TIMP-1 (Tissue Inhibitor of Metalloproteinases-1) is a protein involved in regulating extracellular matrix (ECM) degradation. It is recognized as a significant biomarker for cancer diagnosis. This study aimed to develop and characterize a single-stranded DNA (ssDNA) aptamer targeting human TIMP-1 protein with high affinity and specificity. A magnetic beads-based SELEX process combined with qPCR was used to select aptamers over seven rounds. The enriched ssDNA library was analyzed using high-throughput sequencing to identify candidate sequences, and these sequences were characterized using surface plasmon resonance (SPR) and binding assays to evaluate their affinity and specificity. The selected ssDNA aptamer demonstrated a dissociation equilibrium constant (K) of 0.41 nM and a very slow off-rate, enabling effective capture of TIMP-1 in serum samples. Furthermore, a chemiluminescent aptasensor was developed for TIMP-1 detection, which exhibited high specificity and a broad linear detection range from 1 to 500 ng/mL in human serum. The developed ssDNA aptamer targeting TIMP-1 shows high affinity and specificity, and the chemiluminescent aptasensor demonstrates promising potential for clinical diagnosis of TIMP-1 levels in human serum.

"Liquid gold" has been traditionally used for over a century to decorate ceramicware, but its chemical composition has not been thoroughly investigated. One of the keys to successfully characterizing liquid gold, which is a complex mixture, is to distinguish Au-containing products from other chemicals. In this paper, we propose a separation based on the difference in collision cross section, of which chemicals with heavy atoms are relatively smaller than those without in ion mobility-mass spectrometry (IM-MS). Chemicals containing a single Au atom (and Pt atom) were successfully separated from other species in the two-dimensional distribution map for IM-MS. By a detailed analysis of the spectra obtained by IM-MS/MS with collision-induced dissociation before and after IM separation, we found that liquid gold (gold resinate) was a mixture of a series of (1) Au reacted with α-pinene-related units and (2) Au reacted with abietic acid units. α-Pinene and abietic acid are the main components of turpentine and rosin, the raw materials of liquid gold as reported previously (Anal. Sci. 2024, 40, 133-139). All Au-containing species contain sulfur atoms. Species of Au reacted with α-pinene-related units with different degrees of unsaturation and oxidation have also been identified. Liquid gold, a complex mixture of chemicals containing Au, has been successfully analyzed compositionally.

The diverse functional roles of RNA within cells have led to a growing interest in developing RNA-binding fluorescent probes to investigate RNA functions. In particular, the probes for double-stranded RNA (dsRNA) structures are of significant value given the importance of the secondary and tertiary RNA structures on their biologic functions. This review highlights our recent efforts on the development of triplex-forming peptide nucleic acid (TFP)-based probes for fluorescence sensing of dsRNA structures. We demonstrated that the forced intercalation of asymmetric cyanine dyes integrated as base surrogate within the probes was useful for achieving significant light-up response toward target dsRNAs. We also showed that the TFP probes conjugated with small RNA-binding molecules facilitated the fluorescence sensing of biologic relevant dsRNAs containing unpaired nucleobases. The binding and fluorescence signaling functions of such probes were discussed, emphasizing their potential as analytical tools for studying dsRNA structures.

A digital-movie-based flow colorimetry for pH measurement using a universal indicator has been applied to the end point detection of acid-base titrations. A two-channel flow system of feedback-based flow ratiometry, primarily consisting of two peristaltic pumps, a digital microscope-based detector, and a laptop computer, was constructed; a Visual Basic.NET program written in-house was used for automating the analytical processes. While maintaining the total flow rate (F) constant, a titrand solution was merged with a titrant solution, both containing the same concentration of Van Urk's universal indicator, under varying flow ratios. Downstream, the video image was captured with a digital microscope and its color was expressed as RGB values, hue, and luminance. The end point was determined from the rapid change of hue reflecting the color transition of the universal indicator around the equivalence point. A stepwise titration of multivalent acid (i.e., HPO) was also possible by setting hue values corresponding to the first (1st) and second (2nd) equivalence points as criteria to determine the respective end points. The hue-based approach was validated by the titrations of CHCOOH and HPO (1st and 2nd equivalence points) with 0.1 mol dm NaOH and to those of NaOH and NH with 0.1 mol dm HCl. The method was applied to determine Japanese Pharmacopoeia (JP) borax and JP citric acid. The respective assay results were 100.0 ± 0.0% and 100.0 ± 0.0%, both meeting the JP specifications. The developed method is simple, high throughput (1 titration/min), versatile, and does not require indicator replace depending on the equivalence point pH.

