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The etiology and pathological complexity of acute kidney injury (AKI) pose great challenges for early diagnosis, typing, and personalized treatment. It is an important reason for poor prognosis and high mortality of AKI. In order to provide a relatively noninvasive diagnostic and typing method for AKI, we proposed the pathological changes of albumin permeability after glomerular injury and reabsorption efficiency after tubular injury as potential entry points. Thus, a renal tubule labeling fluorescent dye which features albumin concentration-related fluorescence intensity was used to fit these pathological changes. Utilizing this fluorescence assay, we realized urinary tract obstruction imaging as early as 12 h after morbidity. For glomerular and tubular injury discrimination, compared to a healthy control, membranous nephropathy as a representative glomerular injury resulted in enhanced fluorescence intensity of the kidney due to increased albumin penetration, while renal tubular injury caused insufficient dye reabsorption to exhibit weakened fluorescence intensity. The significant differences demonstrated the feasibility of this approach for fluorescence imaging-based AKI typing in vivo.

A new measuring system based on the already existing Multi-Color-PAM Fluorimeter (Schreiber et al. in Photosynth Res 113:127-144, 2012) was developed that in addition to standard PAM measurements enables pump-and-probe flash measurements and allows simultaneous measurements of the changes in chlorophyll fluorescence yield (F) during application of saturating flashes (ST). A high-power Chip-on-Board LED array provides ST flashes with close to rectangular profiles at wide ranges of widths (0.5 µs to 5 ms), intensities (1.3 mmol to 1.3 mol 440 nm quanta m -2  s -1 ) and highly flexible repetition times. Using a dedicated rising-edge profile correction, sub-µs time resolution is obtained for assessment of initial fluorescence and rise kinetics. At maximal to moderate flash intensities the flash-kinetics (changes of F during course of ST, STK) are strongly affected by 'High Intensity Quenching' (HIQ), consisting of Car-triplet quenching, TQ, and donor-side-dependent quenching, DQ. The contribution of TQ is estimated by application of a second ST after 20 µs dark-time. Upon application of flash trains (ST sequences with defined repetition times) typical period-4 oscillations in dark fluorescence yield (F 0 ) and ST-induced fluorescence yield, F m ST , are obtained which can be measured in vivo both with suspensions and from the surface of leaves. Examples of application with dilute suspensions of Chlorella and an intact dandelion leaf are presented. It is shown that weak far-red light (730-740 nm) advances the S-state distribution of the water-splitting system by one step, resulting in substantial lowering of F m ST and also of the I 1 -level in the polyphasic rise of fluorescence yield induced by a multiple-turnover flash (MT). Based on comparative measurements of STK and the polyphasic rise kinetics with the same Chlorella sample, it is concluded that the generally observed lower values of maximal fluorescence yields using ST-protocols compared to MT-protocols are due to a higher extent of HIQ (mainly DQ) and the contribution of variable PSI fluorescence to F m ST ., (© 2024. The Author(s).)

An indicator displacement assay for colorimetric and fluorometric dual-mode detection of urinary uric acid (UA) was constructed using a water-soluble naphthalene-based tetralactam macrocycle and the phenoxazine dye, resorufin (RF). The visual detection of UA levels of volunteers was successfully realized using modified paper assays, which could be used for the home monitoring of urinary UA.

River biofilms are a suitable indicator of toxic stress in aquatic ecosystems commonly exposed to various anthropogenic pollutants from industrial, domestic, and agricultural sources. Among these pollutants, heavy metals are of particular concern as they are known to interfere with various physiological processes of river biofilm, directly or indirectly related to photosynthetic performance. Nevertheless, only limited toxicological data are available on the mechanisms and toxicodynamics of heavy metals in biofilms. Pulse Amplitude Modulated (PAM) fluorometry is a rapid, non-disruptive, well-established technique to monitor toxic responses on photosynthetic performance, fluorescence-kinetics, and changes in yield in other non-photochemical processes. In this study, a new micro-PAM-sensor was tested to assess potential acute and chronic effects of heavy metals in river biofilm. Toxicity values across the three parameters considered in this study (photosynthetic yield YII, non-photochemical quenching NPQ, and basal fluorescence F 0 ) were comparable, as determined EC50 were within one order of magnitude (EC50 ∼1-10 mg L -1 ). However, the stimulation of NPQ was more clearly associated with early acute effects, especially in illuminated samples, while depression of YII and F 0 were more prevalent in chronic tests. These results have implications for the development of functional indicators for the biomonitoring of aquatic health, in particular for the use of river biofilm as a bioindicator of water quality. In conclusion, the approach proposed seems promising to characterize and monitor the exposure and impact of heavy metals on river periphyton communities. Furthermore, this study provides a fast, highly sensitive, inexpensive, and accurate laboratory method to test effects of pollutants on complex periphyton communities that can also give insights regarding the probable toxicological mechanisms of heavy metals on photosynthetic performance in the river biofilm., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.

DNA damage plays a decisive role in epigenetic effects. The detection and analysis of DNA damages, like the most common change of guanine (G) to 8-oxo-7,8-dihydroguanine (OG), is a key factor in cancer research. It is especially true for G quadruplex structure (GQ), which is one of the best-known examples of a non-canonical DNA arrangement. In the present work, we provided an overview on analytical methods in connection with the detection of OG in oligonucleotides with GQ-forming capacity. Focusing on the last five years, novel electrochemical tools, like dedicated electrodes, were overviewed, as well as different optical methods (fluorometric assays, resonance light scattering or UV radiation) along with hyphenated detection and structural analysis methods (CD, NMR, melting temperature analysis and nanopore detection) were also applied for OG detection. Additionally, GQ-related computational simulations were also summarized. All these results emphasize that OG detection and the analysis of the effect of its presence in higher ordered structures like GQ is still a state-of-the-art research line with continuously increasing interest.

