501 is expected to be a strong tool that enables simultaneous multicolor imaging of multiple targets and elucidation of their relationship in cells.

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Cation/anion-exchange mode switching chromatography utilizing pH-responsive mixed charge polymer-modified silica beads

Kaku T., Deura K., Yoshii T., Citterio D., Hiruta Y.

Molecular Systems Design and Engineering （Molecular Systems Design and Engineering）  9 （ 1 ） 56 - 62 2023年11月

　概要を見る

The separation capacity of a column typically remains constant. By applying stimuli-responsive materials to the stationary phase, the separation capacity in a single column can be tuned; however, the separation mode is not completely switched. In this study, we aimed to develop a cation/anion-exchange mode switching chromatography approach, in which the monomer ratio is adjusted, enabling the surface charge to become either negative or positive in response to mobile phase pH. Three types of beads were prepared, each modified with a pH-responsive mixed-charge polymer combining a cationic monomer, a pH-responsive carboxylic acid monomer, a neutral monomer, and a cross-linking monomer. The composition ratio of the cationic monomer to the pH-responsive carboxylic acid monomer was set at 1 : 2 so that the cation-exchange mode occurs at a pH above the pKa and the anion-exchange mode occurs below the pKa. At a pH below the pKa, the retention factor of the negatively charged compound increased. In contrast, at a pH above the pKa, the retention factor of the positively charged compound increased, confirming the charge switching on the bead surface. Switching to the cation- and anion-exchange mode enabled the separation of five basic antidepressants and acidic non-steroidal anti-inflammatory drugs, respectively. Utilizing a pH-responsive mixed-charge polymer, we attributed a cation/anion-exchange mode to a single column.

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Dual Colorimetric/Electrochemical Detection of Salmonella typhimurium Using a Laser-Induced Graphene Integrated Lateral Flow Immunoassay Strip

Preechakasedkit P., Panphut W., Lomae A., Wonsawat W., Citterio D., Ruecha N.

Analytical Chemistry （Analytical Chemistry）  95 （ 37 ） 13904 - 13912 2023年09月

ISSN  00032700

　概要を見る

Foodborne illnesses caused by the ingestion of contaminated foods or beverages are a serious concern due to the millions of reported cases per year. It is essential to develop sensitive and rapid detection methods of foodborne pathogens to ensure food safety for producers and consumers. Unfortunately, current detection techniques still suffer from time-consuming operations and the need for highly skilled personnel. Here, we introduce a highly sensitive dual colorimetric/electrochemical detection approach for Salmonella enterica serovar typhimurium (S. typhimurium) based on a laser-induced graphene-integrated lateral flow immunoassay (LIG-LFIA) strip. The LIG electrode was fabricated by laser engraving on a polyimide tape containing a pseudo silver/silver chloride reference electrode from silver sintering and chlorination. Using double-sided tape inserted into the strip, automatic sequential reagent delivery was enabled for the dual-mode signal readout by single-sample loading. A gold-deposited gold nanoparticle strategy was first employed to simultaneously obtain a colorimetric signal for early screening and a signal turn-on electrochemical response for high-sensitivity and -quantitative analysis. A superior performance of the strip was established, characterized by a short analysis time (12 min assay +15 min sample preparation), a broad working concentration range (1 cfu/10 mL to 108 cfu/mL), and the lowest limit of detection (1 ± 0.5 cfu/10 mL; mean ± standard deviation, n = 3) among reported multimode S. typhimurium detection schemes. The strip was successfully applied in the analysis of various food products without any bacterial enrichment or amplification required, and the results were comparable to those of the standard culture method.

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Development of prediction model for cloud point of thermo-responsive polymers by experiment-oriented materials informatics

Hayakawa M., Sakano K., Kumada R., Tobita H., Igarashi Y., Citterio D., Oaki Y., Hiruta Y.

