Lateral flow immunoassay device to detect staphylococcal enterotoxin B (SEB) in durian candy.

Staphylococcal enterotoxin B (SEB), a potent enterotoxin produced by Staphylococcus aureus, has been implicated in incidences of Staphylococcal food poisoning in the Philippines. The use of lateral flow immunoassay devices to detect this toxin in solid food samples, like durian candy, at the point of sampling is constrained by the requirement for sample purification (e.g. centrifugation). This problem is also true with the other applications of LFIA devices on food samples. To overcome this challenge, a lateral flow immunoassay (LFIA) device capable of detecting SEB in unpurified durian candy sample was developed in this study. A modified LFIA device was assembled with three layers of glass fiber pads functioning as sample pads instead of a conventional cellulose fiber pad. Unlike with the cellulose fiber pad, the glass fiber sample pads acted as filter and allowed the flow of a 1:5 dilution of durian candy. The LFIA device applied to spiked 1:5 diluted durian candy samples achieved a visual limit of detection of 5 ng/mL for SEB, which is twofold lower than reported for previous LFIA devices designed to detect SEB in food samples.

Paper-based analytical device for point-of-care nucleic acid quantification combining CRISPR/Cas12a and a personal glucose meter.

Although CRISPR-based nucleic acid detection has great potential in point-of-care testing due to its simplicity, it has been rarely integrated into paper-based analytical devices (PADs), which are attractive platforms to simplify assays. This work introduces a CRISPR-assisted nucleic acid quantification approach integrated into a PAD with signal readout by a personal glucose meter (PGM). Retention of magnetic beads by filter paper and pre-deposition of all required reagents by freeze-drying stabilized with trehalose enabled the indirect quantification of human papilloma virus (HPV) DNA through a PGM readout without complicated user intervention and complex reagent handling. The calculated limit of detection was 57 pM, which is comparable with other amplification-free CRISPR-based assays detecting nucleic acids. The fully integrated device exhibited good storage stability for up to 4 weeks, suggesting its applicability toward practical point-of-care nucleic acid quantification.

Current advances in the development of bioluminescent probes toward spatiotemporal trans-scale imaging.

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.

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

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.

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

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.

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

Correction for 'Evaluation of separation performance for eggshell-based reversed-phase HPLC columns by controlling particle size and application in quantitative therapeutic drug monitoring' by Tomoka Yoshii et al., Anal. Methods, 2023, 15, 1790-1796, https://doi.org/10.1039/D3AY00219E.

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

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.

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

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.

Paving the way to the future: Mapping historical patterns and future trends of road material stock in Japan.

Roads are a fundamental component of societal infrastructure, whose decades-long lifespan has far-reaching implications for developmental decisions. The road construction and development have profound impacts on economic growth, social dynamics, and environmental sustainability. Therefore, comprehensive measurement of the current road material stock (MS) and the projection of expected future road scale based on regional socio-economic scenarios that can reflect unique local conditions are necessary. This study examined the historical changes and progression patterns of the road network across Japan from 1965 to 2020 through material flow and material stock analysis. By using the road MS time series, along with explanatory socioeconomic variables, several models including Autoregressive Integrated Moving Average with explanatory variables (ARIMAX), Support Vector Regression (SVR), hybrid ARIMAX-SVR, Multiple Linear Regression (MLR), Artificial Neural Networks (ANN), and Random Forest (RF) were compared. After comparison analysis, ARIMAX and hybrid ARIMAX-SVR models were employed to forecast expected road MS in each prefecture of Japan by 2050 based on national shared socioeconomic pathways (SSP) scenarios. The study found that the total road MS of Japan increased 5.5-fold over 55 years. Aggregate was the dominant material, comprising over 70 % among the four materials of the total road MS. The forecast results for each prefecture were classified into three different patterns. Expected MS in most prefectures still displayed increasing trends in the five scenarios, but the projection of road MS in eight prefectures revealed a notable downward trend across each SSP scenario. For most prefectures, SSP5 displayed the highest expected road MS, followed by SSP1. SSP3 was the scenario with the lowest MS. This approach provided a more thorough understanding of the likely evolution of road MS across different SSP scenarios and could help inform decisions for resource allocation and policy formulation concerning road infrastructure management.

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

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.

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

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.

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

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).

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

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.

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

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).

Hourly future climate scenario datasets for impact assessment of climate change considering simultaneous interactions among multiple meteorological factors.

Assessing the impacts of climate change in multiple fields, such as energy, land and water resources, and human health and welfare is important to find effective strategies to adapt to a changing climate and to reduce greenhouse gases. Many phenomena influenced by climate change have diurnal fluctuations and are affected by simultaneous interactions among multiple meteorological factors. However, climate scenarios with detailed (at least hourly) resolutions are usually not available. To assess the impact of climate change on such phenomena while considering simultaneous interactions (e.g., synergies), climate scenarios with hourly fluctuations are indispensable. However, because meteorological indicators are not independent, the value of one indicator varies as a function of other indicators. Therefore, it is almost impossible to determine the functions that show all relationships among meteorological elements considering the geographical and temporal (both seasonal and time of a day) characteristics. Therefore, generating hourly scenarios that include possible combinations of meteorological indicators for each hourly observation unit is a challenging problem. In this study, we provide secondary future climate scenario datasets that have hourly fluctuations with reasonable combinations of meteorological indicator values that are likely to occur simultaneously, without losing the long-term climate change trend in the existing daily climate scenarios based on global climate models. Historical hourly weather datasets observed from 2017 to 2019 (the reference years) are used to retrieve short-term fluctuations. Bias-corrected daily future climate scenario datasets generated using four global climate models (GFDL CM3, HadGEM2-ES, MIROC5, and MRI-CGCM3) and two Representative Concentration Pathways (RCP8.5 and 2.6) are used to model long-term climate change. A total of 48 different types of hourly future scenario datasets for five meteorological indicators (temperature, solar radiation, humidity, rainfall, and wind speed) were acquired, targeting a projection period from 2020 to 2080, for 10 weather stations in Japan. The generated hourly climate scenario datasets can be used to project the quantitative impacts of climate change on targeted phenomena considering simultaneous interactions among multiple meteorological factors.

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level.

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

Traffic light type paper-based analytical device for intuitive and semi-quantitative naked-eye signal readout.

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.

A novel method for acquiring rigorous temperature response functions for electricity demand at a regional scale.

The demand for electricity affects the future climate through its effect on greenhouse gas emissions in the electricity generation process, but climate change also impacts electricity demand by changing the need for heating and cooling. Developing reliable temperature response functions (TRFs) that illustrate electricity demand as a function of temperature is key for decreasing uncertainty in future climate projections under a changing climate and for impact assessments of climate change on energy systems. However, this task is challenging because electricity demand is determined by multiple factors that interact in complicated ways because demand fluctuations represent timely human responses to given meteorological conditions. We propose a novel method to acquire reliable TRFs at a regional scale based on comprehensive modeling of electricity demand fluctuations. Six candidate algorithms were examined, and multivariate adaptive regression splines (MARS) was selected as the best algorithm with the dataset used. Using MARS, we constructed models with the capacity to precisely reproduce complex electricity demand patterns based on multiple predictors and simulated the impact of temperature on electricity demand while controlling for the effects of other factors. The temporal segments in TRFs are detected and parameters and functional forms of TRFs for 10 regions in Japan were presented.

Colorimetric paper-based sarcosine assay with improved sensitivity.

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.

Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues.

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.