論文

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.]

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.

Dynamic tuning of nanoscale coloration by exploiting localized surface plasmon resonance of gold nanoparticles (AuNPs) combined with an interference coloration mechanism is demonstrated experimentally. When interference between the scattering field from AuNPs and the reflected field from the substrate is observed under back-scattering white-light microscopy, the AuNPs exhibit various colors depending on their distance to the substrate. When the numerical aperture of the microscope objective is optimized, much greater coverage of the color space than was achieved with previously reported plasmon-based approaches is attained. Also, color tunability is examined by exploiting the temperature-induced volume change of a temperature-responsive hydrogel with embedded AuNPs to dynamically modify the distance to the substrate.

Bioluminescence imaging has recently attracted great attention as a highly sensitive and non-invasive analytical method. However, weak signal and low chemical stability of the luciferin are conventional drawbacks of bioluminescence imaging. In this review article, we describe the recent progress on the development and applications of bioluminescent probes for overcoming the aforementioned limitations, thereby enabling spatiotemporal trans-scale imaging. The detailed molecular design for manipulation of their luminescent properties and functions enabled a variety of applications, including in vivo deep tissue imaging, long-term imaging, and chemical sensor.

Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer. Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color complementary metal-oxide semiconductor camera. Overall, this method enables simple and simultaneous observation of multiple biological targets and phenomena.

Nuclear receptors (NRs) play pivotal roles in functionally diverse cell signaling cascades, regulating metabolism and homeostasis. This study introduces a broadly applicable molecular imaging platform for NR activities based on four rationally designed single-chain bioluminescent probes named P1–P4. As all the ligand binding domains (LBDs) of NRs are highly conserved, the probe portfolio was exemplified using the LBD of peroxisome proliferator-activated receptor γ (PPARγ-LBD), i.e., the LBD was centrally positioned and fused with luciferases and/or fluorescent proteins based on the schemes of the protein-fragment complementation assay (PCA), molecular strain (MS) probe, circular permutation (CP) probe, and bioluminescence resonance energy transfer (BRET) system. In the developing process, we identified the optimal dissection sites for the marine luciferase RLuc for PCA systems and validated them in in vitro and in vivo studies in response to various ligands. Among the probes, P3 and P4 series probes exhibited strong BL intensities in response to a PPARγ agonist with signal-to-background ratios of maximally 14-fold. The animal study using furimazine (FMZ) substrate analogs such as Ad-FMZ showed that the probes can sustain agonist-dependent BL signals for up to 24 h in animal models. Considering the biological importance of NRs, the molecular imaging platform with the portfolio of probes developed in this study can contribute to interrogate many NR-related cell signaling pathways by replacing the PPARγ-LBD with LBDs from other NRs in the probes.

© The Royal Society of Chemistry 2020. Bioluminescence is a powerful imaging modality for monitoring biological phenomena bothin vitroandin vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.

Monoclonal antibodies (mAbs) have become essential in modern therapeutics, offering high specificity and efficacy in treating diseases such as cancer, autoimmune disorders, and infectious diseases. With the increasing demand for mAbs, robust analytical and purification technologies are critical to ensuring their safety, efficacy, and consistency. This review highlights recent advances in the application of HPLC for both mAb analysis and purification. In the analytical domain, techniques such as size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEX), and reversed-phase chromatography (RPLC) are explored for their role in evaluating mAb structural attributes and post-translational modifications (PTMs). These methods are indispensable for ensuring stability and functionality while meeting stringent regulatory requirements. In the context of purification, Protein A affinity chromatography remains the gold standard for initial capture of mAb; however, challenges such as high cost and harsh elution conditions have prompted the development of alternative purification technologies. IEX, HIC, multimodal, and membrane chromatography are also critical in achieving high-purity mAb products through multistep workflows, including intermediate purification and polishing stages. This review emphasizes the central role of HPLC in addressing the complex challenges of mAb manufacturing and characterization. By integrating established and emerging chromatographic techniques, it provides insights into the future directions of therapeutic antibody development.

