Ultraviolet-fluorescence detection of E. Coli using threshold-comparison electronics.

We describe an optical system that detects the presence of E. coli bacteria, making use of the bacteria's natural fluorescence properties. The system provides an excitation signal at 365 nm and detects the emission signal, from the bacteria, at approximately 445 nm. The system also allows the intensity of the emitted signal to be compared with a user-programmable threshold. This allows rapid testing of many samples in a laboratory setting. Complete setup and performance details are provided, enabling the experimentalist to tailor the system parameters to other species of microorganisms, which may have fluorescence properties at other wavelengths.

A ratiometric fluorescent dark box and smartphone integrated portable sensing platform based on hydrogen bonding induction for on-site determination of enrofloxacin.

Enrofloxacin (ENR) residues in animal-derived food and water threaten human health. Simple, low-cost and on-site detection methods are urgently needed. Blue emitting carbon quantum dots (CQDs) and orange rhodamine B (RhB) were used as recognition and reference signals, respectively, to construct a ratiometric fluorescence sensor. After the addition of ENR, the color of the sensor changed from orange to blue because hydrogen bonding induced a considerable increase in CQDs fluorescence. Based on this mechanism, a simple and low cost on-site portable sensing platform was constructed, which integrated a stable UV light strip and a smartphone with voice-controlled phototaking function and an RGB app. The t-test results of spiked ENR recoveries for diluted milk, honey and drinking water revealed no significant differences between the ratiometric fluorescent sensor and portable sensing platform. Thus, this portable sensing platform provides a novel strategy for on-site quantification of quinolone antibiotics in foodstuffs and environmental water.

Portable smartphone-based RecJf exonuclease-modulated enhanced ratiometric fluorescence bioplatform for rapid visual detection of As3.

Arsenite (As3+), a highly carcinogenic heavy metal ion and widely distributed in nature, can have serious health implications even with minimal exposure. Herein, a portable smartphone device-based ratiometric fluorescence platform was established for sensitive detection of As3+. The work relied on the use of metal-organic framework-tagged cDNA (PCN-224-cDNA), with high adsorption capability and fluorescence properties, as an internal reference to quench the fluorescence of FAM-anchored aptamer (FAM-Apt) via hybridization. In the presence of As3+, FAM-Apt specifically bound to As3+ leading to conformational changes, which detached from the PCN-224-cDNA surface. Interestingly, a smartphone-based readout equipment engineered using a 3D-printed hardware device administered the portable detection of As3+. The limit of detection (LOD) for the proposed ratiometric biosensor was calculated to be 0.021 ng/mL, significantly below WHO's safety threshold. Hence, it demonstrates significant potential for large-scale screening of As3+ residues in food and the environment.

ChloroSpec: A new in vivo chlorophyll fluorescence spectrometer for simultaneous wavelength- and time-resolved detection.

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer - ChloroSpec - allowing three-dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time-resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three-dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.

Smartphone-assisted mobile fluorescence sensor for self-calibrated detection of anthrax biomarker, Cu2+, and cysteine in food analysis.

Dipicolinic acid (DPA), as a biomarker for Bacillus anthracis, is highly toxic at trace levels. Rapid and on-site quantitative detection of DPA is essential for maintaining food safety and public health. This work develops a dual-channel self-calibrated fluorescence sensor constructed by the YVO4:Eu and Tb-ß-diketone complex for rapid visual detection of DPA. This sensor exhibits high selectivity, fast response time, excellent detection sensitivity, and the detection limit is as low as 4.5 nM in the linear range of 0-16 µM. A smartphone APP and portable ultraviolet lamp can assemble a mobile fluorescence sensor for on-site analysis. Interestingly, adding Cu2+ ions can quench the fluorescence intensity of Tb3+. In contrast, the addition of cysteine can restore the fluorescence, allowing the accurate detection of Cu2+ ions and cysteine in environmental water and food samples. This work provides a portable sensor that facilitates real-time analysis of multiple targets in food and the environment.

In-lab synthesized turn-off fluorescence sensor for estimation of Gemigliptin and Rosuvastatin polypill appraised by Spider diagram, AGREE and whiteness metrics.

