Two-dimensional Fluorescence Correlation Research Articles

Theoretical analysis of the resonance fluorescence correlation spectrum of three-level multi-mer 85Rb atomic system

In a vapor cell, [Formula: see text] is the average velocity of the moving atoms. When [Formula: see text], the in-homogeneous broadening of the atomic spectral lines is important when taking into account thermal Doppler broadening. We also discover that the narrowing mechanism for inter-atomic with different components has more influence on the Doppler line-shape. Thus, the spectral line broadening of two-level and three-level multi-mer [Formula: see text] atom is not only influenced by the in-homogeneous Doppler broadening, but also affected by the Dicke narrowing effect. The fluorescence spectrum affords a good possibility for a detailed study of Dicke narrowing effect and thermal Doppler broadening effect. We analyze the influence of multi-mer [Formula: see text] atomic system component on resonance fluorescence spectrum (RFS). Based on the quantum regression theorem, we develop a method and derive a general analytical expression for the two-dimensional (2D) fluorescence correlation spectrum of three-level [Formula: see text] atomic system.

Separation of the overlapped humic acid and BGP characteristic peaks using two-dimensional correlation fluorescence-UV-vis spectroscopy

Fluorescence spectroscopy has been widely used to detect polycyclic aromatic hydrocarbons (PAHs) in the environment. However, the interference of coexisting humic acids (HA) in the environment poses a great challenge to the qualitative and quantitative detection of PAHs using fluorescence spectroscopy. In this study, the spectral properties of benzo [ghi] perylene (BGP) and HA were investigated based on fluorescence and UV–vis spectroscopy combined with two-dimensional (2D) correlation analysis. Under the external disturbance of HA concentration, the homo-2D (fluorescence, UV–visible) correlation and hetero-2D fluorescence-UV–visible correlation spectral characteristics of the mixed samples of HA and BGP were studied, and the effect of HA on the fluorescence of BGP was investigated. It can be inferred that the fluorescence peak at 478 nm come from BGP, and the fluorescence peaks at 442 nm and 533 nm, UV absorption peak at 233 nm come from HA. Meanwhile, asynchronous two-trace two-dimensional (2T2D) fluorescence correlation slice spectra at 533 nm were obtained. The slice spectral intensity at 478 nm was extracted to quantify the BGP concentration in mixture. The results showed that the slice spectral intensity and BGP concentration had a good linear relationship with the coefficient of determination R2 = 0.96. This research provides a way to further study the separation method of HA and PAHs or explore the correction method of the effect of HA on PAHs.

Differentiation of ethanol–water clusters in Fenjiu by two-dimensional correlation fluorescence and Raman spectra

The taste of different flavor liquor is multifarious, but the same brand liquor with different quality is truth. The essence of supramolecular ethanol–water clusters and their intrinsic structural differences in three kinds of Fenjiu are studied by two-dimensional correlation spectra (2D-COS) of fluorescence and Raman. The 2D-COS of fluorescence reveals the prominent emission peaks of three Fenjiu are apparently different. The central fluorescence peak of Fenjiu (a) is located at 330 nm, corresponding to the cluster of (H2O)m(EtOH)n. In Fenjiu (b), the emission peak appears near 310 nm, while those of Fenjiu (c) appear mainly near 310 and 373 nm, corresponding to the clusters of (H2O)(EtOH)n and (H2O)m(EtOH), respectively. Based on 2D-COS of Raman, the peak of Fenjiu (b) at 3440 cm−1 changes initially, indicating its disorder degree is getting higher with continuous dilution with water. However, along with the dilution of Fenjiu (a) and Fenjiu (c), the peak located near 3200 cm−1 changes in priority, indicating that the degree of association between ethanol and water is high, and the clusters formed there are stable. Therefore, this work provides the combined methods to distinguish different supramolecular sets in Fenjiu, applying liquor differentiation in the future.

