Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Performance evaluation of CA242 by flow fluorescence assay.

ABSTRACT: Carbohydrate antigen 24-2 (CA24-2) is usually used as a biomarker for the diagnosis of pancreatic cancer and colorectal cancer. Currentlly, a new quantitative assay kit for CA242 by flow fluorometry assay (FFA) was developed by Shanghai Tellgen Cooperation Co. Ltd. China. Therefore, we conducted the performance evaluation for it.According to the "Guiding principles on performance analysis of diagnostic reagents in vitro" and "American association of clinical laboratory standardization guidelines EP15-A2", the accuracy, precision, linear range, reportable range, biological reference interval verification, carry-over contamination rate, anti-interference capability and cross reaction of the assay kit used in TESMI F3999-Luminex200 automatic immunoassay system were evaluated. In addition, the assay kit was performed in parallel to CanAg kit (CanAg Diagnostics Products Beijing Co., Ltd.) to analyze the correlation between the 2 kits.The bias of accuracy of the new assay kit was less than 12.5% and the coefficient of variations (CVs) of precision were all less than 10.0%. The linear range of CA242 concentration of the testing kit was between 3.46 U/ml and 434.76 U/ml and the reportable range was 6.00 to 535.13 U/ml. The CA242 reference interval 0.00 to 20.00 U/ml was suitable for use in laboratory. The carry-over contamination rate was -0.14%. Correlation analysis showed a satisfactory relevance and consistency (r = 0.982, P < .001) between the new assay kit and CanAg kit, with a regression equation Y = 1.0012X to 0.878 (R2 = 0.9647, P < .001). No statistically significant difference between serum samples without interferences and samples containing lipemia, bilirubin and hemoglobin. And no cross reaction existed between the assay kit and the other tumor markers, such as carbohydrate antigen 125 (CA125), alpha-fetoprotein (AFP), and cytokeratin-19 soluble fragment (CYFRA21-1).The new CA242 quantitative assay kit possesses good detection performance when it is used in TESMI F3999-Luminex200 automatic immunoassay system, which can be used for the examination of CA242 in clinical practice.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are highly specialized structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In this study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntington's disease and amyotrophic lateral sclerosis, whereas biphasic disruption occurred in a mouse model of Alzheimer's disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

Hemin@carbon dot hybrid nanozymes with peroxidase mimicking properties for dual (colorimetric and fluorometric) sensing of hydrogen peroxide, glucose and xanthine.

The multifunctional hemin@carbon dot hybrid nanozymes (hemin@CD) with simultaneous peroxidase-like activity and fluorescence signalling property was prepared for the first time. Based on these properties, hemin@CD was applied to develop a dual-channel fluorescent probe for H2O2 and H2O2-based biocatalytic systems. By virtue of the peroxidase-like activity, hemin@CD can catalyze the oxidative coupling of 4-aminoantipyrine with phenol in the presence of H2O2 to form a pink-red quinoneimine dye with a maximum absorbance at 505 nm. Under the excitation wavelength of 480 nm, the green fluorescence of hemin@CD peaks at 540 nm and is quenched by the generated quinoneimine dye due to an inner filter effect, and also by H2O2 because of dynamic quenching. Thus, a colorimetric and fluorimetric dual-channel optical probe for H2O2 is obtained. Due to the glucose/xanthine transformations under formation of H2O2 by the relevant oxidase catalysis, the probe can be applied for detection of glucose and xanthine. The colorimetric detection limits for H2O2, glucose and xanthine are 0.11, 0.15, 0.11 µM, and the and fluorimetric detection limits are 0.15, 0.15, 0.12 µM, respectively. Graphical abstractSchematic representation of the colorimetric and fluorimetric dual probe for H2O2, glucose and xanthine based on the multifunctional emin@carbon dot) hybrid nanozymes with simultaneous peroxidase-like activity and fluorescence signalling property.

Fluorimetric determination of histidine by exploiting its inhibitory effect on the oxidation of thiamine by cobalt-containing Prussian Blue nanocubes.

A fluorometric assay for histidine (His) is described. It is based on the inhibitory effect of His on nanocubes consisting of cobalt-containing Prussian Blue analog (CoFe NCbs), which have a strong oxidation effect on thiamine (THI) in the presence of NaOH. THI is nonfluorescent but the oxidized form (thiochrome; ThC) has a strong blue fluorescence, with excitation/emission maxima at 370/445 nm. His inhibits the oxidation effect of the CoFe NCbs due to the strong interaction between its imidazole side chain and the amino groups of the CoFe NCbs. This method is fast and has good sensitivity and selectivity. The lower detection limit is 14.3 nM of His, the linear range extends from 0.05 to 2.5 µM, and the relative standard deviation is calculated to be 1.5%. The method was successfully employed to quantify His in spiked serum samples. Graphical abstractSchematic representation of cobalt-containing Prussian Blue nanocubes (CoFe NCbs)-thiamine (THI)-based fluorometric assay for Histine (His). His inhibits the generation of thiochrome (ThC; the oxidized form of THI). The detection limit is 14.3 nM with the linear range of 0.05-2.5 µM.

Determination of iron(II) and iron(III) via static quenching of the fluorescence of tryptophan-protected copper nanoclusters.

"Tryptophan-coated blue fluorescent copper nanocluster (CuNC@Trp) was prepared by a strategy where Trp acts as both the reducing and capping agent. The fluorescence of the CuNC, with excitation/emission peaks at 340/405 nm, is selectively quenched by iron(II) and iron(III) ions. Studying the mechanism of this interaction revealed that Fe2+ and Fe3+ ions can make a ground state complex with the protecting ligand which can result in quenching of the cluster emission. Structural and optical properties of the modified CuNC were investigated by ESI-MS, DLS, TEM, UV-vis and photoluminescence. The effects of pH value and temperature, time of interaction, and cluster volume were optimized. Under optimized conditions, the probe response is linear in concentration range of 10-1000 µM for Fe(II) and Fe(III) with the relative standard deviations of 0.13 and 0.14% (n = 5) respectively. The respective limits of detection are 3.0 and 2.2 µM. The method was successfully used for determination of trace amount of both ions in spiked water, blood and iron supplement tablets. The results were in good agreement with those obtained by the ICP-AES method." Graphical abstractThe scheme represents the synthesis of CuNC@Trp at basic conditions and at elevated temperature. The emission of the cluster decreases due to static quenching of fluorescence by iron ions.

A terbium(III)-functionalized zinc(II)-organic framework for fluorometric determination of phosphate.

A terbium(III)-functionalized zinc(II)-organic framework (Tb-MOF-Zn) is shown to be a viable fluorescent probe for phosphate. The organic ligands 4,4',4″-[((2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene))tris(oxy)]tribenzoic acid (H3L3) contains multiple carboxyl groups that can react with zinc(II) to yield tubular MOF-Zn. The MOF-Zn was further functionalized with Tb(III) to produce a lanthanide composite of type Tb-MOF-Zn which displays strong fluorescence with excitation/emission maxima at 285/544 nm. Fluorescence is quenched by phosphate because of the specific interaction with Tb(III) in Tb-MOF-Zn. The concentration of Tb-MOF-Zn, reaction time and pH value of the solution were optimized. Fluorescence drops linearly in the 0.01 to 200.0 µM phosphate concentration range, and the detection limit is 4.0 nM. The fluorescent probe was also used to prepare a microdot array on a glass slide for visual detection of phosphate under illumination with UV light. Graphical abstractA terbium(III) functionalized zinc(II)-organic framework was synthesized and used as fluorescent probe for determination of phosphate ions.

Fluorometric determination of lead(II) by using aptamer-functionalized upconversion nanoparticles and magnetite-modified gold nanoparticles.

A fluorescent nanoprobe for Pb(II) has been developed by employing aptamer-functionalized upconversion nanoparticles (UCNPs) and magnetic Fe3O4-modified (MNPs) gold nanoparticles (GNPs). First, aptamer-functionalized UCNPs and aptamer-functionalized magnetic GNPs were synthesized to obtained the fluorescent nanoprobe. The particles were combined by adding a complementary ssDNA. In the absence of Pb(II), the UCNPs, MNPs and GNPs are linked via complementary base pairing. This led to a decrease in the green upconversion fluorescence peaking at 547 nm (under 980 nm excitation). In the presence of Pb(II), the dsDNA between UCNPs and MNPs-GNPs is cleaved, and fluorescence recovers. This effect allows Pb(II) to be quantified, with a wide working range of 25-1400 nM and a lower detection limit of 5.7 nM. The nanoprobe gave satisfactory results when analyzing Pb(II) in tea and waste water. Graphical abstractSchematic representation of fluorescent nanoprobe based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs)-Fe3O4 magnetic nanoparticles (MNPs) for detection of Pb2+.

Green conventional and first-order derivative fluorimetry methods for determination of trimebutine and its degradation product (eudesmic acid). Emphasis on the solvent and pH effects on their emission spectral properties.

In this report, the fluorescence properties of the antimuscarinic drug trimebutine maleate (TRB) were fully studied and characterized. TRB exhibited intrinsic fluorescence that is greatly dependent on the local environmental factors including the solvent nature and the pH. Yet, its fluorescence was not significantly influenced by the existence of some surface active agents and polymer. The outcomes of this investigation verified that TRB fluorescence emission is intense in ethanol: 1.0 M aqueous acetic acid (9:1, v/v) with emission maxima at 357 nm and excitation maxima at 270 nm. Whereas, going towards higher pH causes fluorescence quenching. These conditions permitted ultrasensitive fluorimetric determination of TRB over the concentration range of 2.00-1500.0 ng/mL with a lower detection limit of 0.40ng/mL Application for the determination of TRB in tablets, ampoule and suspension was successfully achieved with %recoveries ranged between 98.21-100.17%. Furthermore, a first order derivative fluorimetric method was validated for resolving and simultaneous determination of TRB and its degradation product and impurity, eudesmic acid (EUA) making use of the pH-mediated fluorescence spectral shift of EUA. An ethanolic solution containing acetate buffer (pH 5.3) was used for this goal with excitation at 255 nm and measurement of the first order derivative peak amplitudes at respective zero-crossing points of 375 and 351 nm over the corresponding concentration ranges of 20.00-500.00 and 10.00-300.00 ng/mL for TRB and EUA, respectively. The two methods were assessed regarding greenness and eco-friendship by the National Environmental Methods Index and analytical eco-scale score approaches which confirmed their excellent greenness and safety.

Sensitive fluorometric determination of glutathione using fluorescent polymer dots and the dopamine-melanin nanosystem.

A bioinspired fluorometric method has been developed for the detection of glutathione (GSH) in biological fluids. It is based on the use of near-infrared fluorescent semiconducting polymer dots (P-dots) and of the dopamine (DA)-melanin nanosystem. The P-dots were prepared from poly(styrene-co-maleic anhydride), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] and the fluorescent dye tetraphenylporphyrin. They have excitation/emission maxima at 458/656 nm, and this enables measurement to be performed with low autofluorescence and scattering background. DA can self-polymerize on the surface of the P-dots to yield a poly-DA coating. This coating, at weak alkaline pH values, causes the quenching of the fluorescence of the P-dots. However, the polymerization of DA is inhibited by GSH. Hence, quenching of fluorescence is prevented. This effect was used to design a fluorometric assay for GSH that has good selectivity and sensitivity. Under optimal conditions, the method has a linear response in the 0.2 to 20 µM GSH concentration range and a 60 nM detection limit. It was successfully applied to the determination of GSH in HepG2 cells and in spiked human serum. Graphical abstract Schematic representation of using a NIR fluorescent P-dots and dopamine (DA)-melanin nanohybrid as a probe for glutathione (GSH) detection. The P-dots were prepared from poly(styrene-co-maleic anhydride) (PSMA), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] (PEPV) and the fluorescent dye tetraphenylporphyrin (TPP).The GSH can inhibit the dopamine self-polymerization and prevented the formation of PDA and fluorescence quenching of P-dots.

A "turnon" aptasensor for simultaneous and time-resolved fluorometric determination of zearalenone, trichothecenes A and aflatoxin B1 using WS2 as a quencher.

A "turn on" time-resolved fluorometric aptasensor is described for the simultaneous detection of zearalenone (ZEN), trichothecenes A (T-2), and aflatoxin B1 (AFB1). Multicolor-emissive nanoparticles doped with lanthanide ions (Dy3+, Tb3+, Eu3+) were functionalized with respective aptamers and applied as a bioprobe, and tungsten disulfide (WS2) nanosheets are used as a quencher of time-resolved fluorescence. The assay exploits the quenching efficiency of WS2 and the interactions between WS2 and the respective DNA aptamers. The simultaneous recognition of the three mycotoxins can be performed in a single solution. In the absence of targets, WS2 is easily adsorbed by the mixed bioprobes via van der Waals forces between nucleobases and the WS2 basal plane. This brings the bioprobe and WS2 into close proximity and results in quenched fluorescence. In the presence of targets, the fluorescence of the bioprobes is restored because the analytes react with DNA probe and modify their molecular conformation to weaken the interaction between the DNAs and WS2. Under the optimum conditions and at an excitation wavelength of 273 nm, the time-resolved fluorescence intensities (peaking at 488, 544 and 618 nm and corresponding to emissions of Dy3+, Tb3+ and Eu3+) were used to quantify ZEN, T-2 and AFB1, respectively, with detection limits of 0.51, 0.33 and 0.40 pg mL-1 and a linear range from 0.001 to 100 ng mL-1. The three mycotoxins can be detected simultaneously without mutual interference. The assay was applied to the quantification of ZEN, T-2 and AFB1 in (spiked) maize samples. This homogeneous aptamer based assay can be performed within 1 h. Conceivably, it can become an alternative to other heterogeneous methods such as the respective enzyme-linked immunosorbent assays. Graphical abstract Schematic presentation of an aptasensor for simultaneous detection of zearalenone, trichothecenes A and aflatoxin B1 using aptamer modified time-resolved fluorescence nanoparticles as signalling probes and tungsten disulfide as the quencher. This assay shows lower detection limit and requires no washing steps.

Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

Electrostatically controlled fluorometric assay for differently charged biotargets based on the use of silver/copper bimetallic nanoclusters modified with polyethyleneimine and graphene oxide.

An electrostatically controlled fluorometric assay is described that is based on the use of silver/copper bimetallic nanoclusters. The nanoclusters were coated with polyethyleneimine (PEI-Ag/CuNCs). At pH 7.4, these particles are positively charged. Their blue fluorescence (with excitation/emission peaks at 341/464 nm) depends on local pH values and temperature. If graphene oxide (which is negatively charged at pH 7.4) is introduced, the fluorescence of the PEI-Ag/CuNCs is quenched. Based on various electrostatic interactions, three kinds of biomacromolecules were detected by fluorometry. These include (negatively charged) heparin, (positively charged) protamine, and (virtually uncharged) trypsin. Heparin is detected by using GO/PEI-Ag/CuNCs, protamine by using GO/heparin/PEI-Ag/CuNCs, and trypsin by using GO/protamine/heparin/PEI-Ag/CuNC. The detection limits and linear ranges are 4.8 nM and 10-450 nM for heparin, 0.09 µg·mL-1 and 0.25-5 µg·mL-1 for protamine, and 0.03 µg·mL-1 and 0.05-1 µg·mL-1 for trypsin. Zeta potentials of the various substances in the system were determined to elucidate the detection mechanism. Comceivably, the method provides a widely applicable approach for electrostatically controlled biomolecular assays. Graphical abstract Schematic presentation of electrostatically controlled fluorometric assay for the detection of heparin, protamine, and trypsin based on the silver/copper bimetallic nanoclusters modified with polyethyleneimine and graphene oxide.

Boron doped carbon dots as a multifunctional fluorescent probe for sorbate and vitamin B12.

Boron doped carbon dots (B-CD) were synthesized by a one-step hydrothermal method using phenylboronic acid as the starting material. They have an average size of about 3.3 nm, with excitation/emission wavelength of 247/323 nm and a quantum yield of 12%. The B-CD is shown to be viable fluorescent probe for sorbate (PS) and vitamin B12 (VB12). The fluorescence (FL) of the B-CD is quenched in the presence of PS or VB12 mainly coming from inner filter effect (IFE), but Förster resonance energy transfer (FRET) from the B-CD (as a donor) to PS/VB12 (as an acceptor) cannot be excluded. The probe enables PS to be detected by fluorometry with a linear response in the 0.20-24 µM concentration range and a 6.1 nM detection limit (at 3σ/slope). For VB12, the data are 0.20-30 µM and 8.0 nM. Graphical abstract Boron doped carbon dots (B-CD) as a probe was prepared by phenylboronic acid as single starting material via one-step hydrothermal method, which has remarkable selectivity and high sensitivity for monitoring PS/VB12. The fluorescence quenching of B-CD by PS/VB12 mainly comes from inner filter effect.

A turn-on fluorescent probe for vitamin C based on the use of a silicon/CoOOH nanoparticle system.

The authors describe a fluorometric method for the turn-on determination of vitamin C (ascorbic acid). The blue fluorescence of silicon nanoparticles (SiNPs; with excitation/emission maxima at 350/450 nm) is found to be quenched by CoOOH nanoparticles (NPs). In the presence of vitamin C, the CoOOH NPs are decomposed by a redox reaction between the diol group of vitamin C and CoOOH NPs. As a result, fluorescence recovers. On the basis of this finding, a fluorometric method was designed for the turn-on detection of vitamin C. Under optimal conditions, the method has a low detection limit (0.47 µM) and a linear response in the 0.5 µM to 20 µM a concentration range. It was successfully applied to the determination of vitamin C in spiked red grape and orange juice, and in vitamin C tablets. Graphical abstract A target-triggered dissociation of quencher-based strategy for the fluorescence "turn-on" detection of vitamin C was developed. It is based on surface energy transfer (SET) and an inner filter effect (IFE) between silicon nanoparticles and CoOOH nanoparticles as well as the redox reaction between vitamin C and CoOOH nanoparticles.

Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters.

A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a -TTA- loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/µL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost. Graphical Abstract A method was developed using chimeric DNA-templated silver nanoclusters to detect telomerase activity directly in cell extracts. The sensitivity of this new method outperforms the traditional TRAP assay, and without the need for amplification.

Fluorometric determination of glucose based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

A method is described for fluorometric detection of glucose. It is based on the finding that silicon nanodots (SNDs) are formed from glucose and aminopropyltriethoxysilane (APTES) under mild experimental conditions. The SNDs thus formed have an average diameter of ∼2 nm, exhibit good water dispersibility, blue fluorescence (with excitation/emission maxima at 410/475 nm), broad pH tolerance, and are photostable. The assay was applied to the quantification of glucose with high sensitivity, good specificity, and over a wide detection range (from 10 µM to 0.9 mM). It was applied to the determination of glucose in spiked serum samples and gave satisfactory results and recoveries. Graphical abstract Schematic presentation of serum glucose detection based on a redox reaction between glucose and aminopropyltriethoxysilane and in-situ formation of blue-green emitting silicon nanodots.

Gold-decorated magnetic nanoparticles modified with hairpin-shaped DNA for fluorometric discrimination of single-base mismatch DNA.

The authors describe the use of gold-decorated magnetic nanoparticles (Au/MNPs) in discriminating DNA sequences with a single-base (guanine) mismatch. The Au/MNPs were characterized through dynamic light scattering, X-ray diffraction, superconducting quantum interference device, and UV/visible spectroscopy. They were then conjugated to a probe oligomer consisting of a hairpin-shaped DNA sequence carrying two signalling fluorophores: fluorescein at its 3' end and pyrene in the loop region. When interacting with the target DNA sequences, the hybridized probe-target duplex renders the pyrene signal (at excitation/emission wavelengths of 345/375 nm) either quenched or unquenched. Quenching (or nonquenching) of the pyrene fluorescence depends on the presence of a guanine (or a nonguanine) nucleotide at the designated polymorphic site. The linear range of hybridization in these Au/MNPs is from 0.1 nM to 1.0 µM of ssDNA. Conceivably, this system may serve as a single-nucleotide polymorphism probe. Graphical Abstract Schematic presentation of probe-conjugated Au/MNP preparation (upper panel) and working principle to discriminate DNA with or without single-base (guanine) mismatch sequences at the polymorphic sites (lower panel). Py denotes pyrene-hooked pyrrolocytidine; F denotes fluorescein.