Simultaneous Determination of Glucose and Choline Based on the Intrinsic Fluorescence of the Enzymes

It has been possible to perform the simultaneous determination of choline and glucose using the intrinsic fluorescence of the corresponding enzyme as an analytical signal. This can be done in two ways. First, for low glucose and choline concentrations (about 0.55 mM and 0.75 μM respectively) two differentiated signals, without mutual interference, are obtained for both analytes in the same measurement. Second, when glucose and choline concentrations are higher, a new model has been designed which permits the concentrations to be accurately determined in samples containing from 0.55 mM to 3.75 mM glucose and from 0.75 μM to 11.0 μM choline; the method has been applied to simultaneous glucose and choline determinations in serum samples with good results. This method gives a better performance than multivariate calibration based on Partial Least Squares Regression. The methodology here shown could be also used for the simultaneous determination of other pairs of analytes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fluorescence study on Interactions of α–Crystallin with the Molten Globule State of 1, 4–β–D–Glucan Glucanohydrolase from Thermomonospora sp. induced by guanidine hydrochloride

In this paper, the interaction between α- crystallin and molten globule structure of 1,4–β–D–Glucan Glucohydrolase (TSC) from an alkalothermophilic Thermomonospora sp. was investigated mainly by fluorescence quenching spectra, circular dichroism and three dimensional fluorescence spectra under simulative physiological conditions. Denaturation studies using GdnCl indicated that TSC folds through a partially folded state that resembles molten globule at 1.8 M GdnCl. The chaperone activity of α- crystallin was employed to study refolding of TSC. Here we studied the refolding of GdnCl denatured TSC from its molten globule state (TSC-m complex) in the presence and absence of α-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and ANS binding studies, suggest that α-crystallin formed a complex with a putative intermediate molten globule – like intermediate in the refolding pathway of TSC. Reconstitution of the active TSC was observed on cooling the α-crystallin • TSC -m complex to 4°C. Addition of α-crystallin to the molten globule – like intermediate of TSC (TSC-m complex) complex initiated the refolding of TSC with 69 % recovery of the biological activity of the enzyme.     

Conformational Transitions in <Emphasis Type="Italic">Ariesaema curvatum</Emphasis> Lectin: Characterization of an Acid Induced Active Molten Globule

Biophysical characterization of a lectin from Ariesaema curvatum (ACL) was carried out using steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. An intermediate with altered tryptophan microenvironment was detected in the phase diagram, which exibited pronounced secondary structure and hemagglutinating activity in presence of 0.25 M Gdn–HCl. An acid induced molten- globule like structure possessing activity and higher thermostability was detected. Transition to the molten globule state was reversible in nature. The lectin retained hemagglutinating activity even after incubation at 95 °C. Both chemical and thermal unfolding of the lectin were found to consist of multistate processes. Fluorescence quenching of ACL was strong with acrylamide and KI. The single tryptophan was found to be surrounded by high density of the positively charged amino acid residues as shown by a ten fold higher K_sv for KI compared to that for CsCl. The average lifetime of tryptophan fluorescence increased from 1.24 ns in the native state to 1.72 ns in the denatured state.     

Conformational and Functional Transitions in Class II α-mannosidase from <Emphasis Type="Italic">Aspergillus fischeri</Emphasis>

The conformational transitions in an oligomeric and high molecular weight class II α-mannosidase from Aspergillus fischeri were examined using fluorescence and CD spectroscopy under chemical, thermal and acid denaturing conditions. The enzyme lost the activity first and then the overall folded conformation and secondary structure. The midpoint values of GdnHCl mediated changes measured by inactivation; fluorescence and negative ellipticity were 0.48 M, 1.5 M and 1.9 M, respectively. The protein almost completely unfolded in 4.0 M GdnHCl but not at 90 °C. The inactivation and unfolding were irreversible. At pH 2.0, the protein exhibited molten-globule like intermediate with rearranged secondary and tertiary structures and exposed hydrophobic amino acids on the surface. This species showed increased accessibility of Trp to the quenchers and got denatured with GdnHCl in a different manner. The insoluble aggregates of a thermally denatured protein could be detected only in the presence of 0.25–0.75 M GdnHCl.     