A reusable optofluidic point-of-care testing platform (OPOCT) was successfully constructed through integrating evanescent wave fluorescence technology into an all-fiber-based optofluidic system. The compact design of the OPOCT allows it to be portable and suitable for on-site sensitive determination of biomarkers in serum without complicated and costly procedures. The sensitivity of 90.9 pM for antibody determination is observed thanks to the high transmission efficiency of excitation light and fluorescence in the OPOCT. The affinity constant between cholylglycine (CG) and anti-CG antibody was quantified using this platform based on the proposed theory. Using the lyophilized fluorescence-labeled specific antibody and reusable fiber optic biosensor, the OPOCT is applied to the one-step sensitive determination of CG in serum, which eliminates the dearth associated with liquid reagent handling, disposable biosensors, and user intervention. A limit of detection of 0.025 μg/mL for CG is obtained, which is far more than adequate for meeting diagnostic requirements. The matrix effect of serum samples on the evanescent wave-based optofluidic biosensor can be effectively reduced by simple dilution of serum samples. The performance of the OPOCT also compared favorably with that of a commercial turbidimetric inhibition immunoassay through analyzing multiple serum samples. This platform is ready to expand to measure any other biomarker by using its specific antibody. The simplicity, sensitivity, cost-effectiveness, and robustness of the OPOCT enable the early diagnosis of disease and making a timely clinical decision. Graphical abstract .

A new imidazole derivative of 1,2-diaminoanthraquinone and fluorene-2-carboxaldehyde was designed as a sensor B2 to selectively detect the cyanide (CN - ) ion through colorimetric and/or fluorometric methods. The photochemical characterizations of sensor B2 were tested using absorption and emission spectral studies in CH 3 CN-H 2 O (8:2) semi-aqueous medium. An excited state proton transfer process (ESIPT) was proved by theoretical and spectral studies. The colorimetric and fluorescence detection limit of CN - ion was found to be 5.3 × 10 - 6  M and 4.11 × 10 -8  M, respectively. 1 H NMR titration, electrochemical and DFT studies were supported the removal of -NH proton from B2. In order to utilize this sensor in real-time applications, we developed a test cassette which is coated with sensor B2 detected the presence of CN - ion in the food sample with endogenous cyanide ion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An eco-friendly method was used to synthesize carbon dots (CDs) from Rosemary leaves, as a carbon source. The as-synthesized CDs was applied as a fluorophore in an optical sensor after modification with molecularly imprinted polymers (MIPs) for determination of thiabendazole (TBZ). For this purpose, a silica shell using tetraethoxysilane (TEOS), as a Si source, was stabilized on the surface of CDs via reverse microemulsion technique. Following, MIPs were synthesized in the presence of TBZ as a template, using 3-aminopropyl triethoxysilane and TEOS as a functional monomer and a crosslinker, respectively. After optimization of the experimental parameters, a linear dynamic range of 0.03-1.73 μg/mL TBZ with a detection limit as 8 ng/mL were obtained for the suggested method. Finally, the proposed sensor was successfully applied for the determination of TBZ in apple, orange, and tomato juices. This sensor is a simple, rapid, selective, and non-expensive method for TBZ measurement., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Skin gas that contains volatile metabolites (volatilome) is emanated continuously and is thus expected to be suitable for non-invasive monitoring. The aim of this study was to investigate the relationship between the regional difference of sweat rate and skin volatilome distribution to identify the suitable site to monitor metabolisms. In this study, we developed a biofluorometric gas-imaging system (sniff-cam) based on nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH) to visualize transcutaneous ethanol (EtOH) distribution. The EtOH distribution was converted to a fluorescence distribution of reduced NAD with autofluorescence property. First, we optimized the solution volume and concentration of the oxidized NAD, which was a coenzyme of ADH. Owing to the optimization, a two-dimensional distribution of EtOH could be visualized from 0.05-10 ppm with good sensitivity and selectivity. Subsequently, transcutaneous EtOH imaging and measurement of sweat rate were performed at the palm, dorsum of hand, and wrist of participants who consumed alcohol. Transcutaneous EtOH from all skin parts was imaged using the sniff-cam; the concentrations initially increased until 30 min after drinking, followed by a gradual decrease. Although the determined peak EtOH concentrations of typical subjects were approximately 1100 ± 35 ppb (palm), which were higher than 720 ± 18 ppb (dorsum) and 620 ± 13 ppb (wrist), the results of sweat rate suggested that the dorsum of hand and the wrist were appropriate sites. Finally, the sniff-cam could visualize the individual difference of alcohol metabolism capacity originating from aldehyde dehydrogenase phenotype by imaging transcutaneous EtOH.

Crop productivity can be expressed as the product of the amount of radiation intercepted, radiation use efficiency and harvest index. Genetic variation for components of radiation use efficiency has rarely been explored due to the lack of appropriate equipment to determine parameters at the scale needed in plant breeding. On the other hand, responses of the photosynthetic apparatus to environmental conditions have not been extensively investigated under field conditions, due to the challenges posed by the fluctuating environmental conditions. This study applies a rapid, low-cost, and reliable high-throughput phenotyping tool to explore genotypic variation for photosynthetic performance of a set of hybrid barleys and their parents under mild water-stress and unstressed field conditions. We found differences among the genotypic sets that are relevant for plant breeders and geneticists. Hybrids showed lower leaf temperature differential and higher non-photochemical quenching, resembling closer the male parents. The combination of traits detected in hybrids seems favorable, and could indicate improved photoprotection and better fitness under stress conditions. Additionally, we proved the potential of a low-cost, field-based phenotyping equipment to be used routinely in barley breeding programs for early screening for stress tolerance.

