Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus

The aim of this study was to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence lifetime of cells in an in vitro cell culture model. Autofluorescence decay functions were measured in 3T3-L1 adipocytes by time-correlated single-photon counting (excitation 370nm, emission 420-480nm). Free NADH had a two-exponential decay but cell autofluorescence fitted best to a three-exponential decay. Addition of 30mM glucose caused a 29% increase in autofluorescence intensity, a significantly shortened mean lifetime (from 7.23 to 6.73ns), and an increase in the relative amplitude and fractional intensity of the short-lifetime component at the expense of the two longer-lifetime components. Similar effects were seen with rotenone, an agent that maximizes mitochondrial NADH. 3T3-L1 fibroblasts stained with the fluorescent mitochondrial marker, rhodamine 123 showed a 16% quenching of fluorescence intensity when exposed to 30mM glucose, and an increase in the relative amplitude and fractional intensity of the short lifetime at the expense of the longer lifetime component. We conclude that, though the effect size is relatively small, glucose can be measured non-invasively in cells by monitoring changes in the lifetimes of cell autofluorescence or of a dye marker of mitochondrial metabolism. Further investigation and development of fluorescence intensity and lifetime sensing is therefore indicated for possible non-invasive metabolic monitoring in human diabetes.     

Determination of the mechanism of photo-ionization of NADH in aqueous solution on laser excitation at 355 nm

The efficiency of photoelectron ejection from the reduced form of nicotinamide adenine dinucleotide (NADH) in aqueous solution of pH 8.6 was studied as a function of the laser energy by measuring the transient e_aq⁻ absorption appearing on nanosecond laser excitation at 355 nm. It was found that this process is due exclusively to consecutive two-photon excitation at the laser energies used. Saturation effects were observed at the higher energies: their origin was discussed. The saturation probably accounts for the results of a recent picosecond study of NADH which seemed to indicate the existence of a monophotonic electron ejection of high quantum yield.     

In vivo monitoring the changes of interstitial pH and FAD/NADH ratio by fluorescence spectroscopy in healing skin wounds

The aim of our study was to evaluate the changes of interstitial pH and flavin adenine dinucleotide (FAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio in healing skin wounds using fluorescence spectroscopy in Sprague Dawley rats. In the experiment, excisional and incisional models of wound healing were used. The florescein as the pH-sensitive probe using excitation spectra (lambda(Em) = 535 nm) was used for the measurement of pH changes, and synchronous fluorescence spectra (Deltalambda = 60 nm) for the monitoring of FAD/NADH ratio changes were measured from the surfaces of healing wounds. Increase of interstitial pH and FAD/NADH ratio was recorded during the time interval from the 15th to the 65th minute after surgery. The decrease of pH between the 48th and the 72nd hour after surgery as well as the increase of FAD/NADH ratio between the 72nd and the 96th hour of wound healing were recorded. The results indicate that the use of fluorescence spectroscopy may be considered as a valuable tool for noninvasive in vivo monitoring of selected redox parameters in the early phases of wound healing.     

Spectroscopy of Na3

The excitation spectrum of Na₃ was systematically investigated from 700 to 330 nm. Four excited states were observed. One of them exhibits fractional quantization of the vibronic pseudorotation which constitutes the first direct verification of the adiabatic sign-change theorem. Photofragmentation is studied by depletion spectroscopy and the 420 nm system is found to be completely predissociated. The structure of the ground state and the ionization potential are also measured.     

Photofragment translational spectroscopy of iodobenzene at 266 nm

The photofragmentation of C_6 H_5I at 266 nm is investigated on the nuiversal crossed molecular beam machine, and the translational spectroseopy as well as the angular distribution of I atom is measured. The results reveal that under the laser intensity of 10~8 W/cm~2 the single-phuton dissociation competes with multi-photon processes. In singlephoton dissociation the anisotropy parameter β is 0.4 and the average translational energy is only 1.04 keal/mol, which indicates that this process is a slow predissociation. In two-photon phutofragmentation the average translational energy is 51.64 kcal/mol, which accounts for about 35% of the available energy. Another photofragmentation channel is even more faster, whose peak in time-of-flight spectra corresponds to four or five photon absorptions. The branching ratio of these three channels is determined to be about 3: 3: 4.     