Gemigliptin-Rosuvastatin single-pill combination is a promising therapeutic tool in the effective control of hyperglycemia and hypercholesterolemia. Organic sensors with high quantum yields have profoundly significant applications in the pharmaceutical industry, such as routine quality control of marketed formulations. Herein, the fluorescence sensor, 2-Morpholino-4,6-dimethyl nicotinonitrile 3, (λex; 226 nm, λem; 406 nm), was synthesized with a fluorescence quantum yield of 56.86% and fully characterized in our laboratory. This sensor showed high efficiency for the determination of Gemigliptin (GEM) and Rosuvastatin (RSV) traces through their stoichiometric interactions and simultaneously fractionated by selective solvation. The interaction between the stated analytes and sensor 3 was a quenching effect. Various experimental parameters and the turn-off mechanism were addressed. The adopted approach fulfilled the ICH validation criteria and showed linear satisfactory ranges, 0.2-2 and 0.1-1 µg/mL for GEM and RSV, respectively with nano-limits of detection less than 30 ng/mL for both analytes. The synthesized sensor has been successfully applied for GEM and RSV co-assessment in their synthetic polypill with excellent % recoveries of 98.83 ± 0.86 and 100.19 ± 0.64, respectively. No statistically significant difference between the results of the proposed and reported spectrophotometric methods in terms of the F- and t-tests. Ecological and whiteness appraisals of the proposed study were conducted via three novel approaches: the Greenness Index via Spider Diagram, the Analytical Greenness Metric, and the Red-Green-Blue 12 model. The aforementioned metrics proved the superiority of the adopted approach over the previously published one regarding eco-friendliness and sustainability. Our devised fluorimetric turn-off sensing method showed high sensitivity, selectivity, feasibility, and rapidity with minimal cost and environmental burden over other sophisticated techniques, making it reliable in quality control labs.

Chip-Based Spectrofluorimetric Determination of Iodine in a Multi-Syringe Flow Platform with and without In-Line Digestion-Application to Salt, Pharmaceuticals, and Algae Samples.

In this work, a flow-based spectrofluorimetric method for iodine determination was developed. The system consisted of a miniaturized chip-based flow manifold for solutions handling and with integrated spectrofluorimetric detection. A multi-syringe module was used as a liquid driver. Iodide was quantified from its catalytic effect on the redox reaction between Ce(IV) and As(III), based on the Sandell-Kolthoff reaction. The method was applied for the determination of iodine in salt, pharmaceuticals, supplement pills, and seaweed samples without off-line pre-treatment. An in-line oxidation process, aided by UV radiation, was implemented to analyse some samples (supplement pills and seaweed samples) to eliminate interferences and release iodine from organo-iodine compounds. This feature, combined with the fluorometric reaction, makes this method simpler, faster, and more sensitive than the classic approach of the Sandell-Kolthoff reaction. The method allowed iodine to be determined within a range of 0.20-4.0 µmol L-1, with or without the in-line UV digestion, with a limit of detection of 0.028 µmol L-1 and 0.025 µmol L-1, respectively.

Ratiometric fluorescence and colorimetric dual-mode sensing platform based on carbon dots for detecting copper(II) ions and D-penicillamine.

A sensing platform with both ratiometric fluorescence and colorimetric responses towards copper(II) ions (Cu2+) and D-penicillamine (D-pen) was constructed based on carbon dots (CDs). o-Phenylenediamine (OPD) was employed as a chromogenic development reagent for reaction with Cu2+ to generate the oxidation product 2,3-diaminophenazine (oxOPD), which not only emits green fluorescence at 555 nm, but also quenches the blue fluorescence of CDs at 443 nm via the inner filter effect (IFE) and Förster resonance energy transfer (FRET). Additionally, oxOPD exhibits obvious absorption at 420 nm. Since the intense chelation affinity of D-pen to Cu2+ greatly inhibits the oxidation of OPD, the intensity ratio of fluorescence at 443 nm to that at 555 nm (F443/F555) and the absorbance at 420 nm (A420) were conveniently employed as spectral response signals to represent the amount of D-pen introduced into the testing system. This dual-signal sensing platform exhibits excellent selectivity and sensitivity towards both Cu2+ and D-pen, with low detection limits of 0.019 µM and 0.092 µM, respectively. In addition, the low cytotoxicity of the testing reagents involved in the proposed sensing platform facilitates its application for live cell imaging.

