Nanochemistry offers stimulating opportunities for a wide variety of applications in the biosciences. Understanding of
the interaction of nanoparticles with biomolecules such as proteins is very important as it can help better design and
fabricate nanocomposites for applications in diagnostics, drug delivery, and cell monitoring. In this work, the interaction
of Bovine Serum Albumin (BSA) and two types of metal oxide nanoparticles (titanium and tin) have been studied using
the intrinsic fluorescence of tryptophan residue from the proteins measured by steady state and time resolved
fluorescence techniques. The nanoparticles which were fabricated using a novel synthetic process have average sizes of
~2 nm (SnO2) and ~6 nm (estimated for TiO2) and have very high solubilities in a variety of solvents. The Stern-Volmer
plots indicate an effective quenching process by TiO2 nanoparticles whereas SnO2 nanoparticles have a lower quenching efficiency for BSA fluorescence. Static quenching is the major contribution in the overall process which may indicate a
high degree of association between protein and nanoparticles. The difference in BSA fluorescence quenching efficiency
between the two types of nanoparticles can be explained by the non-covalent interaction differences and the thermal
stability of protein-nanoparticle associated species for both materials.
KEYWORDS: Luminescence, Energy transfer, Microfluidics, Time resolved spectroscopy, Chemical analysis, Nondestructive evaluation, Statistical analysis, Fluorescence spectroscopy, Quantitative analysis, Time metrology
Hydrocarbon-bearing fluid inclusions (HCFI) are microscopic cavities within rocks that are filled with petroleum oil, the composition of which may not have changed since the trapping event. Thus, the composition of that entrapped oil can provide information about the formation and evolution of the oil reservoir. This type of information is important to the petroleum production and exploration industries. Crude oil fluorescence originates from the presence of cyclic aromatic compounds and the nature of the emission is governed by the chemical composition of the oil. Fluorescence based methods are widely used for analysis of crude oil because they offer robust, non-contact and non-destructive measurement options. The goal of our group is the development of a non-destructive analytical method for HCFI using time-resolved fluorescence methods. In broad terms, crude oil fluorescence behavior is governed by the concentration of quenching species and the distribution of fluorophores. For the intensity averaged fluorescence lifetime, the best correlations have been found between polar or alkane concentrations, but these are not suitable for robust, quantitative analysis. We have recently started to investigate another approach for characterizing oils by looking at Time-resolved Emission Spectra (TRES). TRES are constructed from intensities sampled at discrete times during the fluorescence decay of the sample. In this study, TRES, from a series of 10 crude oils from the Middle East, have been measured at discrete time gates (0.5 ns, 1 ns, 2 ns, 4 ns) over the 450-700 nm wavelength range. The spectral changes in TRES, such as time gate dependent Stokes' shift and spectral broadening, are analyzed in the context of energy transfer rates. In this work, the efficacy of using TRES for fingerprinting individual oils and HCFI is also demonstrated.
The unambiguous identification and quantification of hazardous materials is of increasing importance in many sectors such as waste disposal, pharmaceutical manufacturing, and environmental protection. One particular problem in waste disposal and chemical manufacturing is the identification of solvents into chlorinated or non-chlorinated. In this work we have used Raman spectroscopy as the basis for a discrimination and quantification method for chlorinated solvents. Raman spectra of an extensive collection of solvent mixtures (200+) were collected using a JY-Horiba LabRam, infinity with a 488 nm excitation source. The solvent mixtures comprised of several chlorinated solvents: dichloromethane, chloroform, and 1,1,1-trichloroethane, mixed with solvents such as toluene, cyclohexane and/or acetone. The spectra were then analysed using a variety of chemometric techniques (Principal Component Analysis and Principal Component Regression) and machine learning (Neural Networks and Genetic Programming). In each case models were developed to identify the presence of chlorinated solvents in mixtures at levels of ~5%, to identify the type of chlorinated solvent and then to accurately quantify the amount of chlorinated solvent.
The quantitative analysis of illicit materials using Raman spectroscopy is of widespread interest for law enforcement and healthcare applications. One of the difficulties faced when analysing illicit mixtures is the fact that the narcotic can be mixed with many different cutting agents. This obviously complicates the development of quantitative analytical methods. In this work we demonstrate some preliminary efforts to try and account for the wide variety of potential cutting agents, by discrimination between the target substance and a wide range of excipients. Near-infrared Raman spectroscopy (785 nm excitation) was employed to analyse 217 samples, a number of them consisting of a target analyte (acetaminophen) mixed with excipients of different concentrations by weight. The excipients used were sugars (maltose, glucose, lactose, sorbitol), inorganic materials (talcum powder, sodium bicarbonate, magnesium sulphate), and food products (caffeine, flour). The spectral data collected was subjected to a number of pre-treatment statistical methods including first derivative and normalisation transformations, to make the data more suitable for analysis. Various methods were then used to discriminate the target analytes, these included Principal Component Analysis (PCA), Principal Component Regression (PCR) and Support Vector Machines.
