Published papers

© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This work reports on fully integrated “sample-in-signal-out” microfluidic paper-based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

This journal is © The Royal Society of Chemistry. We developed a biomineral-inspired hybrid material composed of CaCO3 and an organic polymer as a column packing material for HPLC. This material combines a hierarchical mesoporous structure and the functionality of the polymer. The surface of monodispersed mesoporous CaCO3 microspheres was modified with poly(maleic acid-alt-1-octadecene) (PMAcO) comprising hydrophobic alkyl chains and anionic carboxylate groups. PMAcO adsorbed onto the surface of CaCO3 through electrostatic interaction between Ca2+ sites and carboxylate groups, resulting in an octadecene coated microsphere interface. These microspheres were applied as a HPLC column and exhibited reversed-phase retention behavior in the separation of alkylbenzenes. This column showed high alkaline mobile phase resistance compared with the conventionally applied ODS column packing material. Quantitative analysis of the basic antidepressants clomipramine and imipramine spiked into whole blood was achieved with an alkaline mobile phase, demonstrating the potential of the biomineral-inspired material as a HPLC stationary phase for practical applications in routine analyses of basic drugs requiring alkaline mobile phases.

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Stimuli-responsive smart materials are a key to the realization of next-generation medical technologies. Among them, the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) is attracting particular attention because it is easy to use in physiological conditions. PNIPAAm-grafted surfaces can undergo temperature-modulated cell adhesion and detachment without proteolytic enzymes, and can be used as cell-separating materials through selective cell adhesion/detachment. However, cell detachment at reduced temperatures is problematic because it takes several hours. A novel thermoresponsive crosslinked microfiber system that can greatly reduce the cell detachment time is introduced in this study. The crosslinked fibers provide temperature-dependent volume change, and enable cell detachment within 10 min of reducing the temperature, which is one-sixth of the time required in previous studies. The prompt cell detachment is thought to arise from a completely new mechanism derived from fiber swelling. This system will make a significant contribution as a novel cell manipulating system for next-generation medical technology.

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. A challenge for paper-based cation sensors relying on classical carrier-based ion-selective optodes (ISOs) is their pH-cross response caused by the use of H+-sensitive chromoionophores as optical signal transducers. This work demonstrates fully pH-independent fluorescence-based calcium detection with a paper-based plasticizer-free ISO. To achieve a pH-independent assay, a solvatochromic dye (SD) instead of a traditional H+-sensitive chromoionophore has been applied to the paper-based ISO by means of inkjet printing technology. The detection principle depends on an ionophore-driven phase-transfer ion-exchange reaction between target cations and the positively charged SD, which no longer involves H+ in the optical signal transduction process. The developed paper-based ISOs with the SD resulted in Ca2+ concentration-dependent response curves not affected by the sample pH (pH 6.0, 7.0, and 8.0). The dynamic range obtained for Ca2+ detection was from 10−5 to 1 mol L−1 with a detection limit of 19.3 μmol L−1. Additionally, excellent selectivity derived from the used ionophore has been confirmed. As a simple practical application, the determination of Ca2+ in mineral water has been achieved without the pH-buffering process required for conventional cation-exchange ISOs.

© 2019 American Chemical Society. The magnesium ion (Mg2+) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg2+, there is a great need for the development of Mg2+-selective fluorescent probes. However, conventional Mg2+ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg2+-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged β-diketone as a selective binding site for Mg2+ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg2+. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg2+. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg2+ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg2+ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg2+ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multi-color imaging of intracellular Mg2+.

© 2019 The Royal Society of Chemistry. A disposable enzymatic electrochemical biosensor for amperometric organophosphorus pesticide determination based on a screen-printed carbon electrode (SPCE) platform was successfully developed. The working electrode consists of acetylcholine esterase (AChE) immobilized onto the surface of multi-walled carbon nanotube (MWCNT), chitosan (CS) and gold nanoparticle (AuNP)-modified SPCE. Ferricyanide was used as a redox mediator in solution. Two linear dynamic response ranges from 0.01 to 10 μg L-1 and from 10 to 100 μg L-1 were found for paraoxon-ethyl, with a detection limit of 0.03 μg L-1 (calculated as the amount of pesticide resulting in 10% of enzyme inhibition). High sensitivity was achieved due to a synergistic effect of AuNPs and MWCNTs deposited on the SPCE surface. Moreover, 83% of enzyme activity was retained after a dry storage period of 49 days (4 °C). Finally, the results of the electrochemical biosensor for paraoxon-ethyl-spiked spinach sample analysis showed a good agreement with those obtained from conventional HPLC.