A novel ratiometric fluorescent probe for the determination of oxytetracycline (OTC) was developed. The method is based on the use of adenosine monophosphate/Eu(III) nanoscale coordination polymers doped with carbon dots (CDs) (CD@AMP/Eu NCPs). These were fabricated by self-assembly of Eu 3+ and AMP on the surface of CDs containing large amounts of hydroxyl and carbonyl groups. Under the excitation at 310 nm wavelength, the doped NCPs display strong pink emission of Eu 3+ at 615 nm and blue emission of the CDs at 430 nm on exposure to OTC. The ratio of fluorescence intensity (F 615 /F 430 ) of such NCPs displays excellent linear relationship with OTC concentration ranging from 0.2 to 60 μM and the limit of detection (LOD) is 25 nM (3σ). The doped NCPs were evenly immobilized on common filter paper to prepare a visual ratiometric probe for the determination of OTC. Assisted by a digital camera with an APP color detector, the paper-based test strip was applied for the quantitative determination of OTC with a LOD of 0.5 μM and a wide linear scope of 1-100 μM. The method was applied to the determination of OTC in milk samples. Graphical abstractSchematic representation of the principle for oxytetracycline (OTC) determination using carbon dots (CD)@adenosine monophosphate (AMP)/Europium (Eu 3+ ) paper-based ratiometic probe.

In the present study we report a fully automated method for the determination of low levels of the genotoxic impurity hydrazine in allopurinol active pharmaceutical ingredient (API) and formulations based on the concept of zone-fluidics. Hydrazine reacts on-line with o-phthalaldehyde in a unique way, that is in acidic medium (pH < 1.5) and in the absence of nucleophilic reagents, to form a highly fluorescent hydrazone (λ ex/em  = 318 / 376 nm). The adaptation of a 120 s long stopped-flow step at elevated temperature (70 °C) offered adequate sensitivity (LOD =0.9 μg L -1 or 0.1 ppm in the solid API) to meet the pharmacopoeia limits for the selected application. The analyte was separated efficiently from the excess of the API by on line solid phase extraction using a Hydrophilic-Lipophilic technology sorbent that provided direct retention of the more hydrophobic API without the need of wetting/conditioning steps. Percent recoveries ranged between 93.3 and 105.8%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Two types of lateral flow immunochromatographic test strips (LFITS) using gold nanoparticles and fluorescent CdTe quantum dots (QDs) as signal labels, respectively, were developed for Shiga toxin type II (STX2) assays. Under optimal conditions, the corresponding visual detection limits were 25 ng mL-1 and 5 ng mL-1, respectively. The test results of gold based LFITS can be recognized directly by the naked eye, whereas the visualized results of CdTe QDs based LFITS can be observed by the aid of a UV lamp. Both assays showed good specificity and stability. The inexpensive LFITS were promising for the rapid clinical detection of STX2.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

Chlorophyll a fluorescence is the most widely used method to study photosynthesis and plant stress. While several commercial fluorometers are available, there is a need for a low-cost and highly customisable chlorophyll fluorometer. Such a device would aid in performing high-throughput assessment of photosynthesis, as these instruments can be mass-produced. Novel investigations into photosynthesis can also be performed as a result of the user's ability to modify the devices functionality for their specific needs. Motivated by this, we present an open-source chlorophyll fluorometer based on the Kautsky induction curve (OJIP). The instrument consists of low-cost, easy-to-acquire electrical components and an open-source microcontroller (Arduino Mega) whose performance is equivalent to that of commercial instruments. Two 3D printable Open-JIP configurations are presented, one for higher plants and the other for microalgae cells in suspension. Directions for its construction are presented and the instrument is benchmarked against widely used commercial chlorophyll fluorometers.

A sensor array is described that consists of a carbon quantum dot (CQD) and metal ions, including Hg 2+ , Cu 2+ , Fe 3+ , Ag + , Cd 2+ , and Pb 2+ . The CQDs display blue fluorescence with excitation/emission maxima at 370/440 nm. It is shown that the array can be applied to the determination of all natural amino acids (NAAs). Metal ions can quench the fluorescence of the CQDs, while NAAs can take metal ions away or co-bind to the CQD@metal-ion complex, which enhances or depresses the fluorescence of the CQDs. Based on the differential fluorescence variation, the CQD@metal-ion@NAA array exhibits a unique pattern for NAAs. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were carried out to generate visualized datagrams for NAA discrimination. The design and construction of the sensor array is convenient and economical. The sensor array can distinguish NAAs at a concentration of as low as 30 μM, and can distinguish NAAs into acidic, neutral and basic NAAs. Semi-quantitative assay of alanine and threonine was also realized. Based on the low limit of detection and multi-NAA detection capability, the array can differentiate healthy cases from acute leukemia, chronic leukemia and lymphoma by analyzing the NAA status of serum samples. Graphical abstractSchematic representation of a fluorometric (FL) sensor array based on single CQD (carbon quantum dot) interacting with metal ions for differentiating all NAAs (natural amino acids) into acidic, neutral and basic NAAs (ANs, NNs and BNs) through PCA (principle component analysis).

The presence of bacterial pathogens in water can lead to severe complications such as infection and food poisoning. This research proposes a point-of-care electroosmotic flow driven microfluidic device for rapid isolation and detection of E. coli in buffered solution (phosphate buffered saline solution). Fluorescent E. coli bound to magnetic microbeads were driven through the microfluidic device using both constant forward flow and periodic flow switching at concentrations ranging from 2 × 10 5 to 4 × 10 7 bacteria/mL. A calibration curve of fluorescent intensity as a function of bacteria concentration was created using both constant and switching flow, showing an increase in captured fluorescent pixel count as concentration increases. In addition, the use of the flow switching resulted in a significant increase in the capture efficiency of E. coli, with capture efficiencies up to 83% ± 8% as compared to the constant flow capture efficiencies (up to 39% ± 11%), with a sample size of 3 µL. These results demonstrate the improved performance associated with the use of the electroosmotic flow switching system in a point-of-care bacterial detection assay.