Thermo-responsive polymers are widely applied in various fields, including drug delivery and surface coating. Dual thermo-responsive polymers, which exhibit both lower and upper critical solution temperatures (LCST and UCST), enable notable temperature-dependent behaviors within a tunable range. However, the structure–property relationships governing dual temperature-responsiveness are still insufficiently understood, particularly for polymers including pH-sensitive zwitterionic groups. In this study, LCST–UCST polymers, P(MEO2MAxMEOMAyGAASt20), composed of mono/di(ethylene glycol) methyl ether methacrylate (MEOMA/MEO2MA) and a zwitterionic monomer derived from glutamic acid (GAASt), were designed. By varying the ethylene glycol (EG) side chain length, pH, and salts, it was investigated how the balance between hydrophilic and electrostatic interactions governs the thermo-responsive behavior of the copolymers. In 10 mM phosphate buffer (PB, pH 7), UCST behavior was observed with only P(MEOMA79GAASt21), likely because shorter EG chains increased the relative contribution of zwitterionic units. The UCST of this polymer increased with the addition of salt, unlike typical zwitterionic UCST-type polymers. Polymers containing MEO2MA exhibited LCST behavior in 10 mM PB (pH 7) containing 100 mM NaCl, and the LCST decreased with shorter EG chains and kosmotropic ions. Under acidic and basic conditions, the thermo-responsive transition was not observed. Furthermore, P(MEO2MA19MEOMA61GAASt20) exhibited LCST–UCST behavior induced by NaCl. The diameter of the polymer in solution varied with temperature: terpolymer chains below the LCST, the largest aggregates between the LCST and UCST, and small globules above the UCST. Our findings provide insight into the molecular design strategies for thermo-responsive polymers in bioapplications.

Abstract: Near-infrared I (NIR-I: 650–950 nm) fluorescence imaging is a powerful tool for deep-tissue biological imaging, addressing the limitations of photon penetration depth in the visible-light region. Over the past decade, NIR imaging has extended to the near-infrared II (NIR-II: 1000–1700 nm) region, offering high-resolution and low background imaging by suppressing light scattering, and autofluorescence of tissues. Near-infrared fluorescent probes from NIR-I to NIR-II, with diverse functionalities, are increasingly utilized across biological fields to meet various detection needs and to explore physiological events in real time and spatial dimensions. This review discusses recent advancements in small-molecule NIR fluorescent dyes and probes, particularly those based on cyanine and rhodamine scaffolds, highlighting examples of their applications in bioimaging.

The quantification of electrolytes in biological fluids plays an important role for evaluating the human health condition. This work proposes a disposable and simple microfluidic paperbased analytical device (µPAD) for the quantitative colorimetric detection of salivary Na+, K+ and Ca2+, relying on ion-selective optodes (ISOs). Nanoscale ISOs composed of pluronic F-127 surfactant-based micelles were used as a colorimetric indicator system, reducing the assay time compared to the previously reported bulk membrane approach. Devices consist of three functionalized paper layers assembled by lamination, with the optical signal read out from the bottom layer. Saliva was chosen as the biological specimen because it is painlessly collected and easy-to-handle, which makes it suitable for POC assays. Simultaneous Na+, K+ and Ca2+ detection by a single µPAD was successfully confirmed by using aqueous metal ion samples with various concentrations of Na+, K+ and Ca2+ within the biologically relevant range. The simultaneous detection of the three cationic species enabled the correction of the three ion quantities by eliminating the interference from other ions using the calculated selectivity coefficients. For proof-of-concept, Na+ quantification in a human saliva sample was conducted by the developed µPADs. The calibration curve for this assay was prepared by applying artificial saliva to the µPADs. The concentrations of Na+ in spiked human saliva were also measured by the µPADs. The obtained recovery rates were 78-132 %, which indicates the quantification of the added Na+ in the real sample matrix. Finally, this µPAD was successfully integrated into a smartphone-based assay, which enables salivary electrolyte quantification in resource poor settings. The developed µPADs satisfy the criteria of low-cost (< $0.3/device), rapidity (< 5 min) and simple analytical procedure compared to the conventional ion-selective electrode (ISE) method.