Design of a dual-signaling sensing system for fluorescent ratiometric detection of Al3+ ion based on the inner-filter effect

A dual-signal sensing system based on the inner-filter effect (IFE) was demonstrated, in which the combination of two signaling mechanisms allows metal binding to turn on two fluorescence emission bands, independently. A proof-of-concept fluorescent ratiometric assay for Al(3+) in pure aqueous solution is presented. The proposed assay is based on the Al(3+)-induced color and fluorescence changes of Alizarin red S (ARS) and IFE between ARS and meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP). In the absence of Al(3+), the absorption spectrum of the ARS in 0.2 M HAc-NaAc buffer (pH 5.5) has a strong peak at 420 nm, significantly overlapping with the excitation of TMPyP. ARS is expected to be capable of functioning as a powerful absorber to tune the emission of TMPyP on account of the spectral overlap. Binding of Al(3+) with ARS forms a fluorometric ARS/Al(3+) complex and shifts the maximum absorbance from 420 nm to 480 nm, which overlaps negligibly with the excitation of TMPyP and turns on the proper emission spectrum for TMPyP. Under the optimum conditions, The fluorescence intensity ratio, F(585)/F(651), responds to Al(3+) over a dynamic range of 0.1-1.5 μM, with a limit of detection of 40 nM, where F(585) and F(651) are the fluorescence intensity at 585 nm and 651 nm in the absence or presence of Al(3+), respectively. Further application in Al(3+)-spiked water samples suggested a recovery between 95 and 108%. The fluorescence response is highly selective for Al(3+) over other metal ions with the addition of thiourea as the masking agent.     

Visible fluorescence spectrometry using second harmonic emission self-induced in semiconductor laser as a light source

Second harmonic emission (416 or 453 nm) self-induced in a nearinfrared semiconductor laser (832 or 906 nm) is used as a light source for excitation of the fluorescent molecules which have absorption bands in the visible region. The conversion efficiency from fundamental to second harmonic emission is 1.7 × 10^–11 (0.5 pW) for a continuous wave (CW) laser, when it is operated at 30 mW. This value is further improved for a pulsed laser operated at a peak power of 10 W. Perylene is used as a standard sample for construction of an analytical curve. The detection limit is 10^–6 M for CW laser excitation. The present fluorimetric system is used for measurements of pH dependence of the fluorescence intensity for 8-hydroxy-1,3,6-pyrenetrisulfonic acid (HPTS). Neutralization titration is demonstrated by using HPTS as a pH indicator.     

Photodissociation and photoionization of 2,5-dihydroxybenzoic acid at 193 and 355 nm

Photodissociation and photoionization of 2,5-dihydroxybenzoic acid (25DHBA), at 193 and 355 nm were investigated separately in a molecular beam using multimass ion imaging techniques. Two channels competed after excitation by one 193 nm photon. One channel is dissociation from the repulsive excited state along O-H bond distance, resulting in H atom elimination from meta-OH functional group. The other channel is internal conversion to the ground state, followed by H(2)O elimination. Some of the fragments further proceeded to secondary dissociation. On the other hand, absorption of one 355 nm photon gave rise to H(2)O elimination channel on the ground state. Absorption of more than one 355 nm photon resulted in the three-body dissociation which also occurs on the ground state. Dissociation on the excited state does not play a role at 355 nm. The large concentration ratio (2×10(5)), between neutral fragments and cations produced from 355 nm multiphoton excitation indicates that internal conversion followed by dissociation, is the major channel for 355 nm multiphoton excitation. Multiphoton ionization is a minor channel. Multiphoton ionization of 25DHBA clusters only produces 25DHBA cations. Neither anion nor protonated 25DHBA cation were observed. It is very different from the ions produced from solid matrix-assisted laser desorption/ionization (MALDI), experiments. This suggests that protonated 25DHBA and negatively charged 25DHBA generated in MALDI experiments does not simply result from the ionization following proton transfer reactions or charge transfer reactions of the clusters in the gas phase.     

