Synchronous Fluorescence Technique Research Articles

A novel derivative synchronous fluorescence method for the rapid, non-destructive and intuitive differentiation of denitrifying bacteria

It is challenging to differentiate bacteria residing in the same habitat by direct observation. This difficulty impedes the harvest, application and manipulation of functional bacteria in environmental engineering. In this study, we developed a novel method for rapid differentiation of living denitrifying bacteria based on derivative synchronous fluorescence spectroscopy, as exemplified by three heterotrophic nitrification-aerobic denitrification bacteria having the maximum nitrogen removal efficiencies greater than 90%. The intact bacteria and their living surroundings can be analyzed as an integrated target, which eliminates the need for the complex pre-processing of samples. Under the optimal synchronous scanning parameter (Δλ = 40 nm), each bacterium possesses a unique fluorescence spectral structure and the derivative synchronous fluorescence technique can significantly improve the spectral resolution compared to other conventional fluorescence methods, which enables the rapid differentiation of different bacteria through derivative synchronous fluorescence spectra as fast as 2 min per spectrum. Additionally, the derivative synchronous fluorescence technique can extract the spectral signals contributed by bacterial extracellular substances produced in the biological nitrogen removal process. Moreover, the results obtained from our method can reflect the real-time denitrification properties of bacteria in the biological nitrogen removal process of wastewater. All these merits highlight derivative synchronous fluorescence spectroscopy as a promising analytic method in the environmental field.

Interaction between a water-soluble anionic porphyrin and human serum albumin unexpectedly stimulates the aggregation of the photosensitizer at the surface of the albumin

The 5,10,15,20-tetrakis(2,6-difluoro-3-sulfophenyl)porphyrin (TDFPPS4) was reported as a potential photosensitizer for photodynamic therapy. The capacity of the photosensitizers to be carried in the human bloodstream is predominantly determined by its extension of binding, binding location, and binding mechanism to human serum albumin (HSA), influencing its biodistribution and ultimately its photodynamic therapy efficacy in vivo. Thus, the present work reports a biophysical characterization on the interaction between the anionic porphyrin TDFPPS4 and HSA by UV–visible absorption, circular dichroism, steady-state, time-resolved, and synchronous fluorescence techniques under physiological conditions, combined with molecular docking calculations and molecular dynamics simulations. The interaction HSA:TDFPPS4 is spontaneous (ΔG° < 0), strong, and enthalpically driven (ΔH° = −70.1 ± 3.3 kJ mol−1) into subdomain IIA (site I). Curiously, despite the porphyrin binding into an internal pocket, about 50 % of TDFPPS4 structure is still accessible to the solvent, making aggregation in the bloodstream possible. In silico calculations were reinforced by spectroscopic data indicating porphyrin aggregation between bound and unbound porphyrins. This results in an adverse scenario for anionic porphyrins to achieve their therapeutical potential as photosensitizers and control of effective dosages. Finally, a trend of anionic porphyrins to have a combination of quenching mechanisms (static and dynamic) was noticed.

Effects of two food colorants on catalase and trypsin: Binding evidences from experimental and computational analysis

Recently, growing concern has been paid to the toxicity of additives in food. The present study investigated the interaction of two commonly used food colorants, quinoline yellow (QY) and sunset yellow (SY), with catalase and trypsin under physiological conditions by fluorescence, isothermal titration calorimetry (ITC), ultraviolet-vis absorption, synchronous fluorescence techniques as well as molecular docking. Based on the fluorescence spectra and ITC data, both QY and SY could significantly quench the intrinsic fluorescence of catalase or trypsin spontaneously to form a moderate complex driven by different forces. Additionally, the thermodynamics results demonstrated QY bind more tightly to both catalase and trypsin than SY, suggesting QY poses more of a threat to two enzymes than SY. Furthermore, the binding of two colorants could not only lead to the conformational and microenvironmental alterations of both catalase and trypsin, but also inhibit the activity of two enzymes. This study provides an important reference for understanding the biological transportation of synthetic food colorants in vivo, and enhancing their risk assessment on food safety.