Lack of access to clean water is a major global issue that affects millions of people worldwide. Drinking contaminated water can be extremely hazardous, so it is imperative that it is tested sufficiently. One method commonly used to determine the quality of water is testing for both E. coli and total coliform. Here, we present a cost-effective and automated device which can concurrently test drinking water samples for both E. coli and total coliform using an EPA-approved reagent. Equipped with a Raspberry Pi microcontroller and camera, we perform automated periodic measurements of both the absorption and fluorescence of the water under test over 24 hours. In each test, 100 mL of the water sample is split into a custom designed 40-well plate, where the transmitted blue light and the fluorescent light (under UV excitation) are collected by 520 individual optical fibers. Images of these fiber outputs are then acquired periodically, and digitally processed to determine the presence of the bacteria in each well of the 40-well plate. We demonstrate that this cost-effective device, weighing 1.66 kg, can automatically detect the presence of both E. coli and total coliform in drinking water within ∼16 hours, down to a level of one colony-forming unit (CFU) per 100 mL. Furthermore, due to its automated analysis, this approach is also more sensitive than a manual count performed by an expert, reducing the time needed to determine whether the water under test is safe to drink or not.

Technological advances have widely improved the field of research as spectroscopic methods are now flexible in analyzing different sample matrix. There have been various methods used in applications of spectrofluorometer, but some were costly, time consuming or complicated for routine analysis, creating barrier for students to understand the basic concepts of fluorescence. This review focuses on the different fluorometer designs and techniques which promote cost efficiency and/or having modifications without compromise in data gathering, and its applications to various scientific fields. The usage of pesticides has a wide range of effects when it comes to the environment and to human health especially when it enters the food chain. The characteristic of having a low-cost, user-friendly and efficient device can occur in different variations as materials and technology are employed to fluorescence detection which primarily contributes to the different applications of the device such as in food safety and security., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel smartphone-based optical biomimetic sensor based on free-standing molecularly imprinted polymer (MIP) membranes was developed for rapid and sensitive point-of-care detection of aflatoxin B1. The developed MIP membranes were capable of selective recognition of the target analyte and, at the same time, of generation of a fluorimetric sensor response, which could be registered using the camera of a smartphone and analysed using image analysis. The developed system provides a possibility of synchronous detection of aflatoxin B1 in 96 channels. UV irradiation of aflatoxin B1, selectively bound by the MIP membranes from the analysed samples, initiated fluorescence of aflatoxin B1 with intensity directly proportional to its concentration. The composition of the MIP membranes used as a recognition element was optimised taking into account data of computational modelling. Two functional monomers (2-acrylamido-2-methyl-1-propansulfonic acid and acrylamide) were identified as optimal for the formation of aflatoxin B1-selective binding sites in the structure of the MIP membranes. Working characteristics of the smartphone-based sensor system were also estimated. The influence of pH and of buffer and NaCl concentrations on the smartphone-based sensor responses were studied. High selectivity of the developed sensor system towards aflatoxin B1 was confirmed in experiments with the close structural analogue of the target analyte - aflatoxin G2, and ochratoxin A. The detection limit for aflatoxin B1 using the smartphone-based sensor systems was found to be 20 ng mL -1 for the sensor based on MIP membranes synthesised with acrylamide as a functional monomer. The storage stability of the recognition elements of the developed sensors was estimated as one year when stored at 22 °C. The possibility to detect the aflatoxin B1 in contaminated food samples was shown. The MIP-membrane-based sensor system provided a convenient point-of-care approach in food safety testing., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We developed a gas-imaging system (sniff-cam) for gaseous ethanol (EtOH) with improved sensitivity. The sniff-cam was applied to measure the extremely low concentration distribution of breath EtOH without the consumption of alcohol, which is related to the activity of the oral or gut bacterial flora. A ring-type ultraviolet-light-emitting diode was mounted around a camera lens as an excitation light source, which enabled simultaneous excitation and imaging of the fluorescence. In the EtOH sniff-cam, a nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH) was used to catalyze the redox reaction between EtOH and the oxidized form of NAD (NAD + ). Upon application of gaseous EtOH to the ADH-immobilized mesh that was soaked in an NAD + solution and placed in front of the camera, NADH was produced through an ADH-mediated reaction. NADH expresses fluorescence at an emission wavelength of 490 nm and excitation wavelength of 340 nm. Thus, the concentration distribution of EtOH was visualized by measuring the distribution of the fluorescence light intensity from NADH on the ADH-immobilized mesh surface. First, a comparison of image analysis methods based on the red-green-blue color (RGB) images and the optimization of the buffer pH and NAD + solution concentration was performed. The new sniff-cam showed a 25-fold greater sensitivity and broader dynamic range (20.6-300000 ppb) in comparison to those of the previously fabricated sniff-cam. Finally, we measured the concentration distribution of breath EtOH without alcohol consumption using the improved sniff-cam and obtained a value of 116.2 ± 35.7 ppb ( n = 10).

To develop conducting organic polymers (COPs) as luminescent sensors for determination of toxic heavy metals, a new benzene sulfonic acid-doped polypyrrole (PPy-BSA) thin film was electrochemically prepared by cyclic voltammetry (CV) on flexible indium tin oxide (ITO) electrode in aqueous solution. PPy-BSA film was characterized by FTIR spectrometry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The optical properties of PPy-BSA were investigated by ultraviolet (UV)-visible absorption and fluorescence spectrometry in dimethylsulfoxide (DMSO) diluted solutions. PPy-BSA fluorescence spectra were strongly quenched upon increasing copper(II) ion (Cu 2+ ) and lead(II) ion (Pb 2+ ) concentrations in aqueous medium, and linear Stern-Volmer relationships were obtained, which indicated the existence of a main dynamic fluorescence quenching mechanism. BSA-PPy sensor showed a high sensitivity for detection of both metallic ions, Cu 2+ and Pb 2+ , with very low limit of detection values of 3.1 and 18.0 nM, respectively. The proposed quenching-fluorimetric sensor might be applied to the determination of traces of toxic heavy metallic ions in water samples., (© 2019 John Wiley & Sons, Ltd.)