Determination of Tetracaine Hydrochloride by Fluorescence Quenching Method with Some Aromatic Amino Acids as Probes

A novel fluorescence quenching method for the determination of tetracaine hydrochloride (TA·HCl) concentration with some aromatic amino acids as fluorescence probe has been developed. In pH 6.3 acidic medium, tryptophane (Trp), tyrosine (Tyr) or phenylalanine (Phe) can react with tetracaine hydrochloride to form an ion-association complex by electrostatic attraction, aromatic stacking interaction and Van der Waals’ force, which lead to fluorescence quenching of above amino acids. The maximum fluorescence excitation and emission wavelengths of them are located at 278, 274, 258 nm and 354, 306, 285 nm, respectively. The relative fluorescence intensity (F ₀/F) is proportional to the TA·HCl concentration in certain range. The linear ranges and detection limits are 1.2–5.0 μg/mL and 0.37 μg/mL for Tyr-TA·HCl system, 1.3–6.0 μg/mL and 0.38 μg/mL for Trp-TA·HCl system, and 1.4–6.0 μg/mL and 0.41 μg/mL for Phe-TA·HCl system. The optimum reaction conditions, influencing factors and the effect of coexisting substances are investigated. And the results show the method has a good selectivity. Judging from the effect of temperature, the Stern-Volmer plots and fluorescence lifetime determination, the quenching of fluorescence of amino acids by TA·HCl is a static quenching process.     

Ultrasound effects on restricted silica gelation during silica extraction from Pyro-Metallurgical copper slag under acidifying conditions

Pyro-metallurgical copper slag (CS) waste was used as the source material for ultrasound (US) silica extraction under acidification processes with 26 kHz with HCl, HNO₃, and H₂SO₄ at different concentrations at 100, 300, and 600 W. During acidifying extraction processes, US irradiation inhibited silica gel formation under acidic conditions, especially at lower acid concentrations of less than 6 M, whereas a lack of US irradiation led to enhanced gelation. When US stopped, gelation occurred to a considerable degree, suggesting that the gel particle size distribution was aggregated in the 3–400 µm size range. However, with US, the size was mainly in the 1–10 µm range. Results of elemental analysis indicated that US treatment decreased the co-precipitation of other metal ions such as Fe, Cu, and Al sourced from CS for lower acidic medium, whereas the higher concentration medium accelerated silica gelation and the co-precipitation of other metals. With acids of HCl and HNO₃, and H₂SO₄, the gelations were less likely to occur at 6 M and 3 M during US irradiation, but acidic extraction without US was efficient for silica gelation and co-precipitation of other metals in the purified silica. The silica extraction yield with H₂SO₄ concentration of 3 M was 80% with 0.04% of Fe, whereas the silica product from HCl 6 M had a 90% extraction yield with only 0.08% of Fe impurity. In contrast, even though the non-US system of HCl 6 M had a higher yield at 96%, the final product had 0.5% Fe impurity, which was much higher than the US system. Consequently, the US extraction process was quite noticeable for silica recovery from CS waste.     

Ultrasound assisted acid catalyzed lactose hydrolysis: Understanding into effect of operating parameters and scale up studies

The current work deals with the value addition of lactose by transforming into hydrolyzed lactose syrup containing glucose and galactose in major proportion using the novel approach of ultrasound assisted acid catalyzed lactose hydrolysis. The hydrolysis of lactose was performed in ultrasonic bath (33 kHz) at 50% duty cycle at different temperatures as 65 °C and 70 °C and two different hydrochloric acid (HCl) concentrations as 2.5 N and 3 N. It was observed that acid concentration, temperature and ultrasonic treatment were the major factors in deciding the time required to achieve ∼90% hydrolysis. The ultrasonic assisted approach resulted in reduction in the reaction time and the extent of intensification was established to be dependent on the temperature, acid concentration and time of ultrasonic exposure. It was observed that the maximum process intensification obtained by introduction of ultrasound in the lactose hydrolysis process performed at 70 °C and 3 N HCl was reduction in the required time for ∼90% hydrolysis from 4 h (without the presence of ultrasound) to 3 h. The scale-up study was also performed using an ultrasonic bath with longitudinal horn (36 kHz as operating frequency) at 50% duty cycle, optimized temperature of 70 °C and acid concentration of 3 N. It was observed that the reaction was faster in the presence of ultrasound and stirring by axial impeller at rpm of 225 ± 25. The time required to complete ∼90% of hydrolysis remained almost the same as observed for small scale study on ultrasonic bath (33 kHz) at 50% duty cycle. The use of recovered lactose from whey samples instead of pure lactose did not result in any significant changes in the progress of hydrolysis, confirming the efficacy of the selected approach. Overall, the work has presented a novel ultrasound assisted approach for intensified lactose hydrolysis.     