© 2019 The Japan Society for Analytical Chemistry. Flow control-based paper devices have recently shown great potential for point-of-need analysis, since they allow for the easy operation of multi-step assays by minimizing user operation. In this work, a wax printing method was evaluated as a means to control liquid flow in 3D microfluidic paper-based analytical devices (μPADs). The resulting flow controlbased 3D μPADs were applied to determine paraoxon-ethyl as a typical organophosphate pesticide model system. The analytical procedure is as simple as applying a 200-μL sample solution, resulting in reproducible (relative standard deviation of colorimetric signals from 6 independently fabricated devices, 2.63%) colorimetric signals within 1 h of the assay time with the limit of detection (LOD) reaching 25.0 μg/L. Finally, results obtained for pesticide-spiked water samples analyzed by flow control-based 3D μPADs showed good agreement with those from a conventional HPLC analysis with UV detection.

© 2018 American Chemical Society. A simple, portable, and cost-effective paper-based colorimetric assay is developed for ascorbic acid (AA) with excellent properties, such as fast response, high sensitivity, high selectivity, and good stability. AA is an important nutrient for human life, and adequate quantification of AA is required for controlling the intake of AA for health management. Moreover, point-of-care analysis, which is a simple, easy-to-use, and cost-effective analysis method for unskilled users, has been a focus in the medical and health care fields. We fabricate a colorimetric paper-based assay with poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) layer-by-layer self-assembly containing silver ions for the point-of-care analysis of AA. The color of the assay is changed from white to yellow and then to dark-brown after immersion in AA solution because of the reduction of silver ions to silver nanoparticles by AA and the localized surface plasmon resonance of the generated silver nanoparticles. The assay shows a fast response to AA of only 60 s and a sensitivity to AA (limit of detection is 0.4 ppm). Furthermore, the colorimetric assay exhibits a high selectivity for AA compared with other compounds contained in human fluid and various drinks. The concentration of AA in various commercial products is quantified by the assay with the same accuracy as high-performance liquid chromatography. The design of the PAH/PAA multilayers containing silver ions for a fast response, highly sensitive, and selective colorimetric assay will contribute to the development of point-of-care analysis for the early detection of diseases as well as controlling the intake of nutrients.

© 2019 The Royal Society of Chemistry. Although the determination of calcium ions (Ca 2+ ) is of high importance to monitor water hardness, currently available devices for on-site analysis suffer from a lack of user-friendliness and sensitivity. This work demonstrates fully inkjet-printed and low-cost microfluidic paper-based analytical devices (μPADs) for the simple naked-eye colorimetric determination of calcium ions (Ca 2+ ) in drinking and tap water samples. The quantification of Ca 2+ relies on visual readout of the length of a colour-changed detection channel modified with ionophore-doped ion-selective optode nanospheres (nano-optodes), eliminating the requirement of a scanner or a camera. All fabrication steps for deposition of assay reagents have been performed by means of a simple desktop thermal inkjet printer, which is expected to contribute to highly batch-to-batch reproducible device preparation. The detectable Ca 2+ concentrations between 0.05 mmol L -1 and 5 mmol L -1 cover the range recommended by the International Organization for Standardization (0.05-2.5 mmol L -1 ) and the World Health Organization (WHO) guideline for Ca 2+ quantification in drinking water (less than 5 mmol L -1 ). The lowest concentration of Ca 2+ detectable by the naked eye was found to be 0.05 mmol L -1 , which is below the value achieved with previously reported paper-based devices. μPAD quantified Ca 2+ concentrations in tap or drinking waters were within 15% error of the results obtained with a classical complexometric titration. Hence, distance-based μPADs relying on nano-optodes are sensitive and reproducible tools for equipment-free on-site assaying of Ca 2+ in real samples.

