A novel fluorescent assay for oxytetracycline hydrochloride based on fluorescence quenching of water‐soluble CdTe nanocrystals

A novel assay for oxytetracycline hydrochloride (OTC) based on fluorescence quenching was developed from the interaction between functionalized cadmium telluride quantum dots (CdTe QDs) and OTC. Optimum conditions for the detection of OTC were found after investigating all factors. Under optimum conditions, luminescence of CdTe nanocrystals (λ_ex = 365 nm, λ_em = 562 nm) was quenched by OTC in a concentration‐dependent manner best described by a modified Stern‐Volmer type equation. Good linearity was obtained with a regression coefficient of 0.9999 in the range of 1.34 ~ 13.4 x 10^‐5 mol/L and a limit of detection of 3.08 x 10^‐7 mol/L. In addition, the quenching mechanism was also established. The results imply that the close proximity of OTC‐CdTe was driven by electrostatic attraction and the resulting effective electron transfer from OTC to QDs could be responsible for fluorescence quenching of CdTe‐QDs. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

Label‐free,zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

We experimentally demonstrate a label‐free biosensor for the ERBB2 cancer gene DNA target based on the distance‐dependent detection of surface‐enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non‐porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays.

With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label‐free biosensor that makes use of distance‐dependent detection of surface‐enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.     

Novel device for quick measurement of glucose in POCT areas without pre‐analytical steps based on a multi‐way glucose biosensor

Professional Point of Care testing demands rapid analysis and professional quality. To assure rapid analysis of high quality the analytical tool ideally should be able to work without sample pre‐treatment and should offer the opportunity to calibrate and/or control the analytical performance of the tool. In contrast to an enormous number of different disposable‐strips used for patient self monitoring today and based on an extended knowledge with respect to multi‐way biosensors used in laboratory analyzers we decided to develop a professional Point of Care Testing system for glucose analysis based on a multi‐way biosensor. The multi‐way glucose biosensor placed in the instrument for 30 days did reduce the lag time between blood withdrawal and availability of a result of lab quality in a bedside area to about 10 seconds. No pre‐analytical steps are necessary for measuring capillary whole blood, no crossing over was observed, and the data could be transferred into a laboratory information system or a hospital information system. Thus, we were able to realize tools for professional health control able to measure glucose values in laboratory quality at places outside laboratories: e.g., in doctor's offices, hospital wards, critical care units, and training units of athletes. By combining the advantages of laboratory analyzers (high quality and low sample price) and the advantages of disposable strips (simple procedure and immediate results after sample withdrawal) with the Glukometer 3000 and LactatProfi 3000 we did start to fill the gap between the two basic technologies available on the market for diagnosis today. Glukometer 3000 and LactatProfi 3000 are worldwide the first and only mobile glucose and lactate measuring instruments for decentralized locations based on multi‐way biosensors.     

Application of a fluorescent biosensor based‐on magneto‐γ‐Fe2O3–methyldopa nanoparticles for adsorption of human serum albumin

Understanding and controlling the interaction between the polymer methyldopa (2‐amino‐3‐(3,4‐dihydroxyphenyl)‐2‐methyl‐propanoic acid) (PMDP)–γ‐Fe₂O₃ nanoparticles and biological fluids is important if the potential of nanoparticles (NPs) in biomedicine is to be realized. Physicochemical studies on the interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of the NP surface, interactions between human serum albumin (HSA) and PMDP–γ‐Fe₂O₃ NPs were investigated. Here, the adsorption of HSA onto small (10–30 nm diameter) PMDP–γ‐Fe₂O₃ NPs was quantitatively analyzed using spectroscopic methods. The fluorescence quenching data were checked for the inner‐filter effect, the main confounding factor in the observed quenching. The binding constants, K_a, were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ?H, ?S and ?G were given. The obtained thermodynamic signature suggests that hydrophobic interactions at least are present. This result indicates that the structure of the protein turns from a structureless denatured state at pH 3 into an ordered biologically active native state on addition of PMDP–γ‐Fe₂O₃ NPs. Copyright © 2015 John Wiley & Sons, Ltd.     