By combination of miniaturization potential of digital microfluidics (DMF) and sensitivity of fluorescence probe, an integrated sensor device has been initially constructed for mercury detection in coastal waters. The actuation feature of the detecting target, seawater droplet, which remains unclear, was basically explored. To overcome a potential risk of driven failure, induced by diversity ion ingredients in seawater, a feedback control loop was included into control system. Analyzing method for coastal waters was well established on DMF, which showed satisfied stability and selectivity in Hg sensing under high salinity condition, with the sensitivity of Hg 2+ at the parts per billion level and total testing time less than 20s. With the advantages of being fast, amenable to automation and low cost, this device is promising for the formation of simple and rapid sensor device, especially for a routine monitoring and emergency detection of Hg/or other metals in coastal waters., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The authors describe the syntheses and application of glutathione-capped gold nanoclusters (AuNCs) with thermoresponsive properties. The AuNCs have excitation/emission maxima at 430/610 nm and the bright redfluorescence changes along with the temperature in the range from 0 to 90 °C which covers the normal temperature range of living cells. In the range of physiological temperatures (35-42 °C), the temperature resolution is 0.73 °C. The AuNCs display excellent colloidal stability and biocompatibility. They were used for fluorometric temperature detection and imaging of hepatic stellate cells. With such attractive features, the AuNCs are quite promising luminescence nanothermometers. Graphical abstract Schematic presentation of the fluorescence of glutathione-capped gold nanoclusters (AuNCs) as nanothermometers in living cells. The AuNCs have excitation/emission maxima at 430/610 nm and the red fluorescence changes with temperature in a wide range of 0 to 90 °C which covers the normal temperature of living cells.

Little cherry virus 1 (LChV-1) belongs to the genus Velarivirus, family Closteroviridae, is an economically important pathogen affecting mainly cherry around the world emphasizing the impetus for its efficient and accurate on-site detection. This study describes the development of a reliable diagnostic protocol of LChV-1 based on a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The protocol detects LChV-1 isolates in less than 10 min by fluorescence monitoring using a mobile detection device and is most optimal when performed at 67 °C. Sharp melting curves and unique melting temperatures (Tm) were obtained for the positive samples. Both the RT-LAMP and classical RT-PCR methods are capable of specifically detecting LChV-1 in infected leaf tissues. In addition, the RT-LAMP has remarkable advantages in comparison to RT-PCR. It is at least hundred fold more sensitive, significantly faster (allowing on-field leaf-to-result diagnostic) and efficient at minimal cost. In conclusion, this innovative RT-LAMP approach can contribute to the implementation of sustainable integrated management strategies for detection of LChV-1 in commercial orchards or for horticultural research stations. It is also suitable for decision support in phytosanitary epidemiological programs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The emergence of begomovirus infection is one of the most important problems affecting production of a variety of vegetable crops worldwide. Infection by begomoviruses has been detected and spread rapidly on Cucurbitaceae and Solanaceae plants in Indonesia. A rapid and simple detection assay for begomoviruses under field conditions for routine sampling of plants is needed. Primers for a loop-mediated isothermal amplification (LAMP) assay were designed based on the sequences of three Indonesian begomoviruses, namely Tomato leaf curl New Delhi virus (ToLCNDV), Pepper yellow leaf curl Indonesia virus (PepYLCIV), and Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), infecting Cucurbitaceae and Solanaceae plants. LAMP assays using a Genelyzer ™ III portable fluorometer with a toothpick method successfully detected these begomoviruses in infected melon, pepper, and eggplant samples. LAMP assays conducted during a field survey for detection of the three begomoviruses on 104 fresh leaves indicated that most of the samples were positive; the findings were confirmed by PCR using universal primers of begomovirus as a common detection method. These results demonstrate that this simple and rapid LAMP assay using a fluorometer portable device may be used to achieve real-time detection of begomoviruses under field conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An enzyme-free fluorometric assay is described that accomplishes dual signal amplification by making use of a two stirring bars. Two Y-shaped DNA probes were designed and placed on the bars. When the target (with chloramphenicol as model analyte) is added, it triggers target recycling and simultaneously catalyzes hairpin assembly (CHA). A large fraction of DNA primers is released by the analyte from the bar to the supernatant and open hairpins with G-quadruplex DNA sequence. The G-quadruplex can specifically bind thioflavin T (ThT) to emit fluorescence (with excitation/emission maxima at 445 and 485 nm) for quantification of chloramphenicol. An enzyme is not needed. ThT is added to the system as a fluorescent DNA probe. All this strongly reduces the cost for sensor construction and usage. The dual signal amplification steps occur simultaneously which reduces the detection time. The assay was successfully employed to the determination of CAP in spiked milk and fish samples within 60 min and with a 16 pM limit of detection (at S/N = 3). Graphical abstract Schematic representation of a new method for the detection of chloramphenicol by using  two stirring bars. It is based on target recycling and catalyzed hairpin assembly amplification. CAP: chloramphenicol, ThT: thioflavin T, CHA: catalyzed hairpin assembly.

Laser-induced fluorometry (LIF) has achieved the detection of single molecules, which ranks it among the most sensitive of detection techniques, whereas capillary electrophoresis (CE) is known as a powerful separation method with resolution that is beyond the reach of many other types of chromatography. Therefore, a coupling of LIF with CE has established an unrivaled analytical technique in terms of sensitivity and resolution. CE-LIF has demonstrated excellent performance in bioanalytical chemistry for the high-resolution separation and highly sensitive detection of DNAs, proteins, and small bioactive molecules. This review describes the CE-LIF methods developed by the author's group that include indirect and direct detection using diode lasers, post-column derivatization, and Hadamard transformation, as well as applications to the binding assays of specific DNA immunoassays of proteins and to the determination of anticancer drugs., (© 2019 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical pH sensors enable noninvasive monitoring of pH, yet in pure sensing terms, the potentiometric method of measuring pH is still vastly superior. Here, we report a full spectrometer-based optical pH sensor system consisting of sensor chemistry, hardware, and software that for the first time is capable of challenging the performance of an electrode-based pH meter in specific applications such as biopharmaceutical process monitoring and in single-use bioproduction. A highly photostable triangulenium fluorophore emitting at 590 nm was immobilized in an organically modified silicon matrix that allows for fast time-response by rapid diffusion of water in and out of the resulting composite polymer deposited on a polycarbonate substrate. Fluctuations from the fiber optical sensor hardware have been reduced by including a highly photostable terrylene-based reference dye emitting at 660 nm, thus enabling intensity-based ratiometric readouts. The dyes were excited by 505 nm light from a light emitting diode. The sensor was operational within a pH range of 4.6-7.6, and was characterized and demonstrated to have properties that are comparable to those of commercial pH electrodes considering time-response ( t 90 < 90 s), precision (0.03 pH-units), and drift.