A modern, simple, and economical accessory for continuous L-format measurement of steady-state fluorescence anisotropy.

In this study, the pros and cons of the most relevant L-format devices reported in the literature for measuring steady-state fluorescence polarization/anisotropy are identified. Combining all this information, and with the use of modern elements for the acquisition, treatment, and recording of signals, a modern, simple, and economical L-format accessory is implemented to rapidly and continuously record steady-state fluorescence anisotropy. This device can be adapted to the majority of the commercial spectrofluorometers (or fluorometers). During the measurement, the emission polarizer is in permanent rotation by means of a Gimbal brushless DC motor, and as a result the recorded fluorescence signal is sinusoidal. The maximums and minimums of this signal, which are obtained with the help of LabVIEW tools, allow recording the fluorescence anisotropy. The LabVIEW applications developed for this investigation are freely available, so it is not necessary to have LabVIEW software.

Capillary-based fluorescence microsensor with polyoxometalates as nanozyme for rapid and ultrasensitive detection of artemisinin.

A novel capillary-based fluorescence microsensor for artemisinin was developed with functional polyoxometalates (POMs) as nanozyme by a layer-by-layer self-assembly strategy. Vanadomolybdophosphoric heteropoly acid (H5PMo10V2O40, PMoV2) and tungstophosphoric heteropoly acid (Na5PW11O39Cu, PW11Cu) with high peroxidase-like activity were synthesized and immobilized on capillary to catalyze artemisinin/thiamine reaction and generate the amplified fluorescence signal. The wide linear range up to 13.0 µM with the low limit of detection of 0.03 µM (S/N = 3) was achieved for the determination of artemisinin by using the proposed POMs-microsensor. The method has been successfully used to detect artemisinin in human plasma and antimalarial drugs with satisfactory accuracy. This work developed a novel capillary fluorescence microsensor with functional POMs as nanozyme, which can serve as a promising candidate in fluorescence microanalysis.

Nitrogen, boron-doped Ti3C2 MXene quantum dot-based ratiometric fluorescence sensing platform for point-of-care testing of tetracycline using an enhanced antenna effect by Eu3.

The Ti3C2 MXene quantum dots (Ti3C2 MQDs) derived from Ti3C2 MXene have received much attention because of their remarkable advantages in biosensing. Nevertheless, the functionalization of Ti3C2 MQDs to improve their properties is just in its infant stage. Herein, we firstly synthesized nitrogen and boron co-doped Ti3C2 MQDs (N, B-Ti3C2 MQDs) with good water solubility, strong stability, and high optical characteristics. The N, B-Ti3C2 MQDs exhibit excitation wavelength-dependent blue photoluminescence with optimal excitation/emission peaks at 335/439 nm. Nowadays, the development of fast and real-time detection of tetracycline (TC) in animal derived food is very essential. In this work, a novel point-of-care testing (POCT) platform was established based on ratiometric fluorescence method using N, B-Ti3C2 MQDs coupled with Eu3+. Upon addition of TC in the Eu3+/N, B-MQDs system, blue fluorescence emission of N, B-Ti3C2 MQDs was quenched and red fluorescence emission of Eu3+ was enhanced gradually, which was ascribed to the synergistic inner filter effect and antenna effect. Moreover, we prepared test papers with N, B-Ti3C2 MQDs and Eu3+ for TC detection based on the change of fluorescence color, which could be recognized by color recognizer app installed in the smartphone. Therefore, great promise for POCT of TC is given with the merits of simplicity and visible detection possibility. The proposed method demonstrated a low detection limit of 20 nM. Application of the platform for TC quantification in milk samples opened a novel means for the potential use of N, B-Ti3C2 MQDs in food safety.

N, Cl-doped carbon dots for fluorescence and colorimetric dual-mode detection of water in tetrahydrofuran and development of a paper-based sensor.