Polymer Chemistry （Polymer Chemistry）  14 （ 19 ） 2383 - 2389 2023年04月

ISSN  17599954

　概要を見る

Thermo-responsive polymers having a lower critical solution temperature (LCST) have attracted attention for biological applications such as drug delivery, diagnosis, and coating materials. In recent years, research on predicting LCST by utilizing machine learning has been conducted. However, since these methods targeted only copolymers combining specific monomer structures, they are not versatile, and multiple trials are still required to obtain new thermo-responsive polymers with the desired LCST. In this study, a prediction model for cloud point temperature (TCP) was built by a combination of materials informatics and chemical insight, named sparse modeling for small data (SpM-S) using a small dataset of polymers collected from the literature as training data. This approach created a model that is interpretable, easy to calculate, and versatile. The prediction accuracy was validated using data from different literature sources and experimental test data. The model was able to predict the TCP of polymers containing monomers not included in the dataset as well as polymers containing monomers included in the dataset. The predictive model has the potential to guide the design of new thermo-responsive polymers, and to contribute to efficient development of thermo-responsive polymers.

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Evaluation of separation performance for eggshell-based reversed-phase HPLC columns by controlling particle size and application in quantitative therapeutic drug monitoring

Yoshii T., Nakano K., Okuda T., Citterio D., Hiruta Y.

Analytical Methods （Analytical Methods）  15 （ 14 ） 1790 - 1796 2023年03月

ISSN  17599660

　概要を見る

Eggshell-based reversed-phase packing materials were applied to an analytical column for high-performance liquid chromatography. Commercially available eggshell powder was classified by a cyclone system to obtain three types of particles with different diameters (arithmetic mean ± standard deviation: 4.3 ± 3.8, 5.6 ± 3.3, and 9.5 ± 5.5 μm). Sedimentation separation removed tiny particles from each sample, resulting in particles with arithmetic means of 6.6 ± 5.5, 7.3 ± 4.5, and 10.2 ± 5.0 μm, respectively. The unclassified particles and three particle types treated with sedimentation separation were subsequently packed into analytical columns (150 mm × 4.6 mm I.D.), and their separation efficiencies were evaluated by comparing their height equivalent to a theoretical plate (HETP). The column without sedimentation separation exhibited the highest HETP, whereas the columns with sedimentation separation showed better separation efficiency and lower back pressure. The column with the best separation efficiency was applied for the separation of 10 alkylbenzenes and 5 steroids, and all peaks were observed with complete separation (peak resolution: RS > 1.5). Finally, the column was used for quantitative analysis of voriconazole, an azole antifungal agent, and imatinib, a first-generation molecularly targeted drug for cancer treatment, in spiked whole blood. Excellent accuracy (99.1-102.8%) and precision (0.6-1.9%) were observed for the spiked drugs and long-term stability (>3000 column volumes of mobile phase flow) indicated good applicability of the developed eggshell-based column as an analytical column for routine analyses of therapeutic drugs in blood.

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Evaluation of cellophane as platform for colorimetric assays on microfluidic analytical devices

Shigemori H., Maejima K., Shibata H., Hiruta Y., Citterio D.

Microchimica Acta （Microchimica Acta）  190 （ 2 ）  2023年02月

ISSN  00263672

　概要を見る

Due to their low cost, simplicity, and pump-free liquid transport properties, colorimetric assays on paper spots and microfluidic paper-based analytical devices (µPADs) are regarded as useful tools for point-of-care testing (POCT). However, for certain types of colorimetric assays, the “non-transparent” and “white” characters of paper can be a disadvantage. In this work, the possibilities of using cellophane as an alternative platform for colorimetric assays have been investigated. Cellophane is a low cost and easy-to-handle transparent film made of regenerated cellulose. Owing to its hydrophilic character, cellophane-based microfluidic channels fabricated through a print–cut–laminate approach enabled pump-free liquid transport into multiple detection areas, similar to µPADs. In addition, the water absorption characteristics of cellophane allowed the stable immobilization of water-soluble colorimetric indicators without any surface modification or additional reagents. The transparency of cellophane provides possibilities for simple background coloring of the substrates, increasing the dynamic signal range for hue-based colorimetric assays, as demonstrated for two model assays targeting H2O2 (46-fold increase) and creatinine (3.6-fold increase). Finally, a turbidity detection-based protein assay was realized on black background cellophane spots. The lowest limits of detection achieved with the cellophane-based devices were calculated as 7 µM for H2O2, 2.7 mg dL−1 for creatinine, and 3.5 mg dL−1 for protein (human serum albumin). Graphical Abstract: [Figure not available: see fulltext.]