New insights into the autofluorescence properties of cellulose/nanocellulose

This work explored the fluorescence properties of nano/cellulose isolated from bleached softwood kraft pulp by TEMPO oxidation. Fluorescence spectra showed that all samples exhibited a typical emission peak at 574 nm due to the probabilistic formation of unsaturated bonds by glycosidic bonds independent of lignin. Increasing the excitation wavelengths (510–530 nm) caused red shift of fluorescence emission peaks (570–585 nm) with unchanged fluorescence intensity. Conversely, changing acid/alkaline conditions led to an increase of fluorescence intensity with no shifting of fluorescence emission peak. This can be attributed to an increase in the polarity of the solution environment but does not cause interaction of functional groups within the system identified by generalized two-dimensional correlation fluorescence spectroscopy. This study provides new insight in applying nano/cellulose with special luminous characteristics in biomedicine area such as multi-color biological imaging and chemical sensing.

Complexing characteristics between Cu(Ⅱ) ions and dissolved organic matter in combined sewer overflows: Implications for the removal of heavy metals by enhanced coagulation

Enhanced coagulation has been widely used in storm tanks to remove heavy metal ions (HMs) from combined sewer overflows (CSOs), but faces challenges on removing the HMs bound to dissolved organic matter (DOM) with small molecular weight (MW). DOM ubiquitously existing in CSOs generally contains a large distribution range of MW, which can significantly impact the MW distribution of HMs by complexing reaction, thereby adding uncertainties for the removal efficiency of coagulation. Therefore, realizing the potential MW distribution of the HMs bound to CSO-DOM is greatly important for cost-effectively removing HMs from CSOs in the coagulation process. This paper presents a comprehensive approach of ultrafiltration, fluorescence quenching titration, excitation−emission matrix parallel factor analysis, complexation model, and two-dimensional correlation fluorescence spectroscopy for exploring the MW-based complexing characteristics between Cu(II) ions and CSO-DOM components. Results show that: (1) Cu(II) ions that bound to the CSO-DOM were mainly distributed in the MW range of <5 kDa, which makes them very difficult to be removed from CSOs by coagulation technique. (2) Concentration effect and molecular composition exerted great impacts on the MW distribution of the Cu(II) ions bound to CSO-DOM. (3) The humic-like component of terrestrial origin with the MW range of 100 kDa∼0.45 μm possessed high binding stability, capacity, and priority with Cu(II) ions, and they could be used at a high concentration to promote the removal efficiency of coagulation for Cu(Ⅱ) ions of CSOs by competitive complexation and inter-molecular bridging.

Gold nanoclusters fluorescence probe for monitoring chloramphenicol and study of two-dimensional correlation fluorescence spectroscopy

Chloramphenicol (CAP) is widely used in veterinary practice, including treatment of aquaculture species and livestock husbandry because of its effective antibiotic active against gram-positive and gram-negative microorganisms. However, research shows that, for human body, it leads to serious adverse reactions and side effects. In order to effectively monitor the occurrence of residues of CAP, herein a simple fluorescence probe of glutathione stabilized gold nanoclusters (GSH-AuNCs) is synthesized and characterized. The sensing platform shows high sensitivity and selectivity toward CAP though a fluorescence quenching response. The newly prepared GSH-AuNCs exhibits a strong red emission band at 615 nm under excitation at 360 nm. Nitro of CAP with the property of strong electron affinity results in the charge transfer from GSH-AuNCs to the analyte, which inhibits radiative relaxations and drops the fluorescence intensity consequently. On the basis of fluorescence response, the linear range and detection limit are 0.1~1.0 µM and 0.023 µM respectively. In addition, two-dimensional fluorescence correlation spectroscopy (2D-FL-DOS) is applied to investigate the detail information about charge transfer process to deeply understand the mechanism of fluorescence response of GSH-AuNCs. The cross-peaks in asynchronous spectrum suggest three factors affect the interaction between GSH-AuNCs and CAP.