NPQ(T): a chlorophyll fluorescence parameter for rapid estimation and imaging of non‐photochemical quenching of excitons in photosystem‐II‐associated antenna complexes

In photosynthesis, light energy is absorbed by light‐harvesting complexes and used to drive photochemistry. However, a fraction of absorbed light is lost to non‐photochemical quenching (NPQ) that reflects several important photosynthetic processes to dissipate excess energy. Currently, estimates of NPQ and its individual components (q_E, q_I, q_Z and q_T) are measured from pulse‐amplitude‐modulation (PAM) measurements of chlorophyll fluorescence yield and require measurements of the maximal yield of fluorescence in fully dark‐adapted material (F_m), when NPQ is assumed to be negligible. Unfortunately, this approach requires extensive dark acclimation, often precluding widespread or high‐throughput use, particularly under field conditions or in imaging applications, while introducing artefacts when F_m is measured in the presence of residual photodamaged centres. To address these limitations, we derived and characterized a new set of parameters, NPQ_(T), and its components that can be (1) measured in a few seconds, allowing for high‐throughput and field applications; (2) does not require full relaxation of quenching processes and thus can be applied to photoinhibited materials; (3) can distinguish between NPQ and chloroplast movements; and (4) can be used to image NPQ in plants with large leaf movements. We discuss the applications benefits and caveats of both approaches.     

Investigations on the interactions between naphthalimide‐based anti‐tumor drugs and human serum albumin by spectroscopic and molecular modeling methods

The interactions between the three kinds of naphthalimide‐based anti‐tumor drugs (NADA, NADB, NADC) and human serum albumin (HSA) under simulated physiological conditions were investigated by fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results of the fluorescence quenching spectroscopy showed that the quenching mechanisms for different drugs were static and their affinity was in a descending order of NADA > NADB > NADC. The relative thermodynamic parameters indicated that hydrophobic force was the predominant intermolecular force in the binding of NAD to HSA, while van der Waals interactions and hydrogen bonds could not be ignored. The results of site marker competitive experiment confirmed that the binding site of HSA primarily took place in site I. Furthermore, the molecular modeling study was consistent with these results. The study of circular dichroism spectra demonstrated that the presence of NADs decreased the α‐helical content of HSA and induced the change of the secondary structure of HSA. Copyright © 2015 John Wiley & Sons, Ltd.     

A sensitive and regenerable biosensor for organophosphate pesticide based on self‐assembled multilayer film with CdTe as fluorescence probe

A fluorescence biosensor for organophosphorus pesticides was developed. A pH indicator, CdTe quantum dots, were used as an optical transducer of the inhibition of enzyme by analyte. Through the intervening agency of chitosan, the recognition elements (acetylcholinesterase and CdTe) were immobilized onto the surface of quartz by electrostatic attraction to form a self‐assembled multilayer film. In the absence of pesticide, acetylcholine was biocatalytically hydrolysed to yield acetic acid and choline. The released acid resulted in pH decrease, which was sensed by the immobilized pH indicator (CdTe). In the presence of pesticide, the action of acetylcholine was reduced; the fluorescence intensity of the film changed and was related to the concentration of pesticide. This multilayer film could be used as the biosensor for monocrotophos, with a detection limit of 3.20 × 10^?8 mol/L; the sensitivity was 100 times higher than that of CdTe in aqueous solution. The sensor was easily regenerated, and had good stability and selectivity for organophosphorus pesticides. Copyright © 2011 John Wiley & Sons, Ltd.     