Synchronous fluorescence techniques for the detection and monitoring of selected fluorescent dyes in binary and multifluorophoric mixtures

The present study deals with non-destructive conventional emission, constant-wavelength synchronous fluorescence spectroscopy (CWSFS) and first derivative of CWSFS for selective estimation of two fluorescent xanthene dyes viz. rhodamine B (RhB) and fluorescein (Fls) in their binary and multifluorophoric mixtures via spike method in an aqueous environment. Various parameters (Δλ, pH, ionic strength, etc.) affecting these methods were optimized. Based on the results, measurement of RhB and Fls were performed at Δλ, 20.0 nm, pH 7.0, 25.0 ⁰C temperature and scan rate of 240.0 nm/min without their prior separation in mixtures. CWSFS and first derivative CWSFS measurements were carried out at 556.0 nm and 547.0 nm for RhB (R2 = 0.988 and 0.983) and 493.0 nm and 484.0 nm for Fls (R2 = 0.999 and 0.999) over the concentration range of both dyes at 0.10–0.70 mg/L. The mean percentage recoveries from aqueous binary mixtures of RhB and Fls were found to be 98.95, 99.97 (RhB) and 100.68, 100.73 (Fls) for CWSFS and first derivative CWSFS respectively. Results were statistically analyzed in terms of accuracy and precision.

An Ultrasensitive Strand Displacement Signal Amplification-Assisted Synchronous Fluorescence Assay for Surface Proteins of Small Extracellular Vesicle Analysis and Cancer Identification

Small extracellular vesicles (sEVs), often known as exosomes, are expected to be a promising biomarker for the early diagnosis of cancer because they carry enriched proteins that originated from parent cells. Profiling surface proteins of sEVs offers non-invasive access for the early diagnosis of cancer. However, it remains challenging to simultaneously detect surface proteins of sEVs with desired sensitivity. Herein, a dual color DNA nanodevice based on toehold-mediated DNA strand displacement signal amplification and the synchronous fluorescence technique has been developed for simultaneous analysis of surface proteins of sEVs with high sensitivity. As for the DNA nanodevice-based system, the nanoconjugates of aptamer-magnetic beads can recognize surface proteins of sEVs and lead to the release of single-stranded DNA. Then, the released DNA can trigger toehold-mediated DNA strand displacement for signal amplification. In this system, a CD63 aptamer and MUC1 aptamer were used as recognition elements for the detection of surface proteins of sEVs isolated from cancer cells. Under the optimal conditions, the corresponding proteins of sEVs were simultaneously determined with ultrasensitivity by the synchronous fluorescence method. Also, the detection limits of sEVs by two surface proteins were 67 particles/μL by CD63 and 37 particles/μL by MUC1. Of note, the as-constructed method can be applied to recognize sEVs from different tumor cell lines (SGC7901, HepG2, and MCF-7 cells). Furthermore, the system has been successfully applied to precisely identify cancer patients from healthy people by serum analysis. The strategy demonstrates great potential applications in the early diagnosis of cancer.

Excitation, emission, and synchronous fluorescence for astrochemical applications: Experiments and computer simulations of synchronous spectra of polycyclic aromatic hydrocarbons and their mixtures

Synchronous fluorescence spectroscopy is a variation of the fluorescence technique for detection and characterization of many single ring aromatic organic molecules, polycyclic aromatic hydrocarbons, and fluorescent amino acids among other molecules. To our knowledge this technique has not been used in space exploration. In order to demonstrate the utility of this technique for planetary and astrochemical applications, we present several laboratory experiments and computer simulations with pure samples and mixtures. Computer deconvolution of experimental excitation and emission fluorescence bands is presented and used to generate synchronous spectra. The computer simulation successfully predicts the number of synchronous fluorescence (SF) bands, band shapes, and band maximum wavelengths for any constant wavelength difference (Δλ). To test the simulation, emission, excitation, and synchronous spectra were obtained for anthracene in n-hexane. Excellent agreement is obtained reproducing and finding the origin of the experimental SF bands for values of Δλ between 2 and 100. The excitation, emission, and synchronous (Δλ = 10) spectra of toluene, aniline, naphthalene, acenaphthene, pyrene, and anthracene are obtained. Excitation of a mixture of the six compunds at three wavelengths (249, 266, and 355 nm) produce fluorescence emission spectra identifying some compounds. The synchronous spectrum (Δλ = 10) of the same mixture is presented and assigned based on the synchronous bands of the individual compounds. The synchronous fluorescence technique and the computer simulation method are proposed to complement other techniques in the analysis of fluorescent samples from comets, as well as in missions to planets and satellites of the solar system.