The use of designed polymer coatings for specific applications such as drug delivery or modifying cell response is a critical aspect of medical device manufacturing. The chemical composition and physical characteristics of thin polymer coatings need to be analysed in-situ and this can present difficulties for traditional analytical methods. For example, changes in the polarity of polymer coatings are typically measured using the contact angle (CA) method. This is a simple process and gives good results however; it cannot be used to measure very hydrophilic polymers, or to analyse features smaller than a couple of mm in size. There is a need for a non-contact method for polarity measurement that is suitable for hydrophilic polymers on a macro- and microscopic scale. 4'-diethylamino-3-hydroxyflavone (FE), 5, 6-benzo-4'-diethylamino-3-hydroxyflavone (BFE), and 4'-diethylamino-3-hydroxy-7-methoxyflavone (MFE) are fluorescence probes based on 3-hydroxyflavone. They respond to environment perturbations by shift and changes in the relative intensity of two well-separated bands in the emission spectra. These bands originate from an excited state intramolecular proton transfer (ESIPT) reaction. We have incorporated FE, BFE, and MFE into a novel thermoresponsive hydrophilic/hydrophobic copolymer system (NIPAM-NtBA) and studied its fluorescence behaviour. The fluorescence emission spectra depend strongly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (IN*/IT*). This allows for the non-contact, measurement of the exact composition and surface energy of the copolymer system.
The use of fluorescence for cancer detection is currently under investigation. Presently, steady-state fluorescence detection methods are in use, but have limitations due to poor contrast between the fluorescence of the tumor and background autofluorescence. Improved contrast can be obtained with time-resolved techniques because of the differing lifetimes between autofluorescence and exogenous photosensitizers that selectively accumulate within tumor tissue. An imaging system is constructed using a fast-gated (200-ps) charge-coupled device (CCD) camera and a pulsed 635-nm laser diode. To characterize the ability of the system to transfer object contrast to an image, the modulation transfer function (MTF) of the system is acquired by employing an extended knife-edge technique. A knife-edge target is assembled by drilling a rectangular well into a block of polymethyl methacrylate (PMMA). The imaging system records images of the photosensitizer, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS), within the well. AlPcTS was chosen to test the system because of its strong absorption of 635-nm, high fluorescence yield, and relatively long fluorescence lifetime (~7.5 ns). The results show that the system is capable of resolving 10–4 M AlPcTS fluorescence as small as 1 mm. The findings of this study contribute to the development of a time-gated imaging system using fluorescence lifetimes.
Fluorescence based methods are increasingly being used for the analysis of crude oils because they offer high speed, low cost, non-contact, and non-destructive testing options. The fluorescence of crude oils is due to the presence of a wide range of cyclic aromatic compounds, the intensity and temporal behaviour of this fluorescence is directly related to chemical composition. In particular, we have found that the Fluorescence Lifetime (FL) measured at a range of different emission wavelengths is correlated with the concentrations of the aromatic and polar fractions of petroleum oils, and the density (API gravity). We have analysed 22 different crude oils from around the world with API gravities of between 10 to 50. The fluorescence steady-state emission spectra and fluorescence lifetimes at a range of emission wavelengths were measured for 380 nm excitation. It was found that the correlations between the chemical and physical characteristics of the crude oils and the measured fluorescence parameters are highly non-linear. Furthermore, there is a wide degree of scatter in the observed data for medium oils, which have similar physical properties but widely varying chemical compositions. We discuss these findings in the context of developing quantitative methods of analysis for crude petroleum oils based on fluorescence lifetime measurements.
The automated identification and quantification of illicit materials using Raman spectroscopy is of significant importance for law enforcement agencies. This paper explores the use of Machine Learning (ML) methods in comparison with standard statistical regression techniques for developing automated identification methods. In this work, the ML task is broken into two sub-tasks, data reduction and prediction. In well-conditioned data, the number of samples should be much larger than the number of attributes per sample, to limit the degrees of freedom in predictive models. In this spectroscopy data, the opposite is normally true. Predictive models based on such data have a high number of degrees of freedom, which increases the risk of models over-fitting to the sample data and having poor predictive power. In the work described here, an approach to data reduction based on Genetic Algorithms is described. For the prediction sub-task, the objective is to estimate the concentration of a component in a mixture, based on its Raman spectrum and the known concentrations of previously seen mixtures. Here, Neural Networks and k-Nearest Neighbours are used for prediction. Preliminary results are presented for the problem of estimating the concentration of cocaine in solid mixtures, and compared with previously published results in which statistical analysis of the same dataset was performed. Finally, this paper demonstrates how more accurate results may be achieved by using an ensemble of prediction techniques.
Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences. Fluorescence lifetime based pH sensing is potentially more useful than intensity based methods because of the reduced sensitivity to excitation source intensity variations, scattering effects, and photobleaching. In this work, we investigate the variation of fluorescence lifetime with pH for resorufin. The intensity averaged lifetime (τ) of resorufin sodium salt in 0.1M phosphate buffer shows an increase of > 3 ns over the 2 -10 pH range, with 90% of the signal change occurring between pH 4 and 8. The fluorescence is not quenched by chloride or oxygen and was unaffected by the ionic strength of the buffer. Resorufin is relatively insoluble in non-alkaline phosphate buffered solutions, but was estimated to increase by ~2 ns between pH 6 and 8. Resorufin and its sodium salt were both incorporated into sol-gels by either acid or base hydrolysis of tetra-methoxysilane (TMOS). Various surfactants were also added to the sol-gels in an attempt to optimise the fluorescence properties and pH sensitivity of the dyes, and to prevent cracking. The sols were then cast from petri-dishes or dip-coated onto acrylic and glass slides. The dyes retained their pH sensitivity, with showing an increase of approximately 2 ns over the pH range 6 - 8. However, leaching of the dye is observed at higher pH and attempt to minimise dye leaching and sol-gel cracking, poly(vinyl alcohol) (PVA) was cross-linked to the silica gel to form a more flexible matrix.
Phthalocyanine derivatives are currently under investigation for use in Photodynamic Therapy, which is a promising treatment for cancer. These materials, which display preferential uptake in cancerous cells, also exhibit high fluorescence yields, and can be used for tumour detection. Problems with steady-state fluorescence techniques such as background autofluorescence can be eliminated by the use of time-resolved techniques. Improved contrast can be obtained with time-resolved techniques because of the differing lifetimes between endogenous and exogenous photosensitisers. An imaging system was constructed using a fast (200 psec) gated CCD camera and a pulsed 635 nm laser diode. A tissue phantom was assembled to test the system by drilling thirty-six wells of varying diameter and depth (10 mm to 1 mm) into a block of polymethyl methacrylate (PMMA). The system was used to record images of chloroaluminum phthalocyanine tetrasulfonate within the wells at differing concentrations in phosphate buffer. A mixture of 1) Intralipid to mimic tissue scatter, 2) Evans blue to mimic tissue absorption, and 3) zinc phthalocyanine tetrasulfonate to mimic healthy tissue autofluorescence of varying depth was placed on top of the PMMA block. These results contribute to the precision of a time-gated imaging system to image living organisms using fluorescence lifetimes.
Porphycenes are currently under investigation for use in Photodynamic therapy, which is a promising treatment for cancer. These materials, which display preferential uptake in cancerous cells, also exhibit high fluorescence yields, and can be used for tumour detection. Problems with steady-state fluorescence techniques such as background autofluorescence can be eliminated by the use of time-resolved techniques. Improved contrast can be obtained with time-resolved techniques because of the differing fluorescence lifetimes between autofluorescence and longer-living exogenous photosensitisers. An imaging system was constructed using a fast (200 ps) gated CCD camera and a pulsed 635 nm laser diode. A tissue phantom composed of polymethyl methacrylate (PMMA) with thirty-six wells of varying diameter and depth (10 mm to 1 mm) was assembled to test the system. The system was used to record images of a porphycene derivative within the wells at differing concentrations in an organic solvent. A tissue imitator was placed on top of the PMMA block at varying thickness. 10-4 M zinc phthalocyanine tetrasulfonate was also placed on top of the block to mimic autofluorescence. The results indicate that the time-gated imaging system can prevent background excitation scatter and fluorescence from a shorter-lived fluorophore from distorting the fluorescence signal from a longer-lived photosensitiser.
Various chemicals used for photodynamic therapy, a promising treatment for cancer, exhibit high fluorescence yields, which can be used for tumour detection. For a chosen few such compounds fluorescence lifetimes, absorption, and steady state fluorescence spectra were measured at different concentrations (ranging from 10-5 M to 10-8 M) in a variety of solvents. The lifetimes were repeated at three different concentrations (ranging from 10-5 M to 10-6 M) in suspensions of 3T3 fibroblast cells.
Advances in the design and miniaturization of the lasers and electronics required for Time Correlated Single Photon Counting (TCSPC) measurement of fluorescence lifetime have simplified the use of the time domain method. We have assembled a compact portable system that is capable of measuring lifetimes down to approximately 200 ps (with deconvolution) and that can operate at a range of excitation and emission wavelengths. The excitation sources are pulsed LEDs and laser diodes with a maximum pulse rate of 40 MHz and are easily interchanged. Furthermore, the development of violet and blue GaN LEDs and laser diodes is expanding the range of fluorophores available for fluorescence lifetime measurement of ion concentrations. pH sensitive fluorophores have a wide range of biological and clinical applications. The use of fluorescence lifetime rather than intensity to measure pH has a number of advantages including the reduction of effects due to the photobleaching, scattering, and intensity variations in the excitation source. Using our compact TCSPC instrumentation we have measured the dependence of fluorescence lifetimes on pH for a range of dyes in phosphate buffer over the physiologically important range of 6.0 to 8.0. Most dyes exhibit only a small variation in lifetime (<1.0ns) over the 6.0 to 8.0 pH range; however, acridine exhibits a large variation in lifetime and hence shows promise as a pH indicator.
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