Background: Intracranial photometry through chronically implanted optical fibers is a widely adopted technique for measuring signals from fluorescent probes in deep-brain structures. The recent proliferation of bright, photo-stable, and specific genetically encoded fluorescent reporters for calcium and for other neuromodulators has greatly increased the utility and popularity of this technique., New Method: Here we describe an open-source, cost-effective, microcontroller-based solution for controlling optical components in an intracranial photometry system and processing the resulting signal., Results: We show proof-of-principle that this system supports high quality intracranial photometry recordings from dorsal striatum in freely moving mice. A single system supports simultaneous fluorescence measurements in two independent color channels, but multiple systems can be integrated together if additional fluorescence channels are required. This system is designed to work in combination with either commercially available or custom-built optical components. Parts can be purchased for less than one tenth the cost of commercially available alternatives and complete assembly takes less than one day for an inexperienced user., Comparison With Existing Method(s): Currently available hardware draws on a variety of commercial, custom-built, or hybrid elements for both optical and electronic components. Many of these hardware systems are either specialized and inflexible, or over-engineered and expensive., Conclusions: This open-source system increases experimental flexibility while reducing cost relative to current commercially available components. All software and firmware are open-source and customizable, affording a degree of experimental flexibility that is not available in current commercial systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50 × 4.6 mm C 18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10 mM phosphate buffer (pH 7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10 mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30 min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Zearalenone (ZEA), a mycotoxin produced by several Fusarium molds, can be found in many cereals and related products. The toxicity of ZEA has been reported for both humans and animals. Therefore, many countries have adopted regulations in foods and feed materials to limit the exposure to this contaminant. In this paper, we propose a multicommutated flow-through optosensor to quantify ZEA in different cereal samples. ZEA was retained and pre-concentrated on C 18 silica gel, and the use of the multicommutated flow manifold allowed the automated retention/desorption of ZEA on the solid microbeads by the use of appropriate carrier/eluting solutions, hence increasing the selectivity and sensitivity of the system. The native fluorescence of ZEA was recorded on the solid phase at λ exc /λ em of 265/465 nm/nm. A QuEChERS procedure was used to carry out the extraction of ZEA from different cereal samples (feedstuff materials). Recovery studies were performed to assess the accuracy of the method, obtaining recovery yields between 93% and 107% in all the analyzed samples. LC-MS was employed as reference method. The quantitation limit of the proposed method was low enough to fulfill the maximum residue levels established by the Commission of the European Communities, thus demonstrating its potential use for the analysis of ZEA in feedstuffs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: L-histidine (His), L-cysteine (Cys), creatine (Crn), S-methyl-L-cysteine (Me-Cys), DL-homocysteine (Hcy), L-methionine (Met), beta-(3,4-dihydroxyphenyl)-L-alanine (DOPA), L-tyrosine (Tyr), DL-m-tyrosine (m-Tyr), L-a-methyl-DOPA (Me-DOPA), L-phenylalanine (Phe), DL-alpha-methyltyrosine (Me-Tyr), 5-hydroxy-tryptophan (5-HTP), 3-nitro-L-tyrosine (NO 2 Tyr), and L-tryptophan (Trp), as well as of 2 dipeptides L-cystathionine (Cysta) and L-carnosine (Car), and of creatinine (Cre). A multilinear solvent (acetonitrile) gradient elution program, determined by a simple optimization algorithm, is required for the efficient reversed-phase separation of the above mixture of 18 solutes within 27 min at a flow rate of 1.0 mL/min and at 25 °C.

The use of fluorescence-guided surgery for benign and malignant hepatobiliary (HPB) neoplasms has significantly increased and improved imaging methods creating new interesting perspectives. A major challenge in HPB surgery is performing radical resection with maximal preservation of the liver parenchyma and obtaining a low rate of complications. Despite the developments, visual inspection, palpation, and intraoperative ultrasound remain the most utilized tools during surgery today.  In laparoscopic and robotic HPB surgery palpation is not possible. Fluorescence imaging enables identification of subcapsular liver tumors through accumulation of indocyanine green (ICG), after preoperative intravenous injection, in cancerous tissues of hepatocellular carcinoma and in noncancerous hepatic parenchyma, around intrahepatic cholangiocarcinoma and liver metastases, and it can also be used for visualizing extrahepatic bile duct anatomy and hepatic segmental borders, increasing the accuracy and the easiness of open and minimally invasive hepatectomy.