N, Cl-doped carbon dots (N, Cl-CDs) were prepared by hydrothermal method from rhodamine B (RhB) and ethylenediamine (EDA). The resulting N, Cl-CDs exhibited fascinating solvent dependence and strict excitation independence. As the polarity of the solvent increased (from tetrahydrofuran (THF) to water), the emission spectrum of N, Cl-CDs was redshifted and the fluorescence efficiency decreased, which were attributed to hydrogen bond-induced aggregation. Taking advantage of these attributes, the N, Cl-CDs were used as suitable probes for fluorescence and colorimetric dual-mode detection of water in THF. The linear relationship was 0.5-100% water with the detection limit down to 0.093%. Moreover, the sensing platform was converted into a paper-based sensor for handy, real-time, and visible humidity sensing. N, Cl-CDs/PVA films were fabricated and realized continuously tunable solid-state fluorescence, further expanding their practical application.

3D-printed smartphone-based device for fluorimetric diagnosis of ketosis by acetone-responsive dye marker and red emissive carbon dots.

A portable smartphone device is reported that uses 3D printing technology for the primary diagnosis of diseases by detecting acetone. The key part of the device consists of red carbon dots (RCDs), which are used as internal standards, and a sensing reagent (3-N,N-(diacethydrazide)-9-ethylcarbazole (2-HCA)) for acetone. With an excitation wavelength of 360 nm, the emission wavelengths of 2-HCA and RCDs are 443 nm and 619 nm, respectively. 2-HCA effectively captures acetone to form a nonfluorescent acylhydrazone via a condensation reaction occurring in aqueous solution, resulting in obvious color changes from blue-violet to dark red. The detection limit for acetone is 2.62 µM (~ 0.24 ppm). This is far lower than the ketone content in normal human blood (≤ 0.50 mM) and the acetone content in human respiratory gas (≤ 1.80 ppm). The device has good recovery rates for acetone detection in blood and exhaled breath, which are 90.56-109.98% (RSD ≤ 5.48) and 92.80-108.00% (RSD ≤ 5.07), respectively. The method designed here provides a reliable way to provide health warnings by visually detecting markers of ketosis/diabetes in blood or exhaled breath. The portable smart phone device visually detects ketosis/diabetes markers in the blood or exhaled breath through the nucleophilic addition reaction, which effectively captures acetone to form nonfluorescent acyl groups. This will be a reliable tool to warn human health.

A fluorescent and colorimetric dual-channel sensor based on acid phosphatase-triggered blocking of internal filtration effect.

A colorimetric and fluorescent dual-channel detection method for acid phosphatase (ACP) activity has been constructed, based on the internal filtering effect between oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and rhodamine B (RB). Au3+, which in situ form gold nanoparticles (AuNPs), can oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to oxTMB (blue color). The fluorescence of RB can be quenched by oxTMB due to the spectral overlap of emission of RB and absorption of oxTMB. By means of the above process, ACP can be determined because ACP promotes the hydrolysis of 2-phospho-L-ascorbic acid trisodium salt (AAP) to generate ascorbic acid (AA), which can inhibit the internal filtering effect between RB and oxTMB. No material preparation was needed for the determination of ACP. The colorimetric and fluorimetric methods can quantify ACP in the range 0.06-5.0 mU/mL and 0.03-5.0 mU/mL, respectively. Furthermore, a smartphone-assisted sensing platform has been constructed for on-site monitoring of ACP in the range 0.75-50 mU/mL, and the detection limit is 0.3 mU/mL. The methods developed can measure ACP in human serum successfully.

Band-pass filter-assisted ratiometric fluorescent nanoprobe composed of N-(2-aminoethyl-1,8-naphthalimide)-functionalized gold nanoclusters for the determination of alkaline phosphatase using digital image analysis.