Application of two-dimensional correlation fluorescence spectroscopy to detect the presence of trace amount of substances.

Although fluorescence spectroscopy is a highly sensitive method, it is still rather difficult to identify a minor fluorescent component whose fluorescent peak is overlapped and masked by a dominant fluorescent component in a sample solution. Herein, we describe a two-dimensional correlation spectroscopy (2D-COS) approach based on the Kasha's rule to solve the above common problem. We initially suppose that a sample solution contains the major component only, and the spectral behavior of the major component obeys the Kasha's rule. Then, the shapes of emission spectra obtained under excitation lights of different wavelengths remain invariant. Under this condition, the introduction of a minor fluorescent component can be reflected by the changes on the shapes of emission peaks in the series of emission spectra. Moreover, subtle changes, which are difficult to be found in the original spectra, can be clearly visualized as cross peaks in 2D asynchronous spectrum constructed using a series of emission spectra. In addition, we demonstrate that the intensities of cross peaks can be enhanced by changing the sequence of the series of emission spectra. We utilize the approach on an aqueous solution containing eosin Y and a trace amount of bromocresol green. The presence of bromocresol green with the concentration as low as 400nM can be revealed via the cross peaks in the resultant 2D asynchronous spectra. In a preliminary study, we suggest that 2D disrelation spectrum might provide an alternative chance to reveal the presence of small amount bromocresol green.

Detection of pesticide in water using two-dimensional fluorescence correlation spectroscopy and N-way partial least squares.

To determine the concentration of carbaryl and chlorothalonil in water, the potential of two-dimensional (2D) fluorescence correlation spectra with N-way partial least squares (N-PLS) was investigated. A total of 40 mixture solutions of carbaryl and chlorothalonil were prepared and 27 of them were used to form a calibration set. The excitation-emission matrix (EEM) fluorescence spectra of all samples were measured. Under the excitation perturbation, 2D fluorescence correlation spectra of all samples were calculated and formed a 2D correlation spectral matrix. And N-PLS models for determination of carbaryl and chlorothalonil in water were built based on 2D correlation fluorescence spectral matrix and traditional EEM spectral matrix, respectively. Finally, the performances of N-PLS models using both methods were compared. For chlorothalonil, the root mean square error of calibration (RMSEC) were 3.43×10-6gL-1 and 5.08×10-6gL-1, the root mean square errors of prediction (RMSEP) were 5.86×10-6gL-1 and 8.99×10-6gL-1 for 2D correlation spectra and EEM spectra, respectively. For carbaryl, the RMSEC were 5.24×10-7gL-1 and 6.18×10-7, the RMSEP were 9.20×10-7gL-1 and 9.63×10-7gL-1 for 2D correlation spectra and EEM spectra, respectively. From the results of this study, it may conclude that 2D fluorescence correlation spectra is superior over the traditional EEM fluorescence spectra in terms of predictability and can be used as an alternative method for detection of organic pollutants in environment.

Investigation on the effects of sediment resuspension on the binding of colloidal organic matter to copper using fluorescence techniques

Colloidal organic matter (COM), an important component of dissolved organic matter (DOM), plays a significant role in the transport and cycling process of the heavy metals. In this study, COM was fractionated from DOM using 0.2 μm, 100 kDa, and 2 kDa ultrafiltration membranes and the fluorescence spectra of the COM fractions were obtained. Excitation and emission matrix–parallel factor analysis and two-dimensional fluorescence correlation spectroscopy were applied to investigate the effect of sediment resuspension on the heavy metal binding characteristics of COM fractions with different molecular weights. Compared with the DOM fractions, COM exhibited stronger binding affinities and more binding sites for Cu(II), which was attributed to the significant binding effects of the components of COM. Our results suggested that the protein-like components were mainly responsible for binding heavy metals in the high-molecular-weight fraction (>100 kDa), whereas the humic-like components were responsible in the low-molecular-weight fraction (<100 kDa). Furthermore, sediment resuspension significantly influenced the composition and heavy metal binding characteristics of COM. Following resuspension, the binding affinity of COM decreased significantly, which might be attributed to the binding competition from inorganic colloids. Thus, COM plays an important role in the binding and transportation behavior of heavy metals, which is an important consideration in shallow lake ecosystems.