Narcotic Drug Detection and Identification through Synchronous Fluorescence Technique

Illegal production of potentially hazardous narcotics as well as various psychotropic drugs is being carried out in the neighbouring countries and international market which is causing narco-terrorism a global havoc. Compared with traditional/natural drugs the synthetic drugs are more smuggled nowadays. Because of the vulnerability of the borders to drug trafficking, India has increased surveillance at borders and coasts and thus has tackled the problem to some extent. Authors developing a Narcotic drug sensor (point sensor) for detection and identification of Narcotic drugs based on Laser Induced Fluorescence more specifically Synchronous Fluorescence Spectroscopy in an effort. Some narcotic samples viz. Caffeine, Quinine and Tramadol were considered for the study. The fluorescence spectrum was studied using our own laboratory-based sensor incorporating charge coupled device-based Spectrometer and Laser source (266 nm wavelength) and commercial system from ‘Horiba Scientific’ incorporating Photomultiplier tube and Xe lamp source.

Deciphering binding mechanism between bovine serum albumin and new pyrazoline compound K4

The binding mechanism of a new and possible drug candidate pyrazoline derivative compound K4 and bovine serum albumin (BSA) was investigated in buffer solution (pH 7.4) using ultraviolet-visible light absorption and steady-state and synchronous fluorescence techniques. The fluorescence intensity of BSA was quenched in the presence of K4. The quenching process between BSA and K4 was examined at four different temperatures. Decrease of the quenching constants calculated using the Stern-Volmer equation and at increasing temperature suggested that the interaction BSA-K4 was realized through a static quenching mechanism. Synchronous fluorescence measurements suggested that K4 bounded to BSA at the tryptophan region. Fourier transform infrared spectroscopy results showed that there was no significant change in polarity around the tryptophan residue The forces responsible for the BSA-K4 interaction were examined using thermodynamic parameters. In this study, the calculated negative value of ΔG, the negative value of ΔH and the positive value of ΔS pointed to the interaction being through spontaneous and electrostatic interactions that were dominant for our cases. This study provides a very useful in vitro model to researchers by mimicking in vivo conditions to estimate interactions between a possible drug candidate or a drug and body proteins.

Multispectroscopic and molecular modeling studies on the interaction of bile acids with bovine serum albumin (BSA)

Bile acids (BAs) as the main components in two Traditional Chinese Medicine (TCM) of Calculus bovis (niu huang) and Bear bile (xiong dan) have been used for curing hepatic and biliary disorders for decades. However, their severe side effects as injections dramatically hampered their medical development. In this study, the interaction of BAs with bovine serum albumin (BSA) was investigated through multispectroscopic and molecular docking techniques to primarily reveal the physiological effects of BAs at the molecular level. The Stern-Volmer analysis of fluorescence quenching data showed the presence of static quenching mechanism. Thermodynamic parameters of this interaction revealed that the predominant intermolecular force between BAs and BSA was the hydrogen binding or Vander walls interactions. The number of binding sites was calculated by Langmuir adsorption model of single molecule. The combination of BAs-BSA was moderate intensity because the binding constant KLB was lower than the combination of other drugs with BSA. This indicated the safety of BAs. Furthermore, the results from synchronous fluorescence technique and Fourier transform infrared spectroscopy represented that BAs had little effects on the structure of BSA. The diverse binding sites for various BAs with the protein BSA were substantiated by molecular docking studies. These results can contribute to the development and renovation of BAs drugs through revealing the interaction between BAs and BSA.

Molecular Interaction Kinetics and Mechanism Study of Phytohormones and Plant Protein with Fluorescence and Synchronous Fluorescence Techniques

Molecular Interaction Kinetics and Mechanism Study of Phytohormones and Plant Protein with Fluorescence and Synchronous Fluorescence Techniques

Aggregation induced emission enhancement (AIEE) characteristics of quinoline based compound — A versatile fluorescent probe for pH, Fe(III) ion, BSA binding and optical cell imaging