Stress-Induced Alteration of Chlorophyll Fluorescence Polarization and Spectrum in Leaves of <Emphasis Type="Italic">Alocasia macrorrhiza</Emphasis> L. Schott

The value of intrinsic chlorophyll fluorescence polarization, and the intensity in emission spectrum were investigated in leaf segments of Alocasia macrorrhiza under several stress conditions including different temperatures (25–50°C), various concentrations of NaCl (0–250 mM), methyl viologen (MV, 0–25 μM), SDS (0–1.0%) and NaHSO₃ (0–80 μM). Fluorescence emission spectrum of leaves at wavelength regions of 500–800 nm was monitored by excitation at 436 nm. The value of fluorescence polarization (P value), as result of energy transfer and mutual orientation between chlorophyll molecules, was determined by excitation at 436 nm and emission at 685 nm. The results showed that elevated temperature and concentrations of salt (NaCl), photooxidant (MV), surfactant (SDS) and simulated SO₂ (NaHSO₃) treatments all induced a reduction of fluorescence polarization to various degrees. However, alteration of the fluorescence spectrum and emission intensity of F₆₈₅ and F₇₃₁ depended on the individual treatment. Increase in temperature and concentration of NaHSO₃ enhanced fluorescence intensity mainly at F₆₈₅, while an increase in MV concentration led to a decrease at both F₆₈₅ and F₇₃₁. On the contrary, NaCl and SDS did not cause remarkable change in fluorescence spectrum. Among different treatments, the negative correlation between polarization and fluorescence intensity was found with NaHSO₃ treatments only. We concluded that P value being measured with intrinsic chlorophyll fluorescence as probe in leaves is a susceptible indicator responding to changes in environmental conditions. The alteration of P value and fluorescence intensity might not always be shown a functional relation pattern. The possible reasons of differed response to various treatments were discussed.     

Fibrinogen-β-Estradiol Binding Studied by Fluorescence Spectroscopy: Denaturation and pH Effects

Fibrinogen is a blood plasma protein that plays a crucial role in hemostasis. It is known that erythrocyte aggregation increases in the presence of fibrinogen, and that β-estradiol decreases erythrocyte aggregation with a constant fibrinogen concentration. In this work, we have used intrinsic tryptophan fluorescence to obtain information on the conformational changes of fibrinogen upon the recently proposed interaction with β-estradiol. To evaluate the effect on the conformational changes during fibrinogen-β-estradiol binding, fluorescence experiments were performed using guanidine hydrochloride (0–6 M) as denaturant, at different pH values. The results obtained for pH 6.5 and 8.0 showed no effect during the binding. The main differences were observed between pH 4.2 and 7.4, in the absence and in the presence of two different denaturant concentrations (1 and 5 M). A red shift of the fluorescence emission from 344 to 354 nm is observed when denaturant concentration is above 3 M for all studied pH values. This phenomenon may be explained by the loss of compact structure of the protein in the presence of denaturant, with tryptophan residues exposure to the aqueous environment and alteration of fibrinogen-β-estradiol binding. These results demonstrate that the binding sites of fibrinogen are strongly dependent on the conformational state of the protein.     

Spectrofluorimetric Determination of Etodolac, Moxepril HCl and Fexofenadine HCl Using Europium Sensitized Fluorescence in Bulk and Pharmaceutical Preparations

A simple, selective and sensitive luminescence method has been developed for the assay of etodolac (I), moxepril HCl (II) and fexofenadine HCl (III) in bulk drug and pharmaceutical formulations. The method is based on the luminescence sensitization of europium (Eu³⁺) by complexation with the studied drugs. The fluorescence intensities of the products were measured at 667 nm for (I) and at 615 for (II) and (III) while exciting at 276 for all the studied drugs. The fluorescence intensity was directly proportional to the concentration over the range (20–280), (40–240) and (30–80) ng/ml with limits of detection (LOD) = 0.93, 0.92 and 0.95 μg/ml for drugs I, II and III respectively. Optimum conditions for the formation of the complex in methanol were carefully studied. The proposed method was successfully applied for the assay of the studied drugs in pharmaceutical formulations with excellent recovery.     

