Fluorescence Quenching Based Evaluation of Glucose Oxidase

Apr 11, 2018 - Fluorescence Quenching Based Evaluation of Glucose Oxidase ... for the design of bioelectronic devices such as biosensors and biofuel c...
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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Fluorescence Quenching Based Evaluation of Glucose Oxidase Composite with Conducting Polymer - Polypyrrole Urte Bubniene, Raminta Mazetyte, Almira Ramanaviciene, Vidmantas Gulbinas, Arunas Ramanavicius, and Renata Karpicz J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b01610 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Fluorescence Quenching Based Evaluation of Glucose Oxidase Composite with Conducting Polymer - Polypyrrole

Urte Bubniene,1 Raminta Mazetyte,2 Almira Ramanaviciene,3 Vidmantas Gulbinas,2 Arunas Ramanavicius,1,2* and Renata Karpicz2*

1

Department of Physical Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko Str. 24, LT-03225 Vilnius, Lithuania 2

Department of Molecular Compound Physics, Center for Physical Sciences and Technology, Saulėtekio al. 3, LT-10222 Vilnius, Lithuania 3

NanoTechnas – Centre of Nanotechnology and Materials Science, Faculty of

Chemistry and Geosciences, Vilnius University, Naugarduko Str. 24, LT-03225 Vilnius, Lithuania

Correspondance should be addressed to [email protected] and [email protected]

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ABSTRACT: Glucose oxidase (GOx) composites with conducting polymers (e.g. polypyrrole) are excellent nano-biomaterials suitable for the design of bioelectronic devices such as biosensors and biofuel cells. Here we address spectroscopic properties of GOx as well as flavin adenine dinucleotide (FAD), and composites of these compounds with polypyrrole (Ppy) were investigated in this research. The exploration of native GOx and FAD solutions confirmed that about 5% of FAD dissociated from GOx during the period of solution preparation and this fraction remained constant within one month. It has been found that the Ppy, which formed composites with FAD and GOx, facilitated the removal of FAD molecules from GOx and twice reduced the fluorescence decay rate. Differences in the FAD and Ppy average fluorescence relaxation time showed that the FAD composite with Ppy and Ppy effectively quenched the FAD fluorescence and FAD could not freely unfold. The intramolecular electron transfer took place between adenine and isoalloxazine moieties over the first 5 ps seconds after the excitation. The findings are very useful in selection and adaptation of enzyme immobilization strategies, which are applied in the development of biosensors and biofuel cells.

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1.

INTRODUCTION

The development of enzyme-based biosensors, especially those based on glucose oxidase (GOx), still attracts significant attention. Immobilization of GOx is a very important issue affecting enzymatic activity1, 2, biodegradability3, longevity4 and stability1,2,5 which is one the most important and challenging characteristics in practical application of biosensors. Studies devoted to the investigation of glucose oxidase immobilized in the polymer matrix by different methods have been presented6-14. Enhanced performance of biosensors is observed when enzymes are immobilized into electrically conducting polymer matrixes15 because a three-dimensional polymer structure allows one to exclude orientation related effects of the entrapped enzyme and leads to high accessibility of enzymes to the substrate7. In addition, some conducting polymers form a stable porous matrix suitable for diffusion of substrates/products, which are involved in enzymatic reactions16. Many conjugated polymers are well compatible with bio-molecules, and are chemically inert, environmentally and mechanically stable17-21. Some