Novel benzimidazoquinoline derivative (AVT) was synthesized through a substitution reaction and characterized by various spectral techniques. Analyzing the optical properties of AVT under absorption and emission spectral studies in different environments exclusively with respect to solvents and pH, intriguing characteristics viz. aggregation induced emission enhancement (AIEE) in the THF solvent and ‘On-Off’ pH sensing were found at neutral pH. Sensing nature of AVT with diverse metal ions and bovine serum albumin (BSA) was also studied. Among the metal ions, Fe3+ ion alone tunes the fluorescence intensity of AVT probe in aqueous medium from “turn-on” to “turn-off” through ligand (probe) to metal charge transfer (LMCT) mechanism. The probe AVT in aqueous medium interacts strongly with BSA due to Fluorescence Resonance Energy Transfer (FRET) and the conformational change in BSA was further analyzed using synchronous fluorescence techniques. Docking study of AVT with BSA reveals that the active site of binding is tryptophan residue which is also supported by the experimental results. Interestingly, fluorescent AVT probe in cells was examined through cellular imaging studies using BT-549 and MDA-MB-231 cells. Thus, the single molecule probe based detection of multiple species and stimuli were described.

Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV–vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

Synthesis, crystal structure, stability studies, DNA/albumin interactions, and antimicrobial activities of two Cu(II) complexes with amino acids and 5-nitro-1,10-phenanthroline

One-dimensional (1-D) coordination polymer and mononuclear copper(II) complexes, ([Cu(nphen)(asn)]ClO4)n (1) and [Cu(nphen)(gln)(H2O)]ClO4·H2O (2) (nphen = 5-nitro-110-phenanthroline, asn = asparagine, gln = glutamine), have been synthesized and characterized by IR spectroscopy, ESI-MS, CHN analysis, and single-crystal X-ray diffraction. These binary and ternary complexes of copper(II) with nphen, asn, and gln have been investigated using potentiometric methods in 0.1 M KCl aqueous ionic media at 298.2 K. The protonation constants of the ligands and the stability constants of 1 and 2 have been calculated from the potentiometric data using the “BEST” software package. The potentiometric results have been analyzed using the “SPE” software package, and the distribution curves for the copper-containing species have been determined for the ternary systems. The CT-DNA-binding properties of these complexes have been investigated by thermal denaturation measurements and both absorption and emission spectroscopy. Further, the interaction of these complexes with bovine serum albumin (BSA) and human serum albumin (HSA) has been investigated using absorption and emission spectroscopy. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) were calculated by the van’t Hoff equation and discussed. The distances between the serum albumins and 1 and 2 have been obtained according to fluorescence resonance energy transfer (FRET). Conformational changes of serum albumins have been observed from synchronous fluorescence technique. The antimicrobial activity of the complexes has also been tested on some bacteria. The effect of different amino acids on the copper(II) complexes are discussed.

Screening of Antioxidant Properties of the Apple Juice Using the Front-Face Synchronous Fluorescence and Chemometrics

Fluorescence spectroscopy is gaining increasing attention in food analysis due to its higher sensitivity and selectivity as compared to other spectroscopic techniques. Synchronous scanning fluorescence technique is particularly useful in studies of multi-fluorophoric food samples, providing a further improvement of selectivity by reduction in the spectral overlapping and suppressing light-scattering interferences. Presently, we study the feasibility of the prediction of the total phenolics, flavonoids, and antioxidant capacity using front-face synchronous fluorescence spectra of apple juices. Commercial apple juices from different product ranges were studied. Principal component analysis (PCA) applied to the unfolded synchronous fluorescence spectra was used to compare the fluorescence of the entire sample set. The regression analysis was performed using partial least squares (PLS1 and PLS2) methods on the unfolded total synchronous and on the single-offset synchronous fluorescence spectra. The best calibration models for all of the studied parameters were obtained using the PLS1 method for the single-offset synchronous spectra. The models for the prediction of the total flavonoid content had the best performance; the optimal model was obtained for the analysis of the synchronous fluorescence spectra at Δλ = 110 nm (R2 = 0.870, residual predictive deviation (RPD) = 2.7). The optimal calibration models for the prediction of the total phenolic content (Δλ = 80 nm, R2 = 0.766, RPD = 2.0) and the total antioxidant capacity (Δλ = 70 nm, R2 = 0.787, RPD = 2.1) had only an approximate predictive ability. These results demonstrate that synchronous fluorescence could be a useful tool in fast semi-quantitative screening for the antioxidant properties of the apple juices.