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Fabrication process development and basic evaluation of eggshell-based column packing material for reversed-phase preparative separation

Yoshii T., Sakama A., Kanamori K., Nakanishi K., Imai H., Citterio D., Hiruta Y.

Journal of Chromatography A （Journal of Chromatography A）  1688 2023年01月

ISSN  00219673

　概要を見る

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.

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Effect of the side chain composition of mixed-charge polymers on pH-selective cell–membrane interactions

Nakamura Y., Nasu M., Shindo Y., Oka K., Citterio D., Hiruta Y.

Polymer Journal （Polymer Journal）  55 （ 11 ） 1179 - 1188 2023年

ISSN  00323896

　概要を見る

Zwitterionic polymers are characterized by their antifouling ability due to their net-neutral charge and affinity for cancer cell surfaces. We developed six pH-responsive mixed-charge polymers and investigated the effect of their side chain hydrophobicity on their interaction with cancer cell membranes. The polymers were synthesized using [2-(methacryloyloxy)ethyl]trimethylammonium chloride, carboxylic acid monomers, and butyl methacrylate (BMA) as the neutral spacer moiety, and differences in hydrophobicity were created by altering the type of carboxylic acid monomer and ratio of the spacer. The surface charge of the polymers was designed to switch from net-neutral to positive in response to the weakly acidic tumor environment. This tuning occurred in a more cancer-like environment with the usage of a more hydrophobic anionic monomer and a higher ratio of BMA due to the weakening of electrostatic interactions. This trend was further confirmed by evaluation of the interaction between the polymers and heparin, an anionic polysaccharide that is used as a model for cancer cell surfaces. The affinity of the mixed-charge polymers for cells was then evaluated, and both the hydrophobicity and pH-responsive nature of these polymers led to pH-selective toxicity, which was assumed to be caused by the disruption of cell membrane integrity.

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Semi-quantitative microfluidic paper-based analytical device for ionic silica detection

Ogawa M., Katoh A., Matsubara R., Kondo H., Otsuka M., Sawatsubashi T., Hiruta Y., Citterio D.

Analytical Sciences （Analytical Sciences）  39 （ 8 ） 1361 - 1370 2023年

ISSN  09106340

　概要を見る

Silicate ions (SiO32−), or ionic silica, are known to cause silica scaling in industrial water applications when excess amounts are present; hence, concentrations must be monitored and kept at a constant low level. Ionic silica is conventionally measured by spectrophotometry in the form of its silicomolybdic complex based on the molybdenum blue reaction, but the operation process is complicated and not suitable for on-site detection. To solve these issues, microfluidic paper-based analytical devices (µPADs) have been gaining attention as portable, low-cost analytical devices suitable for on-site detection. The foldable origami type device described in this work enabled silica detection based on the molybdenum blue reaction, in the range of 50–1000 mg/L, with a practically detectable lowest concentration of 50 mg/L. The device showed selectivity for silicate ions and stability over 21 days when stored at 4 °C. The semi-quantitative analytical performance makes the proposed paper-based device attractive for on-site industrial monitoring. Graphical abstract: [Figure not available: see fulltext.].

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Nitrogen-Doped Graphene Quantum Dots as “Off-On” Fluorescent Probes in Paper-Based Test Kits for Selective Monitoring of Cyanide in Food

Malahom N., Ma-In T., Naksen P., Anutrasakda W., Amatatongchai M., Citterio D., Jarujamrus P.

ACS Applied Nano Materials （ACS Applied Nano Materials）  6 （ 13 ） 11144 - 11153 2023年