In therapies involving therapeutic antibodies, therapeutic drug monitoring (TDM) has the potential to substantially increase clinical efficacy and to enable more economical drug use, because the clearance from the bloodstream after administration is patient dependent. Since TDM must be performed continuously and over extended time periods, it should ideally be achieved through an economical and easy to perform point-of-care (POC) assay. Here, we introduce a new analytical method for POC TDM of the clinically important therapeutic antibody cetuximab, using microfluidic thread-based analytical devices (µTADs) as a platform for a bioluminescence resonance energy-transfer (BRET) switching sensor protein for simple quantitative antibody detection. The µTAD is pre-treated with NaCl for blood plasma separation and a BRET switching sensor protein and its substrate for cetuximab detection, integrating all elements required for a reagent-free, single-step assay. A change in bioluminescence emission color depending on cetuximab concentrations occurs after whole blood application. The device design enables the quantification of cetuximab within the therapeutically relevant blood concentration range in 2.5 min using only 2 µL of whole blood. The results obtained are independent of the blood volume. This µTAD can be stored for about one month at − 20 °C, a temperature compatible with commonly available freezers. The application of mathematical morphology recognition (MMR)-integrated image processing techniques for the automatic selection of regions of interest (ROIs) yields results with minimal error. The simplicity of use, rapidity, and cost-effectiveness of this µTAD are expected to offer the possibility of achieving TDM of therapeutic antibodies at POC for individual patients.

Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a “traffic light type μPAD”, enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/green & yellow/yellow/yellow & red/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H2O2. User operation is limited to sample application, followed by observing a clear and time-independent traffic light signal after approximately 10-30 min. Multiple factors influencing the H2O2 concentration-dependent appearance of a specific traffic light signal were studied. Making use of the possibilities for customising the concentration threshold levels for traffic light colour appearance, quantification of glucose at 5 levels in a clinically relevant concentration range was demonstrated in artificial urine as a model proof-of-concept. This platform is expected to offer the possibility for the future detection of other important metabolites.

Poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) is a water-soluble polymer in which multiple short ethylene glycol (EG) chains are grafted along the hydrophobic methyl methacrylate backbone. We investigated whether POEGMA could be used as a drug carrier for tumor diagnosis. For this purpose, we synthesized POEGMA derivatives with different molecular weights (11, 21, and 30 kDa) and EG side chain lengths, and labeled them with radioisotopes (indium-111; 111In) or fluorescence dyes (indocyanine green; ICG) as signal emitters. Fluorescence microscopy studies using colon26 tumor cells treated with ICG-labeled POEGMA derivatives revealed that the POEGMA derivatives were efficiently taken up by tumor cells compared to the control, poly(ethylene glycol) (PEG), suggesting the involvement of a POEGMA-specific cellular uptake pathway. The radiolabeled POEGMA derivatives were intravenously injected into colon26 tumor-bearing mice, and their biodistribution was evaluated. As the molecular weight of POEGMA derivatives increased, the circulating time was prolonged, and their accumulation in the liver and spleen increased. In particular, 21 kDa POEGMA showed excellent tumor uptake and tumor-to-normal tissue ratio. Therefore, we investigated the effect of EG chain length on the tumor uptake of POEGMA (21 kDa) and observed high tumor uptake for all POEGMA derivatives; however, relatively higher abdominal uptake was observed for POEGMA derivatives with longer EG chain lengths. Finally, in vivo fluorescence imaging using optimized POEGMA derivatives labeled with ICG clearly visualized the tumor tissues, suggesting that the POEGMA derivatives could be suitable drug carriers for sensitive tumor diagnosis.

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.

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).