Two-dimensional Correlation Fluorescence Spectroscopy Research Articles

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.

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.

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.

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 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.

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 spectropho- tometer (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 pa- rameter 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.

Studies on the binding of a carditionic agent to human serum albumin by two-dimensional correlation fluorescence spectroscopy and molecular modeling

The binding of a novel carditionic agent (2-amino-4-chloro- N-(4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)benzamide (ACPB)) to human serum albumin (HSA) under physiological conditions has been investigated by using UV/vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) in combination with protein–ligand docking study. It was observed that there was a prominent interaction between ACPB and HSA. The interaction was also confirmed by two-dimensional (2D) correction analysis based on the quenching perturbation on the fluorescence spectra and the order of the response of ACPB and HSA to the quenching perturbation was also elucidated based on Noda’s rule. Fluorescence data revealed that the fluorescence quenching was a static quenching process and the binding constants were calculated to be 8.781 × 10 5, 7.310 × 10 5, and 5.358 × 10 5 M −1 at 290, 300, and 310 K, respectively. The thermodynamic parameters were calculated according to the Van’t Hoff equation and the binding mode was determined. In addition, the alterations of protein secondary structure were qualitatively and quantitatively determined by the evidence from synchronous fluorescence, CD and FT-IR. Furthermore, docking studies that corroborate our experimental results revealed that the binding sites were located in subdomain IIA of HSA.

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda’s rule.

Two-dimensional correlation fluorescence spectroscopy V: Polarization perturbation as a new technique to induce intensity changes in fluorescence spectra

Dynamic polarization has been introduced as a new perturbation technique to two-dimensional (2D) correlation fluorescence spectroscopy to bring about intensity changes in the fluorescence spectra. This perturbation technique is based on the fact that the fluorescence intensity of a probe depends on its rotational diffusion time, as well as its fluorescence lifetime, when the relative angle between the excitation-side and emission-side polarizer is changed. Fluorescence spectra of the mixture of anthracene and pyrene in glycerol solutions were analyzed as a model system. We obtained several clearly assignable peaks in correlation maps, although they could not be easily assigned in conventional fluorescence spectra due to heavy overlapping of the component spectra. By comparing the sign of peaks in synchronous and asynchronous maps, we could extract information on the order of the response of the peaks to the polarization perturbation. These results strongly demonstrate the usefulness of dynamic polarization perturbation in 2D correlation fluorescence spectroscopy.

Two-dimensional fluorescence correlation spectroscopy IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein–water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.

Two-dimensional fluorescence correlation spectroscopy II: spectral analysis of derivatives of anthracene and pyrene in micellar solutions

Generalized two-dimensional (2D) correlation spectroscopy has been applied to the analysis of fluorescence spectra in two micellar systems: (1) a mixture of pyrene and 1,3,6,8-pyrenetetrasulfonic acid in the cationic micellar solutions of cetyltrimethylammonium chloride (CTAC) and (2) a mixture of pyrene and 9-anthracencepropionic acid in anionic micellar solutions of sodium dodecyl sulfate (SDS). Fluorescence quenching is employed as a perturbation mode for causing intensity changes in fluorescence bands ( quenching perturbation). Iodide ion (I −) is used as a quencher in the former system, and cetyl pridinium chloride (CPC) is used in the latter. Vibronic bands in the complicated fluorescence spectra of the mixture of the analytes were successfully resolved. It is shown that asynchronous maps are especially useful for spectral resolution enhancement when the quenching perturbation is employed in 2D fluorescence correlation spectroscopy. Furthermore, the information about the order of response of the bands to quenching is obtained by comparing the signs of synchronous and asynchronous cross-peaks.

Two-dimensional fluorescence correlation spectroscopy with modulated excitation.

Overlap of multiple states or multiple species in a chemical system often creates a congested fluorescence spectrum that is difficult to interpret. The resolution of component spectra is essential for the understanding of the structure and dynamics of such multicomponent systems. In this paper, two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented for the dissection of component spectra using the time correlation function. In 2D FCS, the time response of fluorescence intensity is collected at various wavelengths upon an external perturbation. The time correlation function is evaluated between wavelengths. A two-dimensional fluorescence correlation spectrum, or a plot of the correlation intensity as a function of two wavelength axes, resolves the overall spectrum into component spectra. The characteristics of the two-dimensional time correlation function are demonstrated in the frequency domain fluorescence spectroscopy in which the sinusoidally modulated excitation provides the external perturbation. Using 2D FCS, fine vibronic structures of the component fluorescence emission spectra were completely resolved from a strongly overlapped one-dimensional mixture spectrum. The existence of multiple microenvironments of a probe molecule in a biological system is evidenced by nonzero asynchronous correlation intensities. The corresponding spectra are retrieved from correlation analysis. Unlike traditional resolution methods in fluorescence spectroscopy based on statistical fitting of fluorescence decays, 2D FCS can resolve species whose fluorescence decays are linked by the rate constants in chemical reactions and species displaying multiexponential decay kinetics.

Dynamic two-dimensional fluorescence correlation spectroscopy. Generalized correlation and experimental factors.

Dynamic two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented in the general form. Dynamic 2D FCS evaluates the time correlation function between two wavelength axes when an external perturbation is applied to the sample. It displays the vibronic features with similar time response functions in the synchronous correlation spectrum and the features with different time responses in the asynchronous correlation spectrum. The correlation analysis allows detailed assignments of the vibronic spectra of multicomponent samples. The emission-emission 2D FCS has proven to be able to resolve spectra with substantial overlaps, of species in equilibrium with each other, and of reacting species whose kinetic constants are linked and multiexponential. Similarly, the correlation analysis between excitation wavelengths allows the assignment of the excitation bands to fluorescent components. When a sinusoidal light source is used to excite the sample, the excitation-emission correlation requires the collection of only four spectra, two in-phase and two quadrature. The two-dimensional excitation-emission correlation analysis uncovers the association between the excitation and the emission vibronic features, enabling the complete assignment of the component spectra. The band associations and spectral assignments are facilitated by the two-dimensional phase map that is constructed from the synchronous and asynchronous correlation spectra. Spectral resolution can be optimized by varying the frequency of excitation and is not influenced by the detector phase angle used to collect the spectra. The resolution power of the 2D FCS is demonstrated with the retrieval of the anthracene emission spectrum from a pyrene-anthracene mixture when it contributes only 4% to the total fluorescence intensity.