Fluorescence studies on the aggregation behaviors of collagen modified with NHS-activated poly(γ-glutamic acid)

The poly(γ-glutamic acid)-NHS (γ-PGA-NHS) esters were used to endow collagen with both of excellent water-solubility and thermal stability via cross-linking reaction between γ-PGA-NHS and collagen. In the present work, the effect of γ-PGA-NHS on the aggregation of collagen molecules was studied by fluorescence techniques. The fluorescence emission spectra of pyrene in collagen solutions and the intrinsic fluorescence emission spectra of collagen suggested different effects of γ-PGA-NHS on collagen molecules: inhibiting aggregation below critical aggregation concentration (CAC) and promoting aggregation above CAC. The two-dimensional (2D) fluorescence correlation spectra indicated that the intermolecular hydrogen bonding and cross-linking between γ-PGA-NHS and collagen would influence the aggregation of collagen molecules. By the ultra-sensitive differential scanning calorimeter (VP-DSC), it was found that the main denaturational transition temperature (Tm2) of modified collagen increased, while its calorimetric enthalpy changes (ΔH2) decreased compared to those of native collagen, further indicating that the modification of γ-PGA-NHS influenced the aggregation of collagen molecules. The study provide useful information for the utilizing and or the processing of water-soluble collagen in aqueous solution in the fields such as cosmetics, health care products, tissue engineering and biomedical materials, etc.

Feasibility of the simultaneous determination of polycyclic aromatic hydrocarbons based on two-dimensional fluorescence correlation spectroscopy

A new approach for quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in environment was proposed based on two-dimensional (2D) fluorescence correlation spectroscopy in conjunction with multivariate method. 40 mixture solutions of anthracene and pyrene were prepared in the laboratory. Excitation-emission matrix (EEM) fluorescence spectra of all samples were collected. And 2D fluorescence correlation spectra were calculated under the excitation perturbation. The N-way partial least squares (N-PLS) models were developed based on 2D fluorescence correlation spectra, showing a root mean square error of calibration (RMSEC) of 3.50μgL−1 and root mean square error of prediction (RMSEP) of 4.42μgL−1 for anthracene and of 3.61μgL−1 and 4.29μgL−1 for pyrene, respectively. Also, the N-PLS models were developed for quantitative analysis of anthracene and pyrene using EEM fluorescence spectra. The RMSEC and RMSEP were 3.97μgL−1 and 4.63μgL−1 for anthracene, 4.46μgL−1 and 4.52μgL−1 for pyrene, respectively. It was found that the N-PLS model using 2D fluorescence correlation spectra could provide better results comparing with EEM fluorescence spectra because of its low RMSEC and RMSEP. The methodology proposed has the potential to be an alternative method for detection of PAHs in environment.

Using fluorescence quenching combined with two-dimensional correlation fluorescence spectroscopy to characterise the binding-site heterogeneity of dissolved organic matter with copper and mercury in lake sediments