Glutathione (GSH) is a tripeptide and natural reducing agent composed of glutamic acid, glycine, and cysteine. Its level in the human body is closely linked to human health, such as diabetes, Alzheimer's disease, and cancer. The supplementation of exogenous GSH could bring health benefits and GSH detection in food is of considerable importance. However, the existing assays for GSH detection such as high-performance liquid chromatography/mass spectrometry, electrochemiluminescence and fluorescent nanoprobe were not satisfactory because of the disadvantages of equipment and site requirements. In this study, a multiple-colorimetric detection assay for GSH was developed based on GSH's reaction with gold nanorods. During the reaction with varying concentrations of GSH, the gold nanorods degraded into spherical nanoparticles with multiple color changes, which could be used to determine GSH concentrations. The transverse surface plasmon resonance absorption peak of gold nanorods (AuNRs) significantly shifted, indicating a novel mechanism distinct from etching or surface coating, which typically altered the longitudinal surface plasmon absorption peak. Under optimized conditions, the assay exhibited commendable specificity and reliability in actual samples. The assay accurately quantified GSH ranging from 1 to 10 µM, with detection limits of 439 nM and 260 nM for spectrophotometry and visual analysis, respectively. It was firstly to use GSH as a reducing agent to react with AuNRs in the presence of AgNO and the mechanism was different from etching or surface coating. The study's assay shows potential for detecting GSH in food samples and provides an alternative approach for the development of colorimetric detection assays based on AuNRs.

In this research, a green approach utilizing deep eutectic solvent liquid-liquid microextraction is combined with smartphone digital image colorimetry for the determination of boron in nut samples. A smartphone camera was used to capture the image of the analyte extract located in a custom-made colorimetric box. Using ImageJ software, the images were split into RGB channels, with the green channel identified as the optimum. The distance between the cuvette containing the analyte extract and the detection camera was determined to be 8 cm, while the brightness of the light source was 30%. All the images were obtained at 585 nm monochromatic light positioned as a background source. The extraction was achieved with 450 µL of a 1:4 choline-chloride to phenol mole ratio within 60 s and another minute of centrifugation. The limits of detection and quantification were found to be 0.02 and 0.06 µg mL, respectively. The method linearity, as indicated by the relative coefficient, was greater than 0.9955 and the relative standard deviations were below 5.4%. Lastly, the application of chemometrics in the form of artificial intelligence (AI)-based models and hybrid machine learning methodologies has been incorporated with SDIC for the quantitative simulation of SDIC parameters. The results gathered showed that these models are capable of predicting the quantitative SDIC parameters.

As one of the most harmful heavy metal pollutants, hexavalent chromium Cr(VI) is becoming a serious threat to human health. Thus pursuing a remarkably sensitive method to monitor the Cr(VI) concentration in natural conditions is favored for the fast response to prevent harm. In the present work, an ethylenediamine (En) and SiO-modified wool keratin-based carbon quantum dot (CQD)(En@CQDs@SiO) fluorescent sensor is prepared, and the En is found to improve the discrimination ability by binding the Cr(VI) with the surface carboxyl groups. Based on these designs, the En@CQDs@SiO achieves a significant improvement in the Cr(VI) detection ability, with a detection limit of 6.08 × 10 mg/L, which succeeded 6 times over CQDs, and is better than conventional UV-Vis and flame atomic absorption (AAS) techniques. Furthermore, the fluorescent sensor has good relative sensitivity, selectivity, good spectral reproducibility, and excellent structural stability. These properties make the sensor suitable for environmental Cr(VI) detection, which undoubtedly improves the economy and environmental friendliness of the fluorescent sensor.