© 2019 Elsevier B.V. Liposomes composed of egg phosphatidylcholine and cholesterol were modified with the temperature-responsive polymer poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)), and exhibited reversible surface properties with temperature. Completely reversible liposome aggregation due to P(NIPAAm-co-DMAAm) hydration/dehydration was demonstrated over four successive cycles of heating and cooling. The P(NIPAAm-co-DMAAm) polymer was hydrated during cooling, which dispersed the liposomes. The rigidity of the liposomal membrane was one of the factors in the reversible aggregation, as was the modification density of the polymer on the liposomes. A low density on relatively rigid liposomes could maintain the polymer property of reversible hydrated layers below critical solution temperature (LCST) boundary. Above the LCST, temperature-responsive polymers could also transport negatively charged liposomes into cells. The reversible behavior of the temperature-responsive polymer-modified liposomes has not been reported previously and could enable new applications for switching deposit forms as alternative drug carriers.

A pH-responsive mixed-charge polymer consists of uniformly distributed cationic and anionic units in the polymer chain. Carboxylic acid monomers are generally used as anionic monomers. The pH responsive range is confined to a relatively narrow pH of Â5, which is limiting the biological application of pH-responsive polymers. Therefore, we investigated the effect of a series of carboxylic acid and neutral monomers on the pKa value of pH-responsive mixed-charge polymers. We synthesized the polymers using [2-(methacryloyloxy)ethyl]-trimethylammonium chloride, carboxylic acid monomers, and neutral monomers as the spacer moiety. As the monomer hydrophobicity and the composition ratio of neutral monomers increased, the pKa shifted to higher values. Finally, the polymer pKa values were tuned from 4.6 to 6.1. We confirmed that the pH-dependent interaction between anionic tissue constituent and pH-responsive mixed-charge polymers depends on their pKa.

Copyright © 2020 American Chemical Society. This paper introduces the concept of "Drawing-PADs" (Drawing paper-based microfluidic analytical devices) allowing to intuitively evaluate the urinary albumin (Alb) index, a clinically important parameter used for the early detection of renal deficiencies related to diabetes, among others. To enable regular monitoring of the Alb index, a simple examination method suitable for self-diagnosis is highly desirable. The Drawing-PADs rely on the simultaneous naked eye detection of Alb and creatinine (Cre) on a single device according to the distance-based microfluidic PAD (μPAD) approach. The Alb index is visualized by simply drawing a straight line connecting the top of two color-changed assay channel sections (Alb and Cre channels), followed by visually confirming the position of the intercept of the drawn straight line. The semiquantitative Alb index evaluation performed with Drawing-PADs does not require any equipment such as a camera, software, or a color reference chart. The obtained results are independent of the sample volume and are not influenced by changes in the absolute Alb and Cre concentrations caused by urine excretion variations, making spot urine assays possible. Classification of Alb index values according to clinically relevant criteria (normoalbuminuria, microalbuminuria, and macroalbuminuria) is readily achieved within 15 min and has been validated for 15 human urine samples including diabetic patients and healthy volunteers.

Copyright © 2020 American Chemical Society. Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

Copyright © 2020 American Chemical Society. This work describes a colorimetric signaling approach for competitive lateral flow immunoassays (LFIAs) enabling sensitive and semiquantitative direct visual result readout in the form of "text", demonstrated on the example of 8-hydroxy-2′-deoxyguanosine (8-OHdG) detection. The distinctive feature of the developed text-displaying LFIA (TD-LFIA) is the test zone system consisting of a combination of two types of inkjet-deposited capture molecules referred to as "mask antigen"and "text antibody", allowing for sensitive turn-on signaling as opposed to the inverse response of conventional competitive LFIAs. The user operation is limited to sample application, followed by direct reading of assay results written in text after approximately 10 min. TD-LFIAs enabled the visual detection of 8-OHdG at concentrations down to 3 ng/mL, which is a 2-3 orders of magnitude lower visual detection limit than that achieved with the corresponding conventional design and is comparable to the existing LFIAs relying on external signal readout equipment. Highly reproducible observer-independent assay performance was confirmed, and the result interpretation is not influenced by sample color and readout timing. Making use of customizable threshold settings for text appearance, a device for semiquantitative assays was developed and successfully applied to the detection of 8-OHdG at four concentration levels (trace, low, medium, and high) in 54 human urine samples within the clinically relevant concentration range. The sensitive and intuitive signaling method of the developed system offers great potential for an alternative competitive LFIA platform suitable for real-world point-of-care testing applications.