In vitro investigation on the pH dependence of the absorption and fluorescence properties of the photosensitizer mTHPC

Fluorescence excitation efficiency is of great importance for photodynamic diagnosis. Because usually a difference in the interstitial pH between normal and tumor tissue occurs, it is necessary to assess the impact of pH on the fluorescence emission intensity of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) in this context. The results obtained by in vitro fluorescence measurements clearly indicate that pH values below 6 lead to a significant decrease in the fluorescence intensity. In the physiological range of pH 6.5-7.2, however, no pH dependence was found. Besides the decrease in the fluorescence intensity of mTHPC for pH < 6, changes in the spectral shape of the absorption were found. These changes can be utilized for "dual-wavelength ratio imaging," using mTHPC as a pH-sensitive indicator with the excitation pair 405 nm/436 nm in the range of pH 3.5-6.     

A fluorescence sensor for quantifying pH in the range from 6.5 to 8.5

A sensor for quantifying pH values in the physiological range has been prepared by immobilizing the trisodium salt of 8-hydroxyl-1,3,6-pyridine trisulfonic acid (HOPSA) on an anion-exchange membrane. Because electronically excited HOPSA undergoes rapid deprotonation, both acid and base forms of HOPSA lead to fluorescence from the excited state of OPSA^?. However, the acid and base forms of HOPSA can be selectively detected by appropriate choice of excitation wavelengths. The ratio of fluorescence intensities resulting from excitation at 470 and 405 nm can be used to quantify pH values between 6 and 9. The ratio is unaffected by variables such as temperature and ionic strength which affect abslute intensities. At coverages below 15 μg cm^?2, the ratio varies only slightly with the amount of HOPSA immobilized on the membrane. Membranes treated with HOPSA can be stored for extended periods of time without changing characteristics. However, they undergo slow photodegradation which will limit their useful lifetimes.     

Organosilicon Amidophosphates and Their Eu and Er Complexes in Solutions and Films: Spectral and Luminescence Properties

Fluorescence and fluorescence excitation spectra of phosphorus-containing organosilicon ligands O = PX₂NHR (X = NMe₂, OPh; R = CH₂CH₂CH₂Si(Oet)₃ and their Eu(III) complexes in acetonitrile solutions and in films are studied. In UV region (285–420 nm), bis(dimethylamido)triethoxysilylpropylamidophosphate (X = NMe₂) and diphenyltriethoxysilylpropylamidophosphate (X = OPh) exhibit two emission bands, whose position and intensity depend on the nature of substituents at the phosphorus atom. The Eu complexes show the ligand and the cation luminescence. The emission bands of coordinated ligands are shifted to long-wave region. The cation luminescence appears as three or four bands due to f-f transitions from the excited ⁵ D ₀ level to the lower ⁷ F _1–4 levels. The most intense transition is ⁵ D ₀ → ⁷ F ₂. The emission band in a region of 420 nm appears in solutions and films prepared from both pure ligands and their Eu(III) complexes. This band is due to luminescence of spatially crosslinked nanoparticles of sesquioxane structure. The intensity ratio of the Eu³⁺ emission bands changes when going from solutions to films, the emission intensity increases in a range of 420 nm. Films containing incorporated Er complexes with amidophosphates show intense luminescence of a matrix at 430 nm and a series of weak narrow bands due to the Er³⁺ cation at 550–700 nm.__________Translated from Koordinatsionnaya Khimiya, Vol. 31, No. 7, 2005, pp. 550–558.Original Russian Text Copyright © 2005 by Semenov, Cherepennikova, Klapshina, B. Bushuk, S. Bushuk, Douglas.     

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.     

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices.