Multi-wavelength fluorometers, such as the bbe FluoroProbe (FP), measure excitation spectra of chlorophyll a (Chl-a) fluorescence to infer the abundance and composition of phytoplankton communities as well as the concentration of chromophoric dissolved organic matter (CDOM). Experiments were conducted on laboratory cultures and on natural communities of freshwater phytoplankton to determine how the response of phytoplankton to high irradiance might affect fluorometric estimates of community composition and concentrations of Chl-a and CDOM. Cultures of a representative cyanobacterium, bacillariophyte, synurophyte, cryptophyte, and chlorophyte revealed changes in Chl-a excitation spectra as irradiance was increased to saturating levels and non-photochemical quenching (NPQ) increased. The degree of change and resulting classification error varied among taxa, being strong for the synurophyte and cryptophyte but minimal for the cyanobacterium. Acute-exposure experiments on phytoplankton communities of varying taxonomic composition from five lakes yielded variable results on apparent community composition. There was a consistent decrease in CDOM estimates, whereas Chl-a estimates were generally increased. Subsequent exposure to low PAR relaxed NPQ and tended to reverse the effects of high irradiance on composition, total Chl-a, and CDOM estimates. Relaxation experiments on near-surface communities in a sixth, large lake, Georgian Bay, showed that total Chl-a estimates increased by 44% on average when dark treatments were used to relax NPQ, though, in contrast to the findings from the small lakes, there was little effect on CDOM estimates. We observed a statistically-significant, negative linear relationship between the photon flux density of in situ irradiance and the accuracy of taxonomic assignment by FP in Georgian Bay. Not discounting the correlations between light intensity and the accuracy of the FP that were observed in this study, we conclude that the applicability of the reference spectra to the system under investigation is a more important consideration than variability in natural irradiance conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A method is described for sensitive and selective fluorometric determination of morphine. It is based on the effect of morphine on quenching of the fluorescence of fluorescein by gold nanoparticles (AuNPs) via surface energy transfer. When fluorescein is added to solutions of colloidal AuNPs, its fluorescence becomes quenched due to nanometal surface energy transfer (NSET) because the absorption of AuNPs strongly overlaps the emission spectrum of fluorescein. In the presence of morphine, which contains both a tertiary nitrogen ring atom and a phenolic hydroxy group, it will coordinate to the AuNPs, and this causes recovery of fluorescence. The presence of a tertiary nitrogen ring atom and a phenolic hydroxy group (both required for the effect to occur) in morphine make the probe highly selective and sensitive for morphine. A paper strip assay also was developed by utilizing this detection scheme. The turn-on fluorescent probe was successfully applied to the determination of morphine in spiked serum and urine samples. The method has a 53 pM limit of detection. The paper strip was applied to the determination of morphine in sweat, urine and other biological fluids. It is perceived to be useful for early detection of drug abuse by adolescent. Graphical abstract Schematic of the mechanism of fluorescence turn on detection of morphine using Au NPs (gold nanoparticles) acting asquencher of the fluorescence of fluorescein.

Free thyroxine (FT4) quantification is continuing to be a concern. The purpose of the following study was to evaluate the analytical performance of Tosoh AIA900 based on a one-step technique and its comparison to Access 2 (two-step technique) over different clinical contexts (euthyroid, thyroid disorders, uncontrolled diabetes, renal failure and pregnancy). The protocol established by the French society of Clinical Biology was used to evaluate: imprecision, limit of detection, trueness, linearity, interferences and method comparisons. Within-run variation of 3.1%, 5.7% and 4.4% were found for the low, medium and high controls, respectively. Between-run was 5.8% for low control, 5.7% for medium control and 7.1% for high control. Common interferences did not affect one-step immunoassay FT4 results. The linearity was checked up to 86 pmol/L. The limit of detection was 5.5 pmol/L. The concordance correlation coefficient (CCC) showed a low agreement (0.6) between both methods. Bland-Altman plot revealed that AIA 900 one-step immunoassay technique provides a significant higher values ((+2.8 ± 2.7 pmol/L;p < 0.0001). The Passing-Bablok regression demonstrated both proportional and systematic differences in comparison to Access 2. The lowest association was noted in subjects with impaired renal function (CCC = 0.27). At the time of the study, the results of on-step immunoassay are not directly comparable with Access 2., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A fluorescent array based on the use of saccharide-functionalized multicolored quantum dots (s-QDs) and of 4-mercaptophenylboronic acid-functionalized MoS 2 nanosheets (PBA-MoS 2 ) was constructed for multiple identification and quantitation of lectins and bacteria. In this array, the fluorescence of the s-QDs is quenched by the PBA-MoS 2 nanosheets. In the presence of multiple lectins, s-QDs differentially detach from the surface of PBA-MoS 2 nanosheets, producing distinct fluorescence response patterns due to both quenching and enhancement of fluorescence. By analyzing the fluorescence responses with linear discriminant analysis, multiple lectins and bacteria were accurately identified with 100% accuracy. The limits of detection of Concanavalin A, Pisum sativum agglutinin, Peanut agglutinin, and Ricius communis I agglutinin are as low as 3.7, 8.3, 4.2 and 3.9 nM, respectively. The array has further been evidenced to be potent for distinguishing and quantifying different bacterial species by recognizing their surface lectins. The detection limits of Escherichia coli and Enterococcus faecium are 87 and 66 cfu mL -1 , respectively. Graphical abstract Schematic of a fluorometric array based on the use of saccharides-functionalized quantum dots (s-QDs) and 4-mercaptophenylboronic acid-functionalized MoS 2 (PBA- MoS 2 ) nanosheets. This array was successfully applied to simultaneously analysis of lectins, bacteria in real samples with high sensitivity and accuracy.

A dual-color fluorescence resonance energy transfer (FRET) based aptasensor is described for simultaneous determination of the mycotoxins aflatoxin M1 (AFM1) and ochratoxin A (OTA). Aptamers against AFM1 and OTA were labeled with two fluorophores with different excitation wavelengths (Cy5.5; 675 nm; and Alexa 405; 401 nm), respectively. They were used as the signalling probes. A compact dual-color evanescent wave all-fiber detection system with two lasers (635 nm; red; and 405 nm; purple) was used for the simultaneous collection of two-wavelength fluorescence signals. The hybridization of labeled aptamers with complementary sequences (Q-cDNA) labeled with a dark quencher (BHQ 3  or dabcyl) causes fluorescence to be strongly reduced because of the fluorescence resonance energy transfer. In the presence of AFM1 and OTA, they bind to their respective aptamer and result in the dissociation of double stranded DNA, which induce fluorescence recovery. Under the optimum conditions, AFM1 and OTA can simultaneously and selectively be determined ranged from 1 ng·L -1 to 1 mg·L -1 . The detection limits of AFM1 and OTA are 21 and 330 ng·L -1 , respectively (S/N = 3). The FRET-based dual-color detection scheme was applied to the simultaneous detection of AFM1 and OTA in milk with good recovery, precision, and accuracy. Graphical abstract Aptamers against AFM1 and OTA were labeled with two fluorophores with different excitation wavelengths (Cy5.5; 675 nm; and Alexa 405; 401 nm) and then used as signalling probes. A FRET-based aptasensor is described for simultaneous determination of AFM1 and OTA using dual-color evanescent wave system with two lasers (635 nm; red; and 405 nm).