　概要を見る

We successfully developed a fluorometric paper-based test kit for the selective and sensitive determination of cyanide using nitrogen-doped graphene quantum dots (N-GQDs) as the fluorescent probe. Citric acid and tris(hydroxymethyl)aminomethane were precursors for the one-step synthesis of N-GQDs via in situ hydrothermal methods, providing a high quantum yield of 57.9%. The proposed mechanism uses a fluorescence turn-on approach. Specifically, the fluorescence of N-GQDs is quenched by the incorporation of Ag+ via a photoinduced electron transfer (PET). During the detection step, sulfuric acid converts cyanide (CN-) into hydrogen cyanide (HCN). The Ag+ species on the N-GQD surface then react with the evolved HCN via a coordination bond to form a silver cyanide complex, resulting in the fluorescence emission of the N-GQDs being turned back on. As a result, the fluorescence emission intensity of N-GQDs linearly increased with increasing CN- concentrations in the range of 0.5-25 mg L-1, with a limit of detection (LOD) of 0.08 mg L-1. Notably, the developed sensor has advantages in terms of simplicity, rapidity, low cost, and high selectivity toward CN-. The analytical performance of the test kit was also validated the performance of the test kit against a conventional precipitation titration method.

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Microoptics Integrating smartphone applications reader with paper-based analyser for accurate estimation of nickel in water

Nakagawa M., Tong G., Nath P., Citterio D.

28th Microoptics Conference, MOC 2023 2023年

　概要を見る

The estimation of nickel ions in water is critical from a human health perspective. Herein, we demonstrate the usability of a paper strip analyzer coupled with a smartphone application reader for accurate estimation of Ni2+ ions in water. The limit of detection of the proposed technique is found to be 0.764 μM.

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Improvement in sensitivity for lateral flow immunoassay of ferritin using novel device design based on gold-enhanced gold nanoparticles

Preechakasedkit P., Teekayupak K., Citterio D., Ruecha N.

Scientific Reports （Scientific Reports）  12 （ 1 ）  2022年12月

　概要を見る

This work introduces a low-cost adhesive tape combined with a hydroxylamine/polyvinyl alcohol/polyethylene oxide (HA/PVA/PEO) blend film to fabricate novel devices for improving sensitivity of gold nanoparticle (AuNP)-based lateral flow immunoassays (LFIAs) via two platforms: (1) LFIA device with integrated gold enhancement and (2) LFIA device with two independent sample inlets. The detection of ferritin has been used for proof-of-concept. The adhesive tape inserted in the devices assists to separate two solutions independently flowing from two different inlets toward a nitrocellulose membrane. On-device gold enhancement was achieved by the enlargement of AuNPs via the catalytic reaction of KAuCl4 and HA using the HA/PVA/PEO blend film easily prepared via a solution-casting technique, which could delay the flow of HA released from the film for 180s and improve storage stability of the device. Under optimal conditions evaluated by naked eyes, the gold enhancement (LOD = 0.5 ng/mL) and double-sample inlet (LOD = 2 ng/mL) devices exhibited 20-fold and fivefold higher sensitivity respectively than a conventional device, verifying the sensitivity improvement. Furthermore, the proposed device was successfully detected ferritin in human serum samples within 10 min via naked-eye observation, exhibiting rapidity and simplicity of use, and the capability to perform on-site assays.

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Amphiphilic Copolymer-Modified Eggshell-Based Column Packing Materials for the Preparative Separation of Basic Drugs

Yoshii T., Mochida M., Kaizu K., Soda Y., Kanamori K., Nakanishi K., Sato T., Imai H., Citterio D., Hiruta Y.

ACS Applied Polymer Materials （ACS Applied Polymer Materials）  4 （ 10 ） 6949 - 6957 2022年10月

　概要を見る

A way to utilize eggshells is desired because most of the eggshell waste is disposed of in landfills. Despite eggshells having attractive features such as a thermodynamically stable porous crystalline structure, their effective utilization as a functional material has yet to be demonstrated. Herein, we developed an alkali-stable column packing material for reversed-phase preparative high-performance liquid chromatography (HPLC) based on eggshells. Nanocrystals and biopolymers present on eggshell powder were removed by surface treatment with buffered ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite solutions, resulting in bare calcium carbonate with a uniform porous surface. The obtained particles were modified with poly(maleic acid-alt-1-octadecene) (PMAcO) having hydrophobic alkyl chains and anionic carboxylate groups. The prepared PMAcO-modified eggshell-based material was applied to an HPLC column, and its performance is evaluated. The column succeeded in the separation of alkylbenzenes, steroids, and diastereomers. It was also used for the separation of basic tricyclic antidepressants in an alkaline mobile phase, showing excellent retention capacity and alkali stability. This study demonstrates the potential of eggshell utilization, providing not only a practical stationary phase for HPLC but also a solution for recycling calcium carbonate-based biomineral waste.