A smartphone-based dual-wavelength digital imaging platform containing red (539-695 nm) and blue (389-511 nm) band-pass filters was developed for point-of-care (POC) testing of alkaline phosphatase (ALP) activity. The platform was based on dual-emitting fluorescent nanohybrids (AuNC@NAN), the ratiometric probe, which had a fluorescence "on-off-on-off" response. The probe comprised red-emitting gold nanoclusters (AuNCs) acting as the signal report units and blue-emitting N-(2-aminoethyl-1,8-naphthalimide) (NAN) acting as an internal reference. The different responses of the ratiometric probes resulted in a continuous color-multiplexing change from pink-red to dark-purple upon exposure to ALP. The dual-wavelength digital imaging platform was employed to acquire images of AuNC or NAN fluorescence signals without the influence of background light. Unlike the classical one-time digital imaging mode, the accurate red (R) and blue (B) channel values of the generated images can help to directly judge or eliminate the disturbance from unavoidable interfering factors. The R/B values were successfully employed for determining the ALP activity at a range 2.0 to 35.0 mU·mL-1 with the detection limit of 1.04 mU·mL-1. Such sensing imaging platform is also successful in determining ALP activity in human serum with 94.9-105% recoveries and relative standard deviation in the range 4.2-5.6%. A novel dual-wavelength smartphone-based digital imaging platform was proposed for simultaneous readout of the reporting and internal reference signals from dual-emitting ratiometric fluorescence probes, which allowed us to the accurate, reliable, and highly sensitive assay of ALP activity in complex samples.

Intrinsic Dynamics of Protein-Peptide Unbinding.

The dynamics of peptide-protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive light-emitting diodes and laser diodes only. One mutant shows on-off behavior with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least 2 orders of magnitude, compared to that of other variants of the RNase S system. We conclude that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a time scale of a few hundred microseconds in a strongly stretched-exponential manner.

Sensitive recognition of ractopamine using GQDs-DPA as organic fluorescent probe.

A novel spectrofluorimetric sensing platform was designed for Ractopamine measurement in aqueous and plasma samples. d-penicillamine functionalized graphene quantum dots (DPA-GQDs) was utilized as a fluorescence probe, which was synthesized through the pyrolysis of citric acid in the presence of DPA. This one-pot down-top strategy causes to high-yield controllable synthesis method. The reaction time and probe concentration were optimized. Then, the fluorescence intensity of aqueous samples containing different Ractopamine concentrations and 500 ppm DPA-GQDs were measured at 25°C with an excitation wavelength of 274 nm. The sensing platform was also applied to detect Ractopamine in untreated plasma samples. The fluorescence spectroscopy technique responses indicated a linear relationship between the peak fluorescence intensity and ractopamine concentration in the range of 0.25-15 ppm with low limit of quantification of 0.25 ppm was for aqueous and plasma samples, respectively.

Simple fluorescence optosensing probe for spermine based on ciprofloxacin-Tb3+ complexation.

We developed a facile detection method of spermine based on the fluorescence (FL) quenching of the ciprofloxacin-Tb3+ complex, which shows astrong green emission. Ciprofloxacin (CP) makes efficient bondings to Tb3+ ion as a linker molecule through carboxylic and ketone groups to form a kind of lanthanide coordination polymer. The addition of spermine that competes with Tb3+ ions for the interaction with CP due to its positive charge brings about weakened coordination linkage of CP and Tb3+. The probe exhibited high sensitivity, selectivity, and good linearity in the range of 2-180 µM with a low limit of detection of 0.17 µM. Moreover, we applied this method on the paper strip test (PST), along with the integration of a smartphone and Arduino-based device. The practical reliability of the developed probe was evaluated on human serum samples with acceptable analytical results.

Fluorescence tuning behavior of carbon quantum dots with gold nanoparticles via novel intercalation effect of aldicarb.

A simple, sensitive and rapid fluorometric system has been developed for the detection of aldicarb (ALD) based on inner filter effect (IFE) of gold nanoparticles (AuNPs) on fluorescence (FL) intensity of carbon quantum dots (CQDs). Addition of CQDs into AuNPs, gets them aggregated due to electrostatic interaction resulting in quenching the FL intensity of CQDs. With addition of ALD into AuNPs, an intercalated layer was formed between them through Au-N and Au-S bond which reduced IFE of AuNPs. Hence, CQDs FL intensity recovered along with ALD concentration varying between 3.8 and 76 µg L-1 with lower detection limit of 3.02 µg L-1. The spiked real samples study in fruits, vegetables and soft drinks revealed that this sensing platform was repeatable and effective for real samples. The validation of proposed method indicates that the ALD sensor is promising and adaptable for everyday on spot environment and food safety monitoring.

A Heparin based dual ratiometric sensor for Thrombin.

Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T-Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.