Two-photon excitation fluorometry (TPX) is a separation-free bioaffinity assay technique which enables accurate diagnostic testing in microvolumes. The technology is currently commercially applied in an automated mariPOC® test system for rapid phenotypic multi-microbe detection of pathogen antigens. The first TPX applications for diagnostics were intended for respiratory infection testing from nasopharyngeal and oropharyngeal samples. Feces and urine are more complex sample matrices and contain substances that may interfere with immunoassay binding or fluorescence detection. Our objective was to study the suitability of these complex matrices in the TPX technique. As expected, feces and urine elevated fluorescence levels but the methodology has the unique property of compensating for matrix effects. Compensation allows reliable separation of specific fluorescence from the fluorescence caused by the matrix. The studied clinical samples did not contain immunoassay inhibitors. The results suggest that the methodology is robust and may provide reliable testing of feces and urine samples with high accuracy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The study reports synthesis and photophysical studies of a new zinc sensing pyrazole scaffold structurally characterized to be bis(2-hydroxybenzylidene)-1H-pyrazole-3,5-dicarbohydrazide (PHSA). Excitation of the probe at 330 nm results in an emission band at 417 nm which ratiometrically red shifts to 466 nm upon Zn 2+ addition in an unprecedented way. The photo induced electron transfer (PET) coupled intramolecular charge transfer (ICT) working for a dual-channel fluorescence emission pathway is observed and studies were supported with density functional theory and NMR titration experiments. The probe exhibited dissociation constant of 1.2156 and detection limit as low as 992 nM. The cytotoxic effects of the probe on 60 tumour cell lines were tested. The intracellular zinc sensing with reversible binding potential is verified with fluorescence microscopy experiment.

The rich composition of solutes and metabolites in sweat and its relative ease of collection upon excretion from skin pores make this class of biofluid an attractive candidate for point of care analysis. Wearable technologies that combine electrochemical sensors with conventional or emerging semiconductor device technologies offer valuable capabilities in sweat sensing, but they are limited to assays that support amperometric, potentiometric, and colorimetric analyses. Here, we present a complementary approach that exploits fluorometric sensing modalities integrated into a soft, skin-interfaced microfluidic system which, when paired with a simple smartphone-based imaging module, allows for in situ measurement of important biomarkers in sweat. A network array of microchannels and a collection of microreservoirs pre-filled with fluorescent probes that selectively react with target analytes in sweat (e.g. probes), enable quantitative, rapid analysis. Field studies on human subjects demonstrate the ability to measure the concentrations of chloride, sodium and zinc in sweat, with accuracy that matches that of conventional laboratory techniques. The results highlight the versatility of advanced fluorescent-based imaging modalities in body-worn sweat microfluidics platforms, and they suggest some practical potential for these ideas.

The dynamic and efficient coordination of primary photosynthetic reactions with leaf energization and metabolism under a wide range of environmental conditions is a fundamental property of plants involving processes at all functional levels. The present historical perspective covers 60 years of research aiming to understand the underlying mechanisms, linking major breakthroughs to current progress. It centers on the contributions of Ulrich Heber who had pioneered novel concepts, fundamental methods, and mechanistic understanding of photosynthesis. An important first step was the development of non-aqueous preparation of chloroplasts allowing the investigation of chloroplast metabolites ex vivo (meaning that the obtained results reflect the in vivo situation). Later on, intact chloroplasts, retaining their functional envelope membranes, were isolated in aqueous media to investigate compartmentation and exchange of metabolites between chloroplasts and external medium. These studies elucidated metabolic interaction between chloroplasts and cytoplasm during photosynthesis. Experiments with isolated intact chloroplasts clarified that oxygenation of ribulose-1.5-bisphosphate generates glycolate in photorespiration. The development of non-invasive optical methods enabled researchers identifying mechanisms that balance electron flow in the photosynthetic electron transport system avoiding its over-reduction. Recording chlorophyll a (Chl a) fluorescence allowed one to monitor, among other parameters, thermal energy dissipation by means of 'nonphotochemical quenching' of the excited state of Chl a. Furthermore, studies both in vivo and in vitro led to basic understanding of the biochemical mechanisms of freezing damage and frost tolerance of plant leaves, to SO 2 tolerance of tree leaves and dehydrating lichens and mosses.

Purpose: The permeability of the corneal epithelium to fluorescein Pdc is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of Pdc by a spot fluorometer., Methods: Twenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 μL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 μL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma Fs was measured. Permeability was calculated using Pdc = (Q x Fs)/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops., Results: After the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; kd = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in Fs was negligible. However, after the loading drops, the measured Fs was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of kd was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated Pdc was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated Pdc., Conclusions: The new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The Pdc estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient., Competing Interests: The authors have declared that no competing interests exist.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1 -/- , and Cry2 -/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle., Competing Interests: The authors declare no conflict of interest.

Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins) failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking water.

High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. For harmonization of results in a common database, we compared respiration and H 2 O 2 production of permeabilized HEK 293T cells measured in MiR05 (sucrose and K-lactobionate), Buffer Z (K-MES and KCl), MiR07 (combination of MiR05 and Buffer Z), and MiRK03 (KCl). Respiration in a simple substrate-uncoupler-inhibitor titration protocol was identical in MiR05, Buffer Z, and MiR07, whereas oxygen fluxes detected with MiRK03 were consistently lower in all coupling and electron transfer-pathway states. H 2 O 2 production rates were comparable in all four media, while assay sensitivity was comparatively low with MiR05 and MiR07 and higher but declining over time in the other two media. Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H 2 O 2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.

Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O 2 . Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix, as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, converts superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H 2 O.In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.