A simple method for determining elemental sulfur in environmental water was developed and applied to seawater samples collected immediately after the occurrence of blue tides in Tokyo Bay. To investigate the concentration and extraction methods, artificial elemental sulfur was quantitatively produced by oxidizing a sulfide solution with an iodine solution, then used as a standard reagent in the experiments. To concentrate the elemental sulfur in the water sample, glass filter paper (GF/F) was used to filter and collect the elemental sulfur. The elemental sulfur was then extracted using n-hexane, the main component of petroleum ether; however, the recovery of elemental sulfur from the wet glass filter paper was low, and remained so even when the glass filter paper was dried. We, therefore, used a mixture of n-hexane and an acetone solvent, which is a hydrophilic organic solvent, for extraction and succeeded in recovering more than 90% of the elemental sulfur from the wet glass filter paper. Using this solvent mixture, we extracted and quantified elemental sulfur from seawater samples collected after the occurrence of blue tide, and detected 0.36-0.38 mgS L of elemental sulfur in the near-surface layer. We also found that the elemental sulfur concentrations were higher in the surface layer than in the bottom layer. Therefore, we demonstrated that the quantification of elemental sulfur is important to better understand the blue tide phenomenon.

Cartilage is a connective tissue composed of mainly water, collagen (COL) and proteoglycans (PGs) including chondroitin sulfate (CS). Near-infrared (NIR) spectroscopy is adequate for examination of soft and hard tissues with large amount of water non-destructively and non-invasively. We measured tablets containing CS and COL using NIR spectroscopy to develop an evaluation method for PGs in cartilage non-destructively and non-invasively. Calibration curves were constructed using the NIR spectra of the tablets that show the quantitative linear relationship between the concentration and height of the second derivative at 4260 cm for COL and at 5800 cm for COL and CS. An equation to calculate the CS-to-COL ratio was derived from the calibrated slopes at 5800 and 4260 cm, and the utility of the equation was demonstrated by the evaluation of tablets. Moreover, we conducted an evaluation of the CS-to-COL ratio in the aqueous nucleus pulposus and annulus fibrosus, and the results were consistent with the glycosaminoglycans (GAGs)-to-COL ratios obtained through Raman spectroscopy of the same specimens. Thus, this method is adequate for evaluating PGs with large amount of water non-destructively, non-invasively and with less damage.

Oligosaccharides were covalently labeled with 1-pyrenemethylamine (1-PMA) in 15 min at 75 °C with high yield, separated by quantitative HPLC in less than 20 min, and characterized by off-line Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry (MS). A new MALDI mass spectral precursor ion for fragmentation studies was observed from 2,5-dihydroxybenzoic acid matrix (DHB, hydroquinone/benzoquinone redox system) through photo-induced reductive elimination. The resulting high-energy protonated glycosylamine provided excellent fragmentation efficiency (Y-, B-ions, and combination losses), with ions covering almost the entire structure of several oligosaccharides at the low picomol level. With the new method, glycans from commercially available standard glycoproteins and glycans from bioengineered β-lactoglobulin expressed in the bgs13 mutant of Pichia pastoris were analyzed.

We report 532-nm and 1064-nm excited hyper-Raman (HR) spectra of representative non-proteinogenic amino acids, including α-, β-, and γ-amino acids. Different from the common 20 proteinogenic amino acids, natural non-proteinogenic amino acids cannot be incorporated into proteins during translation, while they are indispensable as intermediates in many processes like biosynthesis and neurotransmitters. In 532-nm excited HR spectra, the COO symmetric stretching bands are commonly intense, and the NH bands are clearly observable. In addition, based on the reported IR and Raman study, we found that some HR bands are IR-active but Raman-inactive. In contrast, HR signals with the 1064-nm excitation are much weaker than the 532-nm excitation. Nevertheless, we observed the COO scissoring band unexpectedly, much stronger than other bands with the 1064-nm excitation. Our results suggest that the electronic resonance effect plays a role in enabling us to detect HR signals in the UV region readily. We expect that this study provides a supplementary reference for HR spectroscopy of natural amino acids.