© 2020 Elsevier Ltd In this report, we wish to present the rational design, synthesis, and study of optical characteristics of near-infrared (NIR) molecular probes emitting over 800 nm. The title pH sensing platforms consist of a piperazine moiety serving as a hydronium ion receptor, coupled with a rigidified symmetric heptamethine backbone. The spectrally fine-tuned response of the fluorophores was studied in both organic solvents and aqueous buffer solutions, employing combined UV–Vis and steady state fluorimetric techniques. In parallel, computational approach and optical spectroscopy were employed aiming to ascribe the origin of spectral behavior of the amino-substituted heptamethines. Herein, we also investigated the halochromic effect of the differently substituted piperazines on the spectral properties of the NIR cyanine dyes. Under acidic conditions, the protonation of the piperazine moiety promoted a gradual bathochromic shift in the absorption spectra. The Δλ between the neutral and the protonated forms are indirectly related to the modifications applied to the proton acceptor. The calculated pKa values (5.13–7.06) were found to be dependent on the dye chemical structure. A good linear correlation was established between the pKa of the newly synthesized dyes and that of the reported piperazine moieties. This aspect allows the future expansion of the current facile strategy since sensing chromophores with predicted pH response could readily be designed based on pKa databases for substituted piperazine derivatives. A detailed literature study along with NMR and computational methods were found to be in agreement with our assumption, that both nitrogen atoms of the 1,4-disubstituted amine are involved in the sensing process. The covered pKa range allows us to recommend the title fluorophores as promising potential candidates for qualitative analysis of biological samples. A cytotoxicity study by the MTT assay showed that the NIR dyes are safe and applicable for bioanalytical purposes at dye concentrations up to 10 μM. Fluorescence imaging and cellular applications, including staining and visualization of HeLa cervical cancer cells was successfully demonstrated.

Fluorescence and bioluminescence have been actively applied to various chemical sensors and biosensors, including to cellular/in vivo imaging. On application to the detection of specific targets, it is necessary to develop fluorescent/bioluminescent probes that selectively respond to the target analytes such as molecules, ions or cells by changing their optical properties. The authors have focused on the development of small molecular probes by means of molecular design based on the desired function. In this paper, we describe the approaches to molecular design of fluorescent/bioluminescent probes and their applications, including the introduction of our recent research works. Finally, a future outlook for further functionalization of small molecular probes is provided.

The fabrication of accentuated gold microstructures is demonstrated by multi-photon photoreduction inside an anionic fluorophore-containing hydrogel. We attempted to facilitate gold-ion photoreduction near the focal point of laser pulses, expecting the donation of electrons by the oxidation of fluorophores in the vicinity of gold ions. The presence of anionic FITCdextran also inhibited the spontaneous reduction in untargeted zones, which is attributed to the coordination of gold ions and the anionic FITC-dextran. Simultaneous facilitation and inhibition are promising for the fabrication of dense metal microstructures in the targeted zone while maintaining the hydrogel's light permeability.

The synthesis and application of stimuli-responsive polymer materials have been extensively studied. Among stimuli-responsive polymers, thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) is the most widely investigated. PNIPAAm-based polymers undergo a reversible hydrophilic/hydrophobic phase transition in response to temperature. In addition, by introducing sites that are responsive to physical and chemical stimuli into PNIPAAm-based polymers, they also undergo phase transitions in response to stimuli, such as light, pH, oxidation/reduction, and enzyme activity. In this focus review, recent advancements in the applications of stimuli-responsive polymers based on PNIPAAm in biomedical fields are summarized, with an emphasis on our own research. In particular, a summary of the design of polymers for application in the separation and purification of (bio)pharmaceutical products and controlled cellular uptake is provided. First, temperature-responsive chromatography with PNIPAAm-modified silica beads is introduced. Thereafter, temperature- and pH-responsive polymers based on PNIPAAm used in imaging and drug delivery applications are discussed. Finally, the conclusions are presented, and future perspectives for the biomedical applications of stimuli-responsive polymers are discussed.

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.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed. Graphical abstract: [Figure not available: see fulltext.]