Intensification of delignification of Tectona grandis saw dust as sustainable biomass using acoustic cavitational devices

Delignification of sawdust was studied using ultrasound assisted alkali peroxide approach using longitudinal horn for the first time and the efficacy compared with more commonly used configurations of ultrasonic reactors. Comparison with the conventional approach based on stirring has also been presented to establish the process intensification benefits. Effect of different operating parameters such as sodium carbonate concentration (0.1, 0.15, 0.2, 0.25 M), hydrogen peroxide concentration (0.2, 0.4, 0.6, 0.8, 1 M) and biomass loading (2, 4, 6, 8, 10 wt%), on the efficacy of lignin extraction has been investigated for different ultrasonic reactors. The optimum conditions for probe type ultrasonic horn were established as 150 W, 50% duty cycle and 80% amplitude with optimum process conditions as Na₂CO₃ concentration as 0.2 M, H₂O₂ concentration as 1 M, biomass loading of 10 wt% and operating time of 70 min. Longitudinal horn resulted in best efficacy (both in terms of yield and energy requirements) followed by ultrasonic horn and ultrasonic bath whereas the conventional approach was least effective. The obtained lignin was also analyzed using different characterization techniques. The presence of peaks at wavelength range of 875–817, 1123–1110, and at 1599 cm⁻¹ for the extracted sample confirmed the presence of lignin. Increase in the crystallinity index of the processed sample (maximum for longitudinal horn) also confirmed the lignin removal as lignin is amorphous in nature. Overall it has been concluded that ultrasound can be effectively used for delignification with longitudinal horn as best configuration.     

Spectrophotometric Study of Luminol in Dimethyl Sulfoxide–Potassium Hydroxide

The spectrophotometric study of luminol (LH₂) in dimethyl sulfoxide (DMSO), DMSO-water solutions, and alkaline DMSO and DMSO-water solutions has been done, focusing on the effect of the KOH additon on LH₂ absorption and fluorescence properties. The absorption spectra indicate an acid-base equilibrium, and the luminol dianion (L^2–) formation at 3 × 10^–4 – 2.4 × 10^–3 M KOH. The decrease of the fluorescence intensity and the variation of the excitation spectra of LH₂-DMSO-KOH solutions with KOH concentration have been similarly explained. The acid-base process is reversible. The addition of HCl to the solution with 3.0 × 10^–3 M KOH leads to an increase of the fluorescence intensity to its highest value, observed in pure DMSO. The addition of HCl to the LH₂-DMSO solution leads to the decrease of the fluorescence intensity as a result of the LH⁺ ₃ cation formation. In LH₂-DMSO-water, the fluorescence band is shifted from 405 nm to 424 nm and increased in the intensity. In the presence of KOH (in LH₂-DMSO-water-KOH solution) a new band appears, with the maximum at 485 nm and the band at 405 nm decreased. The changes in fluorescence lifetimes also evidence the different chemical species formed.     

Ultrasound-assisted soil washing processes for the remediation of heavy metals contaminated soils: The mechanism of the ultrasonic desorption

Ultrasound-assisted soil washing processes were investigated for the removal of heavy metals (Cu, Pb, and Zn) in real contaminated soils using HCl and EDTA. The ultrasound-assisted soil washing (US/Mixing) process was compared with the conventional soil washing (Mixing) process based on the mechanical mixing. High removal efficiency (44.8% for HCl and 43.2% for EDTA) for the metals was obtained for the most extreme conditions (HCl 1.0 M or EDTA 0.1 M and L:S = 10:1) in the Mixing process. With the aide of ultrasound, higher removal efficiency (57.9% for HCl and 50.0% for EDTA) was obtained in the same extreme conditions and similar or higher removal efficiency (e.g., 54.7% for HCl 0.5 M and L:S = 10:1 and 50.5% for EDTA 0.05 M and L:S = 5:1) was achieved even in less extreme conditions (lower HCl or EDTA concentration and L:S ratio). Therefore, it was revealed that the US/Mixing was advantageous over the conventional Mixing processes in terms of metal removal efficiency, consumption of chemicals, amount of generated washing leachate, and volume/size of washing reactor. In addition, the heavy metals removal was enhanced for the smaller soil particles in the US/Mixing process. It was due to more violent movement of smaller particles in slurry phase and more violent sonophysical effects. In order to understand the mechanism of ultrasonic desorption, the desorption test was conducted using the paint-coated beads with three sizes (1, 2, and 4 mm) for the free and attached conditions. It was found that no significant desorption/removal of paint from the beads was observed without the movement of beads in the water including floatation, collision, and scrubbing. Thus, it was suggested that the simultaneous application of the ultrasound and mechanical mixing could enhance the physical movement of the particles significantly and the very high removal/desorption could be attained.     