Environmental contextDissolved organic matter (DOM) plays an important role in influencing the migration and ultimate fate of metals. Different sources of pollution resulted in changes in the structure of sediment DOM in Lake Wuliangsuhai. We investigate the binding properties of DOM with CuII and HgII using fluorescence quenching combined with two-dimensional correlation spectroscopy (2D-COS) in order to demonstrate the influence of different sources of DOM on metals. AbstractDissolved organic matter (DOM) was collected from three sampling sites (L1, L2 and L3) in Lake Wuliangsuhai. L1 received upstream industrial wastewater and domestic sewage. L2 had suffered from agricultural non-point source pollution. L3 was situated in the lake outlet area. The complexation of DOM with CuII and HgII was investigated based on fluorescence quenching of the synchronous fluorescence spectra on the addition of CuII and on two-dimensional correlation spectroscopy (2D-COS). The synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of CuII and HgII binding sites, which was not readily detected using only the synchronous fluorescence spectra. CuII and HgII complexation was stronger at shorter wavelengths than at longer wavelengths. Moreover, fluorescence quenching also occurred intensely in the fulvic-like regions (363nm for DOM-Cu in L2 and 365nm for DOM-Hg in L1). The logarithms of the stability constants (log KM) ranged from 4.42 to 6.23, from 4.75 to 4.86, and from 4.80 to 5.73 for DOM-Cu in L1, L2 and L3, respectively, depending on the wavelength. DOM at the longer wavelengths exhibited a higher log KM than that at the shorter wavelengths, and the f values in the protein-like region were clearly high. These results suggest that the influence of the structural and chemical properties of DOM on CuII binding may differ for DOM from different sources. The combined approach of fluorescence quenching and 2D-COS could be applied as a tool for evaluating the metal binding site heterogeneity of DOM.

Changes in aggregation behavior of collagen molecules in solution with varying concentrations of acetic acid

A critical aggregation concentration of 0.30–0.50mg/mL was previously obtained for type I collagen at 0.1M acetic acid (AA). In the present study, the aggregation behavior of collagen in solution (0.5mg/mL) in the presence of 0.1–2.0M AA was investigated. Circular dichroism showed that the three helix structure was maintained across the whole AA concentration range. However, the ratio of positive peak intensity over negative peak intensity varied depending on the conformational state of collagen aggregates. Ultra-sensitive differential scanning calorimetry revealed that transition temperatures Tm1 and Tm2 decreased by 8.35°C and 7.80°C, respectively, between 0.1M and 2.0M, indicating a possible relationship between the aggregation state and the thermal effect. The surrounding polarity of collagen molecules in solution containing pyrene was investigated by fluorescence spectroscopy, which demonstrated that disaggregation of collagen aggregates was enhanced with increasing AA concentration. This observation was correlated with changes in collagen fiber size observed by atomic force microscopy. Furthermore, collagen tyrosine residues were blue-shifted in an intrinsic fluorescence spectra, further indicating changes in aggregation behavior with increasing AA concentration. Finally, the dynamic response of collagen molecules to AA was analyzed by two-dimensional correlation fluorescence spectra.

Fluorescence study on the aggregation of collagen molecules in acid solution influenced by hydroxypropyl methylcellulose

The effect of hydroxypropyl methylcellulose (HPMC) on the aggregation of collagen molecules with collagen concentrations of 0.25, 0.5 and 1.0mg/mL was studied by fluorescence techniques. On one hand, both the synchronous fluorescence spectra and fluorescence emission spectra showed that there was no change in the fluorescence intensity of collagen intrinsic fluorescence when 30% HPMC was added, while it decreased obviously when HPMC content ≥50%. From the two-dimensional fluorescence correlation analysis, it was indicated that collagen molecules in 0.25 and 0.5mg/mL collagen solutions were more sensitive to HPMC than those in 1.0mg/mL collagen solution. On the other hand, the pyrene fluorescence and the fluorescence anisotropy measurements indicated that HPMC inhibited the collagen aggregation for 0.25 and 0.5mg/mL collagen, but promoted it for 1.0mg/mL collagen. The atomic force microscopy images further confirmed the effect of HPMC on collagen with different initial states.

Fast two-dimensional fluorescence correlation spectroscopy technique for tea quality detection.