Single photon burst techniques were used to detect double-stranded DNA molecules in poly(methylmethacrylate) (PM MA) and polycarbonate (PC) microfluidic devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono-intercalating dye, TOPRO-5, which possessed an absorption maximum at 765 nm allowing excitation with a solid-state diode laser and fluorescence monitoring in the near-infrared (IR). Near-IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing lambda-DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 microm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PM MA-based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths > -120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 microm widths compared to those of 50 microm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of lambda-DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near-IR using polymer microfluidic devices, the near-IR chromophore, NN382, wasanalyzed using ourconfocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.     

ENERGY TRANSFER FROM CHLOROPHYLL b TO CHLOROPHYLL a IN A HYDROPHOBIC MODEL SYSTEM

Abstract— Chlorophyll a and chlorophyll b purified by high-performance liquid chromatography (HPLC) were subsequently adsorbed on the surface of a pellicular reverse phase packing normally used in HPLC. The granule surface is reacted with octadecyl groups and furnishes an hydrophobic substrate for pigment adsorption. Reflectance spectra of chlorophyll a and chlorophyll b , each adsorbed at average spacings of about 11 nm² per molecule, had red region maxima at 664 and 643nm respectively. Fluorescence excitation spectra for 740nm emission from these surfaces peaked at about 420nm for chlorophyll a and 460nm for chlorophyll b. Adsorbed pigments excited at either of the two wave lengths had a single fluorescence emission peak at 683nm for chlorophyll a and at 664nm for chlorophyll b. A surface having both pigments adsorbed in approximately equal amounts with an overall average spacing of about 5.6nm² per molecule also had peaks at 420 and 460nm in the excitation spectrum. However, excitation of adsorbed molecules on this (latter) surface, at either 420 or 460nm, produced emission with the single chlorophyll a peak at 683nm. It is concluded that, under the conditions of our experiment, exciting adsorbed chlorophyll b contributes strongly to emission from adsorbed chlorophyll a.     

Potentialities of expanded natural graphite as a new transducer for NAD-dependent dehydrogenase amperometric biosensors

The present work deals with the use of the porous texture of expanded natural graphite (ENG) as transducer in order to design electrochemical biosensors. The sensing element is a NAD⁺-dependent dehydrogenase. An electrochemical pretreatment of the ENG is favorable because it allows on one hand generating functional surface groups that may act as mediators for NADH oxidation and, on the other hand, eliminating enzyme-toxic compounds. The electrocatalytic oxidation of NADH on the pretreated material leads to the formation of enzymatically active NAD⁺. However, some persistent problems, mainly related to enzyme instability, still hamper the development of the biosensors.     

Imaging studies of the photodissociation of H2S+ cations. II

Ion imaging methods have been used to explore the photodissociation dynamics of state-selected H(2)S(+) and D(2)S(+) cations. Predissociation following one photon excitation to the A (2)A(1) state at wavelengths (385< or =lambda(phot)< or =420 nm) in the vicinity of the first dissociation threshold results in formation of ground state S(+) fragment ions; the partner H(2)(D(2)) fragments are deduced to be rotationally "cold." Two photon dissociation processes are also observed, resonance enhanced at the energy of one absorbed photon by the predissociating A state levels. Two photon excitation at these wavelengths is deduced to populate an excited state of (2)A(1) symmetry, which dissociates to electronically excited S(+)((2)D) products, together with vibrationally excited H(2)(D(2)) cofragments. Ground state SH(+)(SD(+)) fragments, attributable to a one photon dissociation process, are observed once lambda(phot)< or =325 nm. Two photon induced production of SH(+)(SD(+)) fragments is also observed, at all wavelengths studied (i.e., at all lambda(phot)< or =420 nm). These SH(+)(SD(+)) fragments are deduced to be formed in their singlet (i.e., a (1)Delta and b (1)Sigma(+)) excited states, with high levels of rotational excitation. The observed product branching and energy disposals are discussed within the context of the (limited) available knowledge relating to the excited electronic states of the H(2)S(+) cation.     