The analysis of Raman spectrum data has gradually transitioned into the era of machine learning. However, it is still constrained by the challenge of acquiring large volumes of raw data and the issue of losing characteristic information from spectral data. In this paper, we propose a strategy that combines data amplification and attention mechanisms for analyzing antibiotic spectral data. Firstly, a Generative Adversarial Network was employed to amplify the SERS spectrum of eight antibiotics by 10 times, to augment the dataset to fulfill the requirements of the neural network. Then, the amplified data is input into a one-dimensional convolutional neural network with an attentional mechanism module, which enables a more accurate capture of spectral feature information. The one-dimensional convolutional neural network achieved a 97.5% accuracy in classifying eight antibiotics. The accuracy of the four mixtures within the same class was 89.4%.

The femtosecond pump-probe technique, i.e. the transient transmission spectroscopy, has been used for the first time, to detect the vibrational spectra of symmetric fundamentals ν and ν in bromoform and chloroform. The spectra were obtained by fast Fourier transforms of the time domain signals. For both, CHCl and CHBr, there are four isotopologues contributing to the spectra, due to the existence of two stable isotopes; chlorine, Cl and Cl, and bromine, Br and Br, respectively. While for chloroform the isotope splitting of the ν spectral band can be observed even in the spontaneous Raman scattering, for bromoform it is not detectable. Herewith we show that using the time domain spectroscopy and the windowed Fourier transform method we can provide the high resolution spectrum of the ν fundamental in bromoform, in which the contributions of all isotopologues are well distinguishable. The data have been collected for few volume concentrations of the studied liquids diluted in the neutral solvent CCl. It is shown that the intensity pattern of the spectra evolves with decreasing concentration and for the ν fundamental it reaches the natural abundance pattern at a very high dilution. The simple theoretical model, which treats the molecules in a liquid as interacting oscillators, allows us to explain the dependence of the shape of the spectrum on the strength of the intermolecular interactions.

The hydration state of the alcohols was investigated using the extended molar absorption coefficient, which redefines the molar absorption coefficient as a differential coefficient of concentration. The extended molar absorption coefficient is a function of the concentration calculated from the difference in absorbance, and is consistent with the conventional molar absorption coefficient, allowing a complete quantitative comparison. The quantitative performance was verified using IR and NIR absorption spectra of aqueous solutions of monovalent alcohols (methanol, ethanol, 1-propanol, 2-propanol, and tert-butanol) that were soluble in water at any mixing ratio. Extended molar absorption coefficient spectra were calculated for the combination bands of water, which were further separated by multivariate curve resolution-alternating least squares (MCR-ALS) into molecular species with different peak wavenumbers: strongly hydrogen-bonded (SHB), weakly hydrogen-bonded (WHB), and free OH species. The number of water species that change when one alcohol molecule increases, i.e., the perturbed hydration number (PHN), was calculated by comparison with the conventional molar absorption coefficient of pure water. The calculated PHN indicates that the numbers of SHB and WHB species are reversed at approximately 20 wt%, and that the free OH species increase at higher alcohol concentrations and are more pronounced for alcohols with bulky alkyl groups. These results provide a quantitative answer to the long-debated question of anomalies in water-alcohol mixing.

Long noncoding RNAs (lncRNAs) are transcripts exceeding 200 nucleotides that do not encode proteins. Despite lacking protein-coding capabilities, lncRNAs play crucial roles in cellular processes, including gene-expression modulation and structural maintenance. The study of lncRNAs has evolved significantly since 2009, with advancements in analytical methodologies providing new insights into their functions and dynamics. Key developments include BRIC-Seq, SLAM-Seq, TUC-Seq, TimeLapse-seq, and Dyrec-Seq. These methodologies have enabled researchers to investigate lncRNA behavior under various conditions, including cellular stress responses and complex biologic systems. Future challenges include developing comprehensive techniques for identifying lncRNA-interacting proteins and advancing in vivo methodologies using model organisms. As the field progresses, integrating these technologies will enhance our understanding of lncRNA biology, potentially leading to novel therapeutic strategies and deeper insights into gene-regulation mechanisms.