Synthesis and crystal structures of novel copper(II) complexes with glycine and substituted phenanthrolines: reactivity towards DNA/BSA and in vitro cytotoxic and antimicrobial evaluation.

New copper(II) complexes-dimeric-[Cu(nphen)(gly)(H2O)]+ (1) and [Cu(dmphen)(gly)(NO3)(H2O)] (2) (nphen=5-nitro-1,10-phenanthroline, dmphen=4,7-dimethyl-1,10-phenanthroline, and gly=glycine)-have been synthesized and characterized by CHN analysis, single-crystal X-ray diffraction techniques, FTIR, EPR spectroscopy, and cyclic voltammetry. The CT-DNA-binding properties of these complexes have been investigated by thermal denaturation measurements and both absorption and emission spectroscopy. The DNA cleavage activity of these complexes has been studied on supercoiled pUC19 plasmid DNA by gel electrophoresis experiments in the absence and presence of H2O2. Furthermore, the interaction of these complexes with bovine serum albumin (BSA) has been investigated using absorption and emission spectroscopy. The thermodynamic parameters, free-energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) for BSA+complexes 1 and 2 systems have been calculated by the van't Hoff equation at three different temperatures (293.2, 303.2, and 310.2K). The distance between the BSA and these complexes has been determined using fluorescence resonance energy transfer (FRET). Conformational changes of BSA have been observed using the synchronous fluorescence technique. In addition, in vitro cytotoxicities of these complexes on tumor cell lines (Caco-2, A549, and MCF-7) and healthy cells (BEAS-2B) have been examined. The antimicrobial activity of the complexes has also been tested on certain bacteria cells. The effect of mono and dimeric in the above complexes is presented and discussed. New copper(II) complexes-dimeric-[Cu(nphen)(gly)(H2O)]+ (1) and [Cu(dmphen)(gly) (NO3)(H2O)] (2) (nphen = 5-nitro-1,10-phenanthroline, dmphen = 4,7-dimethyl-1,10-phenanthroline and gly = glycine)-have been synthesized and characterized by CHN analysis, single-crystal X-ray diffraction techniques, FTIR and EPR spectroscopy. They have been tested for their in vitro DNA/BSA interactions by the spectroscopic methods. These complexes exhibited higher cytotoxic and antimicrobial activities. Complex 1 shows better DNA / BSA interactions in comparison to complex 2.

Binding interaction of phosphorus heterocycles with bovine serum albumin: A biochemical study

Interaction between bovine serum albumin (BSA) and phosphorus heterocycles (PHs) was studied using multi-spectroscopic techniques. The results indicated the high binding affinity of PHs to BSA as it quenches the intrinsic fluorescence of BSA. The experimental data suggested the fluorescence quenching mechanism between PHs and BSA as a dynamic quenching. From the UV–vis studies, the apparent association constant (Kapp) was found to be 9.25×102, 1.27×104 and 9.01×102L/mol for the interaction of BSA with PH-1, PH-2 and PH-3 respectively. According to the Förster's non-radiation energy transfer (FRET) theory, the binding distances between BSA and PHs were calculated. The binding distances (r) of PH-1, PH-2 and PH-3 were found to be 2.86, 3.03, and 5.12nm, respectively, indicating energy transfer occurs between BSA and PHs. The binding constants of the PHs obtained from the fluorescence quenching data were found to be decreased with increase of temperature. The negative values of the thermodynamic parameters ΔH, ΔS and ΔG at different temperatures revealed that the binding process is spontaneous; hydrogen bonds and van der Waals interaction were the main force to stabilize the complex. The microenvironment of the protein-binding site was studied by synchronous fluorescence and circular dichroism (CD) techniques and data indicated that the conformation of BSA changed in the presence of PHs. Finally, we studied the BSA-PHs docking using Autodock and results suggest that PHs is located in the cleft between the domains of BSA.

