Simple and Cost-effective "Turn-on" Fluorescence Sensor for the Determination of Xanthine.

A simple and selective 'turn-on' fluorescence sensor have been developed for the determination of xanthine (XA) based on glutathione (GSH) capped copper nanoclusters (CuNCs) as the fluorescent probe. The proposed sensor possess several advantages such as sensitivity, short analysis time and requires no sample pretreatment. The conditions for the performances of the sensor have been optimized and good linear relationship was obtained between concentration and relative fluorescence intensity in the concentration range 9.0[Formula: see text]10-3 M to 8.0[Formula: see text]10-5 M with a detection limit 6.0[Formula: see text]10-6 M. The mechanism behind the fluorescence enhancement may be ascribed to the binding of XA on the surface of GSH CuNCs. The sensor have been successfully applied to determine XA in spiked physiological samples.

A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells.

Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (<1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.

Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.

What are the Main Sensor Methods for Quantifying Pesticides in Agricultural Activities? A Review.

In recent years, there has been an increase in pesticide use to improve crop production due to the growth of agricultural activities. Consequently, various pesticides have been present in the environment for an extended period of time. This review presents a general description of recent advances in the development of methods for the quantification of pesticides used in agricultural activities. Current advances focus on improving sensitivity and selectivity through the use of nanomaterials in both sensor assemblies and new biosensors. In this study, we summarize the electrochemical, optical, nano-colorimetric, piezoelectric, chemo-luminescent and fluorescent techniques related to the determination of agricultural pesticides. A brief description of each method and its applications, detection limit, purpose-which is to efficiently determine pesticides-cost and precision are considered. The main crops that are assessed in this study are bananas, although other fruits and vegetables contaminated with pesticides are also mentioned. While many studies have assessed biosensors for the determination of pesticides, the research in this area needs to be expanded to allow for a balance between agricultural activities and environmental protection.

Developed a novel sensor based on fluorescent graft conjugated polymer for the determination of aristolochic acid in traditional Chinese medicine.

A novel fluorescent graft conjugated polymer (poly (2, 5-bis (Polyethylene glycol oxybutyrate)-1, 4-phenylethynylene-alt-1, 4-phenyleneethynylene, PPE-OB-PEG) has been designed and synthesized for the determination of aristolochic acid (AA). The detection conditions and detection characters of PPE-OB-PEG were systematically explored in this work. The fluorescence intensity of PPE-OB-PEG changes with the different concentration of AA. PPE-OB-PEG has a good linear range towards AA from 1.00 × 10-7 to 8.00 × 10-5 mol L-1 and the limit of detection (LOD) is 3.00 × 10-8 mol L-1 (S/N = 3). PPE-OB-PEG have been applied to detect AA in traditional Chinese medicine samples and the results are satisfactory. The experimental results show that PPE-OB-PEG can be used as a fluorescence probe for rapid and sensitive detection of AA.

Fluorescence-Based High-Throughput Salt Screening.

The present study reports a high-throughput screening method for the salt formation of amine-containing active pharmaceutical ingredients (APIs) based on fluorescence measurements. A free form amine API was alkynylated by a solid-vapor reaction using propargyl bromide, and a fluorescent compound was produced by a subsequent reaction using 9-azidomethylanthracene. In contrast, salts were inert to propargyl bromide; thus, no fluorescence was observed. Samples for salt screening were prepared by grinding haloperidol with various counter acids, and these mixtures were derivatized in a 96-well microplate to determine whether the salt formation had occurred between haloperidol and the counter acids. Samples that turned into fluorescent and nonfluorescent were confirmed to be free form and salt form, respectively, using powder X-ray diffraction and Raman spectroscopy. In conclusion, our method adequately functions as an indicator of the salt formation of amine APIs. Further, this method allows for the rapid evaluation of the salt formation of APIs using 96-well microplates without the need for special reagents or techniques; thus, it is valuable for the discovery of an optimal salt form of newly developed amine APIs in the pharmaceutical industry.

Stability-indicating spectrofluorimetric method with enhanced sensitivity for determination of vancomycin hydrochloride in pharmaceuticals and spiked human plasma: Application to degradation kinetics.