A fast two-dimensional fluorescence correlation spectroscopy technique based on light emitting diodes is developed, which uses light intensity and excitation wavelength as quickly changeable and easily controllable external perturbations. A compact and automatic system is set up to detect tea quality. A partial least square regression method is used to create predictive models for tea grades. Compared to the traditional fluorescence spectroscopy method, this convenient two-dimensional correlation spectroscopy technique is more accurate according to our experimental results and is promising for practical applications.

Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase

Abstract AzoR is a homodimeric, flavin mononucleotide (FMN)-containing, NADH-dependent azoreductase from Escherichia coli . In this paper, we investigated the effect of the concentration of both AzoR and R59G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra. Two cross peaks (530, 490) and (580, 530) were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR, while only one cross peak appeared at (600, 530) for R59G mutant. This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates. The specific activity of both AzoR and R59G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L, which suggested that no significant dissociation of FMN occurred at low concentrations. Additionally, in order to explore the origin of these intermediates, we carried out a 2D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy. The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm, respectively. Taken together, these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.

The response of collagen molecules in acid solution to temperature

The response of collagen molecules in acid solution (0.25, 0.50 and 1.0 mg/mL) to temperature (20–50 °C) was studied by ultra-sensitive differential scanning calorimeter (US-DSC), atomic force microscopy (AFM) and fluorescence spectrophotometer. The enthalpy changes (ΔH) for collagen varied with collagen concentrations, indicating that the aggregation state of collagen molecules was correlated with its thermal effect. Fluorescence measurements of pyrene in collagen solutions, which showed the polarity of the microenvironment in collagen molecules, suggested different behaviors: aggregation (20–32 °C) and disaggregation (38–50 °C), which was manifested by the changes in morphology of collagen fibers as observed by AFM. Furthermore, emission intrinsic fluorescence spectra of collagen at different temperatures showed that the fluorescence intensity of collagen decreased with increasing temperature, and the dynamic response of collagen molecules to temperature was further explored from the corresponding two-dimensional correlation fluorescence spectra.

Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy

The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions.

The thermal behavior of collagen in solution: Effect of glycerol and 2-propanol

The thermal stability of different solutions of collagen (Col), collagen mixed with glycerol (Col-G) and collagen mixed with 2-propanol (Col-P) was studied by differential scanning calorimetry (DSC), viscosity and fluorescence. The DSC and viscosity methods showed that glycerol increased the denaturation temperature of collagen about 2 °C, while 2-propanol decreased it about 2 °C. The values of intrinsic viscosity ([ η]) for Col, Col-G and Col-P were 21.67, 20.20 and 24.71 dl/g, respectively. Huggins coefficient ( k H) increased in the presence of glycerol and decreased in the presence of 2-propanol. It was suggested that glycerol promoted the dissolution of collagen molecular aggregates while 2-propanol enhanced the aggregation. Fluorescence spectra were investigated within the temperature ranging from 15 to 45 °C. By comparing the sign of peaks in the two-dimensional (2D) fluorescence correlation maps, the orders of peak response were ∼360, ∼410 > 297 nm for Col and Col-G, and 297 > ∼360, ∼410 nm for Col-P, respectively. These indicated that the respondences of tyrosine residues, excimer-like species and bityrosine on the perturbation of temperature were different in the presence of glycerol and 2-propanol.

The application of two-dimensional fluorescence correlation spectroscopy on the interaction between bovine serum albumin and prulifloxacin

The interaction between bovine serum albumin (BSA) and prulifloxacin was investigated by ultraviolet spectrophotometer (UV) and fluorescence spectroscopy in this paper. Two-dimensional (2D) correlation spectroscopy was applied to the analysis of fluorescence spectra. The results of spectroscopic measurements suggested that prulifloxacin (PL) have a strong ability to quench the intrinsic fluorescence of bovine serum albumin through static quenching procedure. Thermodynamic parameter enthalpy changes (ΔH) and entropy changes (ΔS) were calculated. Owing to the spectral resolution enhancement in 2D correlation spectroscopy, the structure change of prulifloxacin can be observed.