Quenching of Myosin Intrinsic Fluorescence Unravels the Existence of a High Affinity Binding Site for Decavanadate

Decavanadate, one of the aggregated species of vanadate, is a potent inhibitor of several enzymes, including skeletal muscle myosin. However, its putative binding sites in myosin are largely unknown. Titration of the intrinsic fluorescence of myosin, purified from rabbit skeletal muscle, have been carried out in 0.3 M KCl, 5 mM CaCl₂ and 25 mM Tris-HCl (pH 7.0), with 0.1 mg/ml myosin. In the 0-200 M total vanadate concentration range, decavanadate produced approximately 25% quenching of the intrinsic fluorescence of myosin, with an apparent dissociation constant in the micromolar range. This effect was found to be specific of decavanadate, because titration with metavanadate up to 200 M did not produce a significant quenching of the intrinsic fluorescence of myosin. This quenching was accompanied by a parallel decrease of the accessibility of myosin tryptophans to the water-soluble collisional quencher KI, with an apparent dissociation constant also in the micromolar range. It is concluded that the binding of decavanadate to high-affinity sites in myosin produces local conformational change(s) near the tryptophans more accessible to water in the three-dimensional structure of this protein.     

Comparative Studies of Two Araceous Lectins by Steady State and Time-Resolved Fluorescence and CD Spectroscopy

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2–1.4 and 6.3–6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0–12.0 was observed in CD spectra.     

Influence of Prototropic Reactions on the Absorption and Fluorescence Spectra of Methyl p-dimethylaminobenzoate and Its Two Ortho Derivatives

The influence of prototropic reactions on the spectral characteristics of methyl p-dimethylaminobenzoate (I) and its o-methoxy (II) and o-hydroxy (III) derivatives has been studied using steady-state spectroscopic technique and quantum-chemical calculations. This study concerns the solvent-induced shift of the absorption, locally excited (LE) and intramolecular charge transfer (ICT) fluorescence bands in the neat tetrahydrofuran (THF) and its hydrochloric acid solutions at different HCl concentrations. On the basis of the experimental results and quantum-chemical calculations, it was shown that in a hydrochloric acid solution the studied molecules exist as a mixture of neutral, mono-, and dicationic forms. Additionally, the results of spectroscopic measurements were used to calculate, according to the Benesi-Hildebrand method, the equilibrium constants of protopropic reactions in the ground, S ₀, and excited, S ₁, states. Our findings predestine molecules I and II to be used as acid fluorescence probes in a region of 0–2.5 M of [H⁺] concentrations.     

Investigation of the effect of acid dopant on the physical properties of polyaniline prepared using microwave irradiation

The microwave (MW) synthesis of polyaniline (PANI) is performed using potassium iodate (KIO₃) as oxidizing agent in different concentrations of aqueous hydrochloric acid (HCl) at 8 and 93 W applied microwave power for duration of 10 min. The morphological and structural changes in synthesized MW PANI samples are investigated using Scanning Electron Microscopy (SEM) and Fourier transform Infrared Spectroscopy (FTIR). With decreasing pH of the reaction medium the morphology of MW PANI samples changed from slab-like with a small amount of fibrils to porous products which consist of short, rod-like structures. The FTIR spectra confirm that the microwave generated materials structurally consist of PANI, but aniline oligomer peaks are observed in the FTIR at 725 and 686 cm⁻¹ for MW PANI synthesized using 0.5 M aqueous HCl. The influence of acid dopant on the spin concentration of MW PANI synthesized at 8 and 93 W are examined.     