Based on investigating the relative fluorescence intensity of vancomycin hydrochloride (VCM) in methanol, a simple, highly sensitive, time-saving and specific spectrofluorimetric method was developed and validated. VCM fluorescence was measured at 335 nm when excited at 268 nm. Excellent linearity is obeyed in the concentration range 1-100 ng/mL with a detection limit of 5.94 pg/mL, a quantitation limit of 18.03 pg/mL and a very good correlation coefficient (r = 0.9999). Our method was applied to analyze VCM in pharmaceuticals as well as spiked human plasma. Moreover, VCM stability was studied when exposed to various degradation conditions such as oxidative, alkaline as well as acidic stress. Acidic and alkaline degradation kinetics of VCM was studied for the first time. The degradation follows pseudo-first-order kinetics. The apparent rate constants and half-life times were calculated. The Arrhenius equation was assessed and the activation energies of the degradation were also calculated. The developed method can be easily applied in quality control laboratories due to its sensitivity, specificity, simplicity and low cost.

A near-infrared fluorescent sensor with large Stokes shift for rapid and highly selective detection of thiophenols in water samples and living cells.

The development of simple methods with high sensitivity and selectivity to differentiate toxic aromatic thiols (thiophenols) from aliphatic thiols (cysteine, homocysteine, and glutathione) and hydrogen sulfide (H2S) is of great significance. Herein, we report on the fabrication of a novel near-infrared (NIR) fluorescent sensor for rapid and highly selective detection of thiophenols through the photoinduced electron transfer (PET) mechanism. In the presence of the thiophenols, an obvious enhancement of NIR fluorescence at 658 nm could be visualized with the aid of nucleophilic aromatic substitution (SNAr) reaction. The sensor displays large Stokes shift (~ 227 nm), fast response time (< 30 s), high sensitivity (~ 8.3 nM), and good biocompatibility. Moreover, the as-prepared sensor possesses an excellent anti-interference feature even when other possible interferents exist (aliphatic thiols and H2S) and has been successfully utilized for thiophenol detection in both water samples and living cells. Graphical abstract Illustration of the sensor for thiophenol imaging in living cells.

Biosensors for rapid and sensitive detection of Staphylococcus aureus in food.

Foodborne illness outbreaks caused by the consumption of food contaminated with harmful bacteria has drastically increased in the past decades. Therefore, detection of harmful bacteria in the food has become an important factor for the recognition and prevention of problems associated with food safety and public health. Staphylococcus aureus is one of the most commonly isolated foodborne pathogen and it is considered as a major cause of foodborne illnesses worldwide. A number of different methods have been developed for the detection and identification of S. aureus in food samples. However, some of these methods are laborious and time-consuming and are not suitable for on-site applications. Therefore, it is highly important to develop rapid and more approachable detection methods. In the last decade, biosensors have gained popularity as an attractive alternative method and now considered as one of most rapid and on-site applicable methods. An overview of the biosensor based methods used for the detection of S. aureus is presented herein. This review focuses on the state-of-the-art biosensor methods towards the detection and quantification of S. aureus, and discusses the most commonly used biosensor methods based on the transducing mode, such as electrochemical, optical, and mass-based biosensors.

Switch-on fluorescent strategy based on crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione in water and urine.

The concentration of L-cysteine (Cys) and glutathione (GSH) is closely related to the critical risk of various diseases. In our study, a new rapid method for the determination of Cys and GSH in water and urine samples has been developed using a fluorescent probe technique, which was based on crystal violet (CV)-functionalized CdTe quantum dots (QDs). The original QDs emitted fluorescence light, which was turned off upon adding CV. This conjugation of CV and QDs could be attributed to electrostatic interaction between COO- of mercaptopropionic acid (MPA) on the surface of QDs and N+ of CV in aqueous solution. In addition, Förster resonance energy transfer (FRET) also occurred between CdTe QDs and CV. After adding Cys or GSH to the solution, Cys or GSH exhibited a stronger binding preference toward Cd2+ than Cd2+-MPA, which disturbed the interaction between MPA and QDs. Thus, most MPA was able to be separated from the surface of QDs because of the participation of Cys or GSH. Then, the fluorescence intensity of the CdTe QDs was enhanced. Good linear relationships were obtained in the range of 0.02-40 µg mL-1 and 0.02-50 µg mL-1, and the detection limits were calculated as 10.5 ng mL-1 and 8.2 ng mL-1, for Cys and GSH, respectively. In addition, the concentrations of biological thiols in water and urine samples were determined by the standard addition method using Cys as the standard; the quantitative recoveries were in the range of 97.3-105.8%, and relative standard deviations (RSDs) ranged from 2.5 to 3.7%. The method had several unique properties, such as simplicity, lower cost, high sensitivity, and environmental acceptability. Graphical abstract Crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione with switch-on fluorescent strategy.

Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells.

Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40µM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements.

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a "gold-standard" instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.

Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay.

The detection and profiling of microRNAs are of great interest in disease diagnosis and prognosis. In this paper, we present a method for the rapid amplification-free detection of microRNAs from total RNA samples. In a two-step sandwich assay approach, fluorescently labeled reporter probes were first hybridized with their corresponding target microRNAs. The reaction mix was then added to a microarray to enable their specific capture and detection. Reporter probes were Tm equalized, enabling specificity by adjusting the length of the capture probe while maintaining the stabilizing effect brought about by coaxial base stacking. The optimized assay can specifically detect microRNAs in spiked samples at concentrations as low as 1 pM and from as little as 100 ng of total RNA in 2 h. The detection signal was linear between 1 and 100 pM (R2 = 0.99). Our assay data correlated well with results generated by qPCR when we profiled a select number of breast cancer related microRNAs in a total RNA sample.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

2-Aminopurine-modified DNA homopolymers for robust and sensitive detection of mercury and silver.

Heavy metal detection is a key topic in analytical chemistry. DNA-based metal recognition has advanced significantly producing many specific metal ligands, such as thymine for Hg2+ and cytosine for Ag+. For practical applications, however, robust sensors that can work in a diverse range of salt concentrations need to be developed, while most current sensing strategies cannot meet this requirement. In this work, 2-aminopurine (2AP) is used as a fluorescence label embedded in the middle of four 10-mer DNA homopolymers. 2AP can be quenched up to 98% in these DNA without an external quencher. The interaction between 2AP and all common metal ions is studied systematically for both free 2AP base and 2AP embedded DNA homopolymers. With such low background, Hg2+ induces up to 14-fold signal enhancement for the poly-T DNA, and Ag+ enhances up to 10-fold for the poly-C DNA. A detection limit of 3nM is achieved for both metals. With these four probes, silver and mercury can be readily discriminated from the rest. A comparison with other signaling methods was made using fluorescence resonance energy transfer, graphene oxide, and SYBR Green I staining, respectively, confirming the robustness of the 2AP label. Detection of Hg2+ in Lake Huron water was also achieved with a similar sensitivity. This work has provided a comprehensive fundamental understanding of using 2AP as a label for metal detection, and has achieved the highest fluorescence enhancement for non-protein targets.

A simple, fast and low-cost turn-on fluorescence method for dopamine detection using in situ reaction.

A simple, fast and low-cost method for dopamine (DA) detection based on turn-on fluorescence using resorcinol is developed. The rapid reaction between resorcinol and DA allows the detection to be performed within 5 min, and the reaction product (azamonardine) with high quantum yield generates strong fluorescence signal for sensitive optical detection. The detection exhibits a high sensitivity to DA with a wide linear range of 10 nM-20 µM and the limit of detection is estimated to be 1.8 nM (S/N = 3). This approach has been successfully applied to determine DA concentrations in human urine samples with satisfactory quantitative recovery of 97.84%-103.50%, which shows great potential in clinical diagnosis.

Ultra-portable, wireless smartphone spectrometer for rapid, non-destructive testing of fruit ripeness.

We demonstrate a smartphone based spectrometer design that is standalone and supported on a wireless platform. The device is inherently low-cost and the power consumption is minimal making it portable to carry out a range of studies in the field. All essential components of the device like the light source, spectrometer, filters, microcontroller and wireless circuits have been assembled in a housing of dimensions 88 mm × 37 mm × 22 mm and the entire device weighs 48 g. The resolution of the spectrometer is 15 nm, delivering accurate and repeatable measurements. The device has a dedicated app interface on the smartphone to communicate, receive, plot and analyze spectral data. The performance of the smartphone spectrometer is comparable to existing bench-top spectrometers in terms of stability and wavelength resolution. Validations of the device were carried out by demonstrating non-destructive ripeness testing in fruit samples. Ultra-Violet (UV) fluorescence from Chlorophyll present in the skin was measured across various apple varieties during the ripening process and correlated with destructive firmness tests. A satisfactory agreement was observed between ripeness and fluorescence signals. This demonstration is a step towards possible consumer, bio-sensing and diagnostic applications that can be carried out in a rapid manner.

Thiazole orange as a fluorescent probe: Label-free and selective detection of silver ions based on the structural change of i-motif DNA at neutral pH.

Silver ions have been widely applied to many fields and have harmful effects on environments and human health. Herein, a label-free optical sensor for Ag(+) detection is constructed based on thiazole orange (TO) as a fluorescent probe for the recognition of i-motif DNA structure change at neutral pH. Ag(+) can fold a C-rich single stranded DNA sequence into i-motif DNA structure at neutral pH and that folding is reversible by chelation with cysteine (Cys). The DNA folding process can be indicated by the fluorescence change of TO, which is non-fluorescent in free molecule state and emits strong fluorescence after the incorporation with i-motif DNA. Thus, a rapid, sensitive, and selective method for the detection of Ag(+) and Cys is developed with a detection limit of 17 and 280nM, respectively. It is worth noting that the mechanism underlying the increase of the fluorescence of thiazole orange in the presence of i-motif structure is explained. Moreover, a fluorescent DNA logic gate is successfully designed based on the Ag(+)/Cys-mediated reversible fluorescence changes. The proposed detection strategy is label-free and economical. In addition, this system shows a great promise for i-motif/TO complex to analyze Ag(+) in the real samples.

Molecular beacon-based enzyme-free strategy for amplified DNA detection.

We report an enzyme-free, sensitive strategy for DNA detections through fluorescence amplification. The sensing method employs molecular beacons (MBs) and two single-stranded helper DNA probes. In the presence of a DNA target, it binds and opens an MB. This triggers the hybridizations between the MB and helper probes, and consequently releases the DNA target, which becomes available to react with another MB and enhances the fluorescence emission of the MBs. The detection limit of the proposed strategy is 0.58 pM, which is about 3 orders of magnitude better than the conventional MB-based method. This method is also fast and exhibits good selectivity. It is superior to previous MB-based amplification approaches employing enzymes or nanomaterials.

A portable fluorescence spectroscopy imaging system for automated root phenotyping in soil cores in the field.

Root architecture traits are a target for pre-breeders. Incorporation of root architecture traits into new cultivars requires phenotyping. It is attractive to rapidly and directly phenotype root architecture in the field, avoiding laboratory studies that may not translate to the field. A combination of soil coring with a hydraulic push press and manual core-break counting can directly phenotype root architecture traits of depth and distribution in the field through to grain development, but large teams of people are required and labour costs are high with this method. We developed a portable fluorescence imaging system (BlueBox) to automate root counting in soil cores with image analysis software directly in the field. The lighting system was optimized to produce high-contrast images of roots emerging from soil cores. The correlation of the measurements with the root length density of the soil cores exceeded the correlation achieved by human operator measurements (R (2)=0.68 versus 0.57, respectively). A BlueBox-equipped team processed 4.3 cores/hour/person, compared with 3.7 cores/hour/person for the manual method. The portable, automated in-field root architecture phenotyping system was 16% more labour efficient, 19% more accurate, and 12% cheaper than manual conventional coring, and presents an opportunity to directly phenotype root architecture in the field as part of pre-breeding programs. The platform has wide possibilities to capture more information about root health and other root traits in the field.