An improved biosensor for acetaldehyde determination using a bienzymatic strategy at poly(neutral red) modified carbon film electrodes

Improved biosensors for acetaldehyde determination have been developed using a bienzymatic strategy, based on a mediator-modified carbon film electrode and co-immobilisation of NADH oxidase and aldehyde dehydrogenase. Modification of the carbon film electrode with poly(neutral red) mediator resulted in a sensitive, low-cost and reliable NADH detector. Immobilisation of the enzymes was performed using encapsulation in a sol-gel matrix or cross-linking with glutaraldehyde. The bienzymatic biosensors were characterized by studying the influence of pH, applied potential and co-factors. The sol-gel and glutaraldehyde biosensors showed a linear response up to 60 μM and 100 μM, respectively, with detection limits of 2.6 μM and 3.3 μM and sensitivities were 1.7 μA mM⁻¹ and 5.6 μA mM⁻¹. The optimised biosensors showed good stability and good selectivity and have been tested for application for the determination of acetaldehyde in natural samples such as wine.     

Imaging the molecular channel in acetaldehyde photodissociation: roaming and transition state mechanisms

The roaming dynamics in the photodissociation of acetaldehyde is studied through the first absorption band, in the wavelength interval ranging from 230 nm to 325 nm. Using a combination of the velocity-map imaging technique and rotational resonance enhanced multiphoton ionization (REMPI) spectroscopy of the CO fragment, the branching ratio between the canonical transition state and roaming dissociation mechanisms is obtained at each of the photolysis wavelengths studied. Upon one photon absorption, the molecule is excited to the first singlet excited S(1) state, which, depending on the excitation wavelength, either converts back to highly vibrationally excited ground S(0) state or undergoes intersystem crossing to the first excited triplet T(1) state, from where the molecule can dissociate over two main channels: the radical (CH(3) + HCO) and the molecular (CO + CH(4)) channels. Three dynamical regions are characterized: in the red edge of the absorption band, at excitation energies below the T(1) barrier, the ratio of the roaming dissociation channel increases, largely surpassing the transition state contribution. As the excitation wavelength is increased, the roaming propensity decreases reaching a minimum at wavelengths ～308 nm. Towards the blue edge, at 230 nm, an upper limit of ～50% has been estimated for the contribution of the roaming channel. The experimental results are interpreted in terms of the interaction between the different potential energy surfaces involved by means of ab initio stationary points and intrinsic reaction coordinate paths calculations.     

Sub-picosecond laser ionization of free C60 and C70 at 248 nm

Delayed ionization is found to be absent for sub-picosecond laser excitation of free C₆₀ and C₇₀ at 248 nm. The autocorrelation trace obtained for C ₆₀ ⁺ in a laser time-of-flight (TOF) mass spectrometer using two time-delayed and collinear 248 nm ultrashort laser pulses has a width of 1.1 ps (715 fs for sech² pulses), in agreement with the laser pulse duration measurement in NO gas. Both above observations can be explained by direct ionization of C₆₀ via coherent two-photon absorption by the high intensity sub-picosecond 248 nm laser excitation avoiding the channel leading to delayed ionization.     

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δI_f) due to microinjection of glucose-6-phosphate (G6P) into EL2 ascites cells. Above 510 nm, δI_F is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δI_F level. The difference between δI_f and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δI_F can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component. The questions raised by the lack of such a component are answered using a new microspectrofluorometer, which aiiows correlated monitoring of NAD(P)H and flavins with excitations at 365 and 436 nm, respectively. As detected by excitation at 436 nm, injections of G6P, malate, ADP, and treatments with azide, cyanide or partial anaerobiosis, all indeed show a redox change of flavins, in the sense of decreased emission. It is understandable, however, that such a change which is not very large even using 436 nm excitation should remain undetected when flavins are excited at 365 nm, i.e. using the tail of their excitation spectrum. In contrast to the increased δI_F spectrum recorded in response to injected substrate, the initial spectrum (I_f) of the cell prior to a metabolic perturbation reveals a third component, even with 365 nm excitation. The position and reactivity of this component shows flavin-like properties. The structural resolution attainable makes it possible to obtain the evaluation of free vs. bound NAD(P)H and flavin fluorochromes in the mitochondrial and cytosolic compartments of the intact cell.