High-performance nonenzymatic electrochemical glucose biosensor based on AgNP-decorated MoS2 microflowers

A facile hydrothermal route was used to synthesize silver nanoparticle (AgNP)-decorated microflower molybdenum disulfide (MoS₂-MF) for bio-electrochemical platform fabrication to detect nonenzymatic glucose concentration. The morphologies of the materials were studied by scanning electron microscopy, and their structural characteristics were analyzed by X-ray diffractometry and energy-dispersive X-ray spectroscopy. The electrochemical characteristics of the AgNPs/MoS₂-MF/PtE biosensor were studied by cyclic voltammetry. The obtained data indicated that the developed nonenzymatic glucose sensor has a large linear response between 1.0 and 15.0 mM, a limit of detection of as low as 1.0 mM, and a sensitivity of 46.5 μA nM⁻¹ cm⁻². The biosensor also displayed outstanding selectivity, stability, reproducibility, and repeatability. Additionally, the AgNPs/MoS₂-MF/PtE biosensor was utilized to detect glucose concentration in real sample and showed practical application potential for glucose detection.     

Flow effects on phenol degradation and sonoluminescence at different ultrasonic frequencies

Current literature shows a direct correlation between the sonochemical (SC) process of iodide oxidation and the degradation of phenol solution. This implies phenol degradation occurs primarily via oxidisation at the bubble surface. There is no work at present which considers the effect of fluid flow on the degradation process. In this work, parametric analysis of the degradation of 0.1 mM phenol solution and iodide dosimetry under flow conditions was undertaken to determine the effect of flow. Frequencies of 44, 300 and 1000 kHz and flow rates of 0, 24, 228 and 626 mL/min were applied with variation of power input, air concentration, and surface stabilisation. Phenol degradation was analysed using the 4-aminoantipyrine (4-AAP) method, and sonoluminescence (SL) images were evaluated for 0.1, 20 and 60 mM phenol solutions. Flow, at all frequencies under certain conditions, could augment phenol degradation. At 300 kHz there was excellent correlation between phenol degradation and dosimetry indicating a SC process, here flow acted to increase bubble transience, fragmentation and radical transfer to solution. At 300 kHz, although oxidation is the primary phenol degradation mechanism, it is limited, attributed to degradation intermediates which reduce OH radical availability and bubble collapse intensity. For 44 and 1000 kHz there was poor correlation between the two SC processes. At 44 kHz (0.01 mM), there was little to suggest high levels of intermediate production, therefore it was theorised that under more transient bubble conditions additional pyrolytic degradation occurs inside the bubbles via diffusion/nanodroplet injection mechanisms. At 1000 kHz, phenol degradation was maximised above all other systems attributed to increased numbers of active bubbles combined with the nature of the ultrasonic field. SL quenching, by phenol, was reduced in flow systems for the 20 and 60 mM phenol solutions. Here, where the standing wave field was reinforced, and bubble localisation increased, flow and the intrinsic properties of phenol acted to reduce coalescence/clustering. Further, at these higher concentrations, and in flow conditions, the accumulation of volatile phenol degradation products inside the bubbles are likely reduced leading to an increase SL.     

Label–free Fluorescence Turn on Trypsin Assay Based on Gemini Surfactant/heparin/Nile Red Supramolecular Assembly

In this research, we designed a label-free fluorometric turn-on assay for trypsin and inhibitor screening, based on a spherical cationic gemini surfactant ethylene-bis (dodecyl dimethyl ammonium bromide) (EDAB)/heparin/Nile red (NR) supramolecular assembly system. The introduction of gemini surfactant EDAB as template greatly enhanced its salt resistance and resulted in the supramolecular assemblies with diameters ranging from 20 to 100 nm. The fluorometric assay for trypsin was performed by firstly disassembling with protamine (a heparin-binding protein) and then re-assembling through hydrolysis of protamine. The disassembly and reassembly of the system resulted in a turn-off first and then a turn-on behavior of the corresponding fluorescence. The overall processes were characterized by fluorescence spectra, TEM measurements and zeta potential tests. The detection level of this assembly system for trypsin was as low as 4.2 ng mL^?1. Also, the EDAB/heparin/NR assembly could be used to screen the trypsin inhibitors. The assembly system was easily-fabricated and cost-effective, but also exhibited good salt tolerance in NaCl solution at the concentration of 0–500 mM. At last, the supramolecular assembly was successfully applied to detect trypsin in human urine, demonstrating its great potential on clinical diagnosis applications.