Conjugation of cytochrome c with ferrocene-terminated hyperbranched polymer and its influence on protein structure, conformation and function

Interaction mechanism of a new hyperbranched polyurethane-based ferrocene (HPU-Fc) with cytochrome c (cyt c) and cyt c structure and conformation change induced by HPU-Fc were investigated using cyclic voltammogram(CV), differential pulse voltammetry (DPV), circular dichroism (CD), fluorescence, synchronous fluorescence and absorbance spectroscopy technique. The peroxidase activity of cyt c in the presence of HPU-Fc was also studied. The structure and conformation of protein are relatively stable at moderate concentration of HPU-Fc without obvious perturbation of the heme pocket and significant changes in protein secondary structure. Conjugation of cyt c with excessive HPU-Fc (over about 3 times of cyt c) slightly changed the α-helix structure in protein, disturbed the microenvironment around heme as well as away from the heme crevice, which caused the changes of the electrochemical behavior and the absorption spectra. Reasonable amount of HPU-Fc has no significant influence on the protein enzymatic activity, while excess HPU-Fc may cause a conformation not suitable for H2O2 activation and guaiacol oxidation. The interaction of HPU-Fc with cyt c and the conservation of protein function at suitable HPU-Fc amount make prepared complex promising for the synergistic anticancer therapy.

Application of Fluorescence Spectrometry With Multivariate Calibration to the Enantiomeric Recognition of Fluoxetine in Pharmaceutical Preparations.

Fluoxetine is the most prescribed antidepressant chiral drug worldwide. Its enantiomers have a different duration of serotonin inhibition. A novel simple and rapid method for determination of the enantiomeric composition of fluoxetine in pharmaceutical pills is presented. Specifically, emission, excitation, and synchronous fluorescence techniques were employed to obtain the spectral data, which with multivariate calibration methods, namely, principal component regression (PCR) and partial least square (PLS), were investigated. The chiral recognition of fluoxetine enantiomers in the presence of β-cyclodextrin was based on diastereomeric complexes. The results of the multivariate calibration modeling indicated good prediction abilities. The obtained results for tablets were compared with those from chiral HPLC and no significant differences are shown by Fisher's (F) test and Student's t-test. The smallest residuals between reference or nominal values and predicted values were achieved by multivariate calibration of synchronous fluorescence spectral data. This conclusion is supported by calculated values of the figure of merit.

Human hemoglobin structural and functional alterations and heme degradation upon interaction with benzene: A spectroscopic study

Here, the effect of benzene on hemoglobin structure, stability and heme prosthetic group integrity was studied by different methods. These included UV–vis absorption spectrophotometry, normal and synchronous fluorescence techniques, and differential scanning calorimetry (DSC). Our results indicated that benzene has high hemolytic potential even at low concentrations. The UV–vis spectroscopic results demonstrated that benzene altered both the globin chain and the heme prosthetic group of hemoglobin increasing met- and deoxy-Hb, while decreasing oxy-Hb. However, with increasing benzene the concentration of all species decreased due to heme destruction. The spectrophotometric results show that benzene has a high potential for penetrating the hydrophobic pocket of hemoglobin. These results were consistent with the molecular docking simulation results of benzene-hHb. Aggregation and thermal denaturation studies show that the increased benzene concentration induced hemoglobin aggregation with a decrease in stability, which is consistent with the DSC results. Conventional fluorescence spectroscopy revealed that the heme degradation species were produced in the presence of benzene. The results of constant wavelength synchronous fluorescence spectroscopy (CWSFS) indicated that at least five heme-degraded species were produced. Together, our results indicated that benzene has adverse effects on hemoglobin structure and function, and heme degradation.

Determination of human albumin in serum and urine samples by constant-energy synchronous fluorescence method.

A sensitive spectrofluorimetric method using constant-energy synchronous fluorescence technique is proposed for the determination of human albumin without separation. In this method, no reagent was used for enhancement of the fluorescence signal of albumin in the solution. Effects of some parameters, such as energy difference between excitation and emission monochromators (ΔE), emission and excitation slit widths and scan rate of wavelength were studied and the optimum conditions were established. For this purpose factorial design and response surface method were employed for optimization of the effective parameters on the fluorescence signal. The results showed that the scan rate of the wavelength has no significant effect on the analytical signal. The calibration curve was linear in the range 0.1-220.0 µg mL(-1) of albumin with a detection limit of 7.0 × 10(-3) µg mL(-1). The relative standard deviations (RSD) for six replicate measurements of albumin were calculated as 2.2%, 1.7% and 1.3% for 0.5, 10.0 and 100.0 µg mL(-1) albumin, respectively. Furthermore the proposed method has been employed for the determination of albumin in human serum and urine samples.