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Quantitative evaluation of reversed-phase packing material based on calcium carbonate microspheres modified with an alternating copolymer

Kaizu K., Mochida M., Imai H., Citterio D., Hiruta Y.

Journal of Chromatography A （Journal of Chromatography A）  1677 2022年08月

ISSN  00219673

　概要を見る

Considering the vulnerability of silica gel to alkaline mobile phases, a highly alkaline stable stationary phase for HPLC is required to separate basic compounds with high separation efficiency. To address this issue, we have developed a high alkaline stable packing material (CaCO3-PMAcO) based on mesoporous calcium carbonate microspheres modified with poly(maleic acid-alt-1-octadecene). In this study, we report further investigation of the separation performance of CaCO3-PMAcO column by systematically evaluating the effects of particle size and chromatographic conditions. Based on the theory of the van Deemter equation, the separation efficiency was related to the size of CaCO3-PMAcO particles (2.9 – 5.7 µm). The evaluation of thermodynamics of retention by changing the column temperature from 20 °C to 45 °C implied that the retention mode was dominated by hydrophobic interaction associated with the exothermic enthalpy changes (-11.1 to -12.5 kJ/mol). The results of column selectivity tests revealed that the CaCO3-PMAcO column had hydrophobic selectivity comparable to C18 silica gel columns (αP/B; CaCO3-PMAcO column: 1.53, C18 column: 1.69), and higher shape/steric selectivity (αTri/Ter; CaCO3-PMAcO column: 1.56, C18 column: 0.955). In practice, the CaCO3-PMAcO column could be applied to the separation of not only alkylbenzenes and polycyclic aromatic hydrocarbons, but also to basic tricyclic antidepressants by using an alkaline mobile phase (pH 12).

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Organelle-selective near-infrared fluorescent probes for intracellular microenvironment labeling

Kurutos A., Shindo Y., Hiruta Y., Oka K., Citterio D.

Dyes and Pigments （Dyes and Pigments）  204 2022年08月

ISSN  01437208

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The design, synthesis, and bio-applicability of a series of 8 near-infrared (NIR) molecular probes with tunable hydrophilic/hydrophobic properties are described in this paper. The title dyes comprise of symmetric heptamethine cyanines with various N-quaternary heterocyclic moieties including indole, benzo[e]indol, benzothiazole and naphtho[1,2-d]thiazole, along with an extra positive charge in close proximity to the polymethine backbone. Combined UV–Vis and spectrofluorimetric techniques in both organic and aqueous-buffered solutions were employed to investigate the optical properties of the NIR chromophores. Two of the fluorophores emerged remarkable fluorescence quantum yield (Φ = 0.083), while their brightness was found up to 3-fold that of the FDA approved Indocyanine green – ICG (Φ = 0.027). What is more, two of the dyes appear to outperform the later clinical benchmark fluorophore in terms of photostability. While the ICG was completely faded after 1 h of constant irradiation, nearly 70% of the original emission signal could be observed for one of the novel molecular probes. The log Pow values determined by a combination of HPLC and UV–Vis (partition between 1-octanol/water) cover a relatively large range, from −2.14 up to 7.92. Investigation on the cytotoxicity by means of the MTT assay revealed, that present dyes are suitable for bioanalytical purposes and are safe to use. This paper also includes fluorescence imaging and cellular applications, such as labeling of HeLa cervical cancer cells. The results from the confocal microscopy reveal the superior staining capability of current heptamethines since they show significantly brighter signal than the commercial ICG probe. An organelle-selective fluorescent staining was achieved depending on the dye chemical structure. Co-localization within the intracellular microenvironment (linear or granular - cytosol, mitochondria, lysosome, endoplasmatic reticulum or Golgi apparatus) was confirmed without interference, employing several conventional organelle dye trackers. To the best of our knowledge, heptamethine dyes represent the first example of a series NIR emitting probes, that exhibit rational structural modifications aiming to fine-tune the hydrophilic/hydrophobic properties furnishing organelle-targeted staining.

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