In recent years, wearable sweat sensors have garnered significant attention for real-time monitoring of human physiological information because of their ability to continuously and non-invasively detect multiple sweat biomarkers. Among these, potentiometric sensors stand out for their low power consumption, low cost, compact design, and real-time monitoring capabilities, making them an ideal alternative for sweat analysis. However, enhancing the sensitivity of ion-selective electrodes (ISEs), a critical parameter of potentiometric sensors, remains a challenging research focus. In this work, the sensitivity of K ISEs was significantly enhanced by doping two-dimensional nanoparticles graphitic carbon nitride (g-C₃N₄) into the ion-to-electron transducer of the electrode via electrodeposition. The calibration curve slope of the K potentiometric sensors with doped g-CN reached 59.6 mV/dec, representing a 33% increase in sensitivity compared to the control sensor without g-C₃N₄. Furthermore, the developed sensors demonstrated excellent repeatability, and anti-interference capabilities. Finally, the feasibility of the prepared sensors was further validated in artificial sweat. The large specific surface area of g-C₃N₄ combined with the excellent conductivity of PEDOT: PSS, significantly improved the sensitivity of ISEs in this study. This innovative approach paves a new avenue for the application of two-dimensional materials in potentiometric sensors, potentially advancing the field of real-time sweat analysis.

The human BK channel (hBK) is an essential membrane protein that regulates various biological functions, and its dysfunction leads to serious diseases. Understanding the biophysical properties of hBK channels is crucial for drug development. Artificial lipid bilayer recording is used to measure biophysical properties at the single-channel level. However, this technique is time-consuming and complicated; thus, its measurement efficiency is very low. Previously, we developed a novel technique to improve the measurement efficiency by rapidly forming lipid bilayer membranes and incorporating ion channels into the membrane using a hydrophilically modified gold probe. To further improve our technique for application to the hBK channel, we combined it using the gold probe with a liposome fusion method. Using a probe on which liposomes containing hBK channels were immobilized, the channels were efficiently incorporated into the lipid bilayer membrane, and the measured channel currents showed the current characteristics of the hBK channel. This technique will be useful for the efficient measurements of the channel properties of hBK and other biologically important channels.

A biosensor for biochemical oxygen demand (BOD) was developed based on intracellular 5'-adenosine triphosphate (ATP) measurements in Saccharomyces cerevisiae. Intracellular ATP was measured using an engineered protein named ATeam, comprising a bacterial FF-ATP synthase ε subunit sandwiched between cyan fluorescent protein and mVenus, a modified yellow fluorescent protein. Because the binding of ATP to ATeam induces changes in the fluorescence spectra owing to Fӧrster resonance energy transfer, S. cerevisiae expressing ATeam is expected to show spectral changes owing to the intracellular ATP produced by the metabolism of the BOD sample. A glycogen phosphorylase knockout S. cerevisiae strain expressing ATeam was prepared, and the fluorescence spectra of the strain were analyzed. Changes in the fluorescence spectra of glucose in the medium were observed, which exhibited a linear relationship with the glucose concentration (0-100 mg/L, R = 0.970). Responses to lactose, fructose, sucrose, Glu, Asp, His, and Gly were evaluated and compared with typical BOD measurements. The results of this comparison suggest that a BOD biosensor based on intracellular ATP can be used for BOD measurements. A BOD standard solution comprising glucose and glutamic acid (GGA) was calibrated across a concentration range of 0 to 100 mg/L. Finally, simulated real samples were prepared using real pond water and GGA was measured. The correlation between the BOD value evaluated using intracellular ATP and that evaluated using the 5-day BOD test showed a linear relationship with R = 0.944.

We synthesized a squaraine dye (F-0) to develop a method for detecting pyrophosphate (PPi) and alkaline phosphatase (ALP) by modulating the fluorescence of F-0. The fluorescence intensity of the F-0 system was quenched upon the addition of Cu ions; however, it was restored when PPi was introduced due to the formation of a complex between PPi and Cu. Since ALP can hydrolyze PPi, the fluorescence of the system was quenched again upon the addition of ALP. Based on these principles, we established a fluorescent probe that exhibits an "off-on-off" fluorescence response. The detection limits of this method for PPi and ALP were 103 nmol dm and 0.18 U dm, respectively. Moreover, this method demonstrates good selectivity and specificity and can be applied to the detection of PPi in actual samples.