Published papers

© 2018 The Authors Purpose: To elucidate the correlation between lactoferrin concentration in the tear film and signs and symptoms of severe dry eye disease (DED) using a novel microfluidic paper-based analytical device (μPAD) and enzyme-linked immunosorbent assay (ELISA). Methods: Twenty-four patients were recruited at the Keio University Hospital. Using a novel μPAD, lactoferrin concentrations were measured in 4 patients with GVHD-related DED, 3 patients with other types of DED and 2 controls (Group A). For validation by ELISA, 22 patients (7 patients from Group A) comprising 9 patients with GVHD-related DED, 6 patients with other types of DED and 7 controls were examined (Group B). The link between lactoferrin concentration and clinical data about the severity of aqueous tear deficient DED was also investigated by both μPAD and ELISA. Results: The lactoferrin concentration in tear fluid of the DED patients was positively correlated between μPAD and ELISA (p = 0.006, r = 0.886). The tear fluid of the GVHD patients showed low or undetectable lactoferrin concentration. Analysis by ELISA demonstrated that lactoferrin concentrations in the tear film from the GVHD patients were significantly lower than those from the non-GVHD patients (p = 0.010576). ELISA revealed lactoferrin concentration correlated with the value of Schirmer test and tear film breakup time, whereas it was inversely correlated with OSDI, fluorescein and rose bengal scores. Conclusions: The novel μPAD may pave the way for measuring lactoferrin concentration in tear fluid from DED patients. Our results suggested that lactoferrin concentration in tear fluid reflect the severity of DED.

© 2018 American Chemical Society. Native coelenterazine (nCTZ) is a common substrate to most marine luciferases and photoproteins. In this study, nine novel dye- and azide-conjugated CTZ analogues were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of the nCTZ backbone to obtain bulkiness-driven substrate specificity and potential chemiluminescence/bioluminescence resonance energy transfer (C/BRET). The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly biased bioluminescence to ALucs and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living animal cells or lysates. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than nCTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). No or less spectral overlap occurs among [Furimazine-NanoLuc], [6-N 3 -CTZ-ALuc26], [6-pi-OH-CTZ-RLuc8.6], and [6-N 3 -CTZ-RLuc8.6] pairs, because of the substrate-driven luciferase specificity and color shifts, providing a crosstalk-free multiplex bioassay platform. The unique bioluminescence system appends a new toolbox to bioassays and multiplex molecular imaging platforms. This study is the first example that systematically synthesized fluorescent dye-conjugated CTZs and applied them for a bioluminescence assay system.

© 2019 American Chemical Society. A distance-based analysis of potassium ion (K + ) is introduced that is performed on a microfluidic paper-based analytical device (μPAD) coupled to an ion-selective capillary sensor. The concept is based on two sequential steps, the selective replacement of analyte ion with an ionic dye, and the detection of this dye in a distance-based readout on paper. To achieve the first step, the capillary sensor holds a poly(vinyl chloride) (PVC) membrane film layer plasticized by dioctyl sebacate (DOS) that contains the potassium ionophore valinomycin, a lipophilic cation-exchanger and the ionic indicator Thioflavin T (ThT) on its inner wall. Upon introduction of the sample, K + in the aqueous sample solution is quantitatively extracted into the film membrane and replaced with ThT. To convert the ion exchange signal into a distance-based analysis, this solution was dropped onto the inlet area of a μPAD to flow the ThT along a channel defined by wax printing, resulting in the electrostatic binding of ThT to the cellulose carboxylic groups. The initial amount of K + determines the amount of ThT in the aqueous solution after ion-exchange, and consequently the distance of ThT-colored area reflects the sample K + concentration. The ion exchange reaction was operated in a so-called "exhaustive sensing mode" and gave a distinct response in a narrow range of K + concentration (1-6 mM) that cannot be achieved by the classical optode sensing mode. The absence of hydrogen ions from the equilibrium competition of the capillary sensor contributed to a complete pH-independence, unlike conventional optodes that contain a pH sensitive indicator. A very high selectivity for K + over Na + and Ca 2+ has been confirmed in separate solutions and mixed solutions tests. K + measurements in pooled serum samples at concentrations between 2 and 6 mM are successfully demonstrated on a temperature controlled support.

© 2018 The Royal Society of Chemistry. In this work, an application of a design of experiments approach for the optimization of an isoniazid assay on a single-area inkjet-printed paper-based analytical device (PAD) is described. For this purpose, a central composite design was used for evaluation of the effect of device geometry and amount of assay reagents on the efficiency of the proposed device. The factors of interest were printed length, width, and sampling volume as factors related to device geometry, and amounts of the assay reagents polyvinyl alcohol (PVA), NH 4 OH, and AgNO 3 . Deposition of the assay reagents was performed by a thermal inkjet printer. The colorimetric assay mechanism of this device is based on the chemical interaction of isoniazid, ammonium hydroxide, and PVA with silver ions to induce the formation of yellow silver nanoparticles (AgNPs). The in situ-formed AgNPs can be easily detected by the naked eye or with a simple flat-bed scanner. Under optimal conditions, the calibration curve was linear in the isoniazid concentration range 0.03-10 mmol L -1 with a relative standard deviation of 3.4% (n = 5 for determination of 1.0 mmol L -1 ). Finally, the application of the proposed device for isoniazid determination in pharmaceutical preparations produced satisfactory results.

© 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.

© 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.

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 American Chemical Society. There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 μM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.

© 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.

© Ivyspring International Publisher. Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ∼300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named “Bottle Blue (BBlue)” and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ∼50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

Lactoferrin is an abundant glycoprotein in human body fluids and is known as a biomarker for various diseases. Therefore, point-of-care testing (POCT) for lactoferrin is of interest. Microfluidic paper-based analytical devices (µPADs) have gained a lot of attention as next-generation POCT device candidates, due to their inexpensiveness, operational simplicity, and being safely disposable. This work presents a colorimetric sensing approach for quantitative lactoferrin analysis. The detection mechanism takes advantage of the high affinity of lactoferrin to ferric ions (Fe3+). Lactoferrin is able to displace an indicator from a colorimetric 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-Fe3+ complex, resulting in a color change. A 5-Br-PADAP-Fe3+ complex was encapsulated into water-dispersible poly(styrene-block-vinylpyrrolidone) particles, whose physical entrapment in the cellulosic fiber network results in the immobilization of the complex to the paper matrix. The complex-encapsulating particles showed a color change response in accordance with lactoferrin concentration. Both color intensity-based paper well plates and distance readout-based µPADs are demonstrated. Color intensity-based devices allowed quantitative analysis of lactoferrin concentrations with a limit of detection of 110 µg/mL, using a smartphone and a color readout app. On the other hand, distance readout-based µPADs showed changes of the length of colored sections in accordance with lactoferrin concentration. In summary, we successfully developed both colorimetric intensity-based paper wells and distance-based µPADs for lactoferrin detection. This work demonstrates a user-friendly colorimetric analysis platform for lactoferrin without requiring lab equipment and expensive antibodies.

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.

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+

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.

Nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) are demonstrated as superior peroxidase mimic antibody labels alternative to horseradish peroxidase (HRP) in an immunoassay, potentially overcoming some of the inherent disadvantages of HRP and other enzyme mimicking nanomaterials. They revealed efficient peroxidase-like activity and catalyzed the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to form the intense blue product (at 620 nm) in the presence of hydrogen peroxide (H2O2). Using low-density lipoprotein (LDL) as a model target, an ultra-low limit of detection (0.0051 mg/dL) and a linear range of 0.0625–0.750 mg/dL were achieved, exhibiting higher sensitivity than the HRP-based immunoassay. Thus, the proposed N-CDs/Ni-MnFe-LDHs can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensors for detection of biomarkers, as well as trace chemical analysis. Graphical abstract: [Figure not available: see fulltext.]

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.

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.

A disposable and simple PPAD (microfluidic paper-based analytical device) for the quantitative colorimetric detection of salivary cation species, relying on ISOs (ion-selective optodes) is introduced. This work demonstrates the applicability of ISO-integrated PPADs to real saliva samples. Calibration enabling accurate salivary cation quantification was achieved by using artificial saliva. Multiple sensing areas on a single device can detect three different types of ions within 3 min by simple application of 20 PL sample. Three kinds of ISOs were optimized for salivary Na+, K+ and Ca2+ quantification. For proof-of-concept, salivary Na+ concentration was accurately quantified by the developed PPAD.

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.

Recent advances in bioluminescence technology rely on the discovery of luciferin-luciferase pairs having excellent luminescence activity. Here, we focus on coelenterazine (CTZ) and its analogues and quantitatively investigate reaction factors, including bioluminescence reaction quantum yield (φBL) and enzyme reaction kinetics (turnover rate, kcat), determining the luminescence intensity to design brighter reaction systems. Initially, it was confirmed that the CTZ was kept stable during the storage and the φBL value measuring experiment in terms of comparative study using benzyl-protected CTZ. Using a luminometer whose absolute responsivity is calibrated for each luminescence reaction spectrum, we absolutely measured φBL and other reaction factors of CTZ and 3 types of blue-shifted CTZ analogues, CTZ 400a, 6-pi-H-CTZ and 6-pi-Ph-CTZ. The results suggested that there was no significant difference in reaction kinetics, but a clear difference in φBL (up to 22-fold), which was presumably due to an improvement in fluorescence quantum yield (φFL) of the corresponding coelenteramide (CTMD), oxidized form of CTZ. Our evaluation suggests that the design of CTZ analogues considering φFL is important for developing a bright luminescent reaction system.