Spectrofluorimetric study on fluorescence quenching of tyrosine and l‐tryptophan by the aniracetam cognition enhancer drug: quenching mechanism using Stern–Volmer and double‐log plots

A novel approach on fluorescence quenching of tyrosine and l ‐tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern–Volmer plots and ultraviolet spectra figures of quencher–fluorophore mixtures. Binding constant and stoichiometry were calculated using double‐log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH‐rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5–8. Fluorescence quenching of tyrosine and l ‐tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5–6.5. Aniracetam quenched the fluorescence of tyrosine and l ‐tryptophan in the concentration range 1–20 μg/ml and 0.3–20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine–aniracetam and 0.14 μg/ml for l ‐tryptophan–aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco‐friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.     

Reproducible fashion of the HSP70B' promoter‐induced cytotoxic response on a live cell‐based biosensor by cell cycle synchronization

Live cell‐based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high‐throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell‐based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum‐starved conditions and we then investigated the cadmium chloride‐induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell‐based biosensors with microdevices, such as a single cell array. Biotechnol. Bioeng. 2010;107: 561–565. © 2010 Wiley Periodicals, Inc.     

Synthesis of F16 conjugated with 5‐fluorouracil and biophysical investigation of its interaction with bovine serum albumin by a spectroscopic and molecular modeling approach

5‐Fluorouracil (5‐FU) has been widely used as a chemotherapy agent in the treatment of many types of solid tumors. Investigation of its antimetabolites led to the development of an entire class of fluorinated pyrimidines. However, the toxicity profile associated with 5‐FU is significant and includes diarrhea, mucositis, hand–foot syndrome and myelosuppression. In aiming at reducing of the side effects of 5‐FU, we have designed and synthesized delocalized lipophilic cations (DLCs) as a vehicle for the delivery of 5‐FU. DLCs accumulate selectively in the mitochondria of cancer cells because of the high mitochondrial transmembrane potential (ΔΨ_m). Many DLCs exhibited anti‐cancer efficacy and were explored as potential anti‐cancer drugs based on their selective accumulation in the mitochondria of cancer cells. F16, the DLC we used as a vehicle, is a small molecule that selectively inhibits tumor cell growth and dissipates mitochondrial membrane potential. The binding of the conjugate F16–5‐FU to bovine serum albumin (BSA) was investigated using spectroscopic and molecular modeling approaches. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between F16–5‐FU and BSA. The activation energy of the interaction between F16–5‐FU and BSA was calculated and the unusually high value was discussed in terms of the special structural block indicated by the molecular modeling approach. Molecular modeling showed that F16–5‐FU binds to human serum albumin in site II, which is consistent with the results of site‐competitive replacement experiments. It is suggested that hydrophobic and polar forces played important roles in the binding reaction, in accordance with the results of thermodynamic experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

A real‐time fluorescence assay for protease activity and inhibitor screening based on the aggregation‐caused quenching of a perylene probe

We have established a real‐time and label‐free fluorescence turn‐on strategy for protease activity detection and inhibitor screening via peptide‐induced aggregation‐caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly‐l ‐glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective.     

Characterization of a highly Al3+‐selective fluorescence probe based on naphthalimide‐Schiff base and its application to practical water samples

A new fluorescent Al³⁺‐probe, N‐allyl‐4‐[3,3′‐((2‐aminoethyl)azanediyl)‐bis(N´‐(2‐hydroxybenzylidene)propanehy‐drazide)]‐1,8‐naphthalimide ( L ), was designed and synthesized based on 1,8‐naphthalimide. The probe L contains 1,8‐naphthalimide moiety as the fluorophore and a Schiff base as the recognition group. The structure of L was determined by single crystal X‐ray. L emission at 526 nm increased on addition of Al³⁺ under excitation wavelength at 350 nm. L exhibited high selectivity and sensitivity fluorescence emission towards to Al³⁺ in ethanol/Tris–HCl buffer solution (1:1, v/v, pH = 7.2) as compared with other tested metal ions. A good linearity with a correlation coefficient (R²) of 0.99 was observed in the concentration range 2–10 μM. The binding constant and the detection limit of L for Al³⁺ were calculated to 2.6 × 10⁴ M^?1 and 0.34 μM, respectively. The results of experiments that including Job plot, ultraviolet–visible (UV–Vis) light titration, fluorescence titration, ESI‐MS and ¹H NMR titration, indicated a 1:1 stoichiometric complex between L and Al³⁺. L was highly effective in monitoring Al³⁺ in real‐life Yellow River and tap water samples.     

Support and challenges to the melanosomal casing model based on nanoscale distribution of metals within iris melanosomes detected by X‐ray fluorescence analysis

Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X‐ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery‐based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis‐based test of the ‘casing model’ predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes – including one with a Cu‐rich shell surrounding a Ca/Zn‐rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co‐exist.     

Inhibiting the color formation by gradient temperature‐elevating Maillard reaction of soybean peptide‐xylose system based on interaction of l‐cysteine and Amadori compounds

Light color and savory flavor enhancer are attractive for consumers and food producers. The effect of addition time of l ‐cysteine on inhibiting color formation was investigated in soybean peptide‐xylose system, and the possible pathway was explored. Once dicarbonyl compounds were formed during the Maillard reaction, the addition of l ‐cysteine had no color‐inhibiting effect; if l ‐cysteine was added immediately after the Amadori compound was formed, the extraordinary color‐inhibiting effect was observed. Therefore, an improved way to inhibit color formation was proposed on the basis of the interaction of l ‐cysteine and Amadori compounds by controlling the addition time of l ‐cysteine through gradient temperature‐elevating Maillard reaction. The system was heated at 80 °C for 60 min to form Amadori compounds, followed by the addition of L‐cysteine, and the temperature was raised to 120 °C and held for 110 min. Compared with traditional products, the lightest color product was found desirable by GC/MS analysis and sensory evaluation. The novel method proposed can be a guide for the industrial preparation of light‐colored products. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Solar‐induced chlorophyll fluorescence is strongly correlated with terrestrial photosynthesis for a wide variety of biomes: First global analysis based on OCO‐2 and flux tower observations

Solar‐induced chlorophyll fluorescence (SIF) has been increasingly used as a proxy for terrestrial gross primary productivity (GPP). Previous work mainly evaluated the relationship between satellite‐observed SIF and gridded GPP products both based on coarse spatial resolutions. Finer resolution SIF (1.3 km × 2.25 km) measured from the Orbiting Carbon Observatory‐2 (OCO‐2) provides the first opportunity to examine the SIF–GPP relationship at the ecosystem scale using flux tower GPP data. However, it remains unclear how strong the relationship is for each biome and whether a robust, universal relationship exists across a variety of biomes. Here we conducted the first global analysis of the relationship between OCO‐2 SIF and tower GPP for a total of 64 flux sites across the globe encompassing eight major biomes. OCO‐2 SIF showed strong correlations with tower GPP at both midday and daily timescales, with the strongest relationship observed for daily SIF at the 757 nm (R² = 0.72, p < 0.0001). Strong linear relationships between SIF and GPP were consistently found for all biomes (R² = 0.57–0.79, p < 0.0001) except evergreen broadleaf forests (R² = 0.16, p < 0.05) at the daily timescale. A higher slope was found for C₄ grasslands and croplands than for C₃ ecosystems. The generally consistent slope of the relationship among biomes suggests a nearly universal rather than biome‐specific SIF–GPP relationship, and this finding is an important distinction and simplification compared to previous results. SIF was mainly driven by absorbed photosynthetically active radiation and was also influenced by environmental stresses (temperature and water stresses) that determine photosynthetic light use efficiency. OCO‐2 SIF generally had a better performance for predicting GPP than satellite‐derived vegetation indices and a light use efficiency model. The universal SIF–GPP relationship can potentially lead to more accurate GPP estimates regionally or globally. Our findings revealed the remarkable ability of finer resolution SIF observations from OCO‐2 and other new or future missions (e.g., TROPOMI, FLEX) for estimating terrestrial photosynthesis across a wide variety of biomes and identified their potential and limitations for ecosystem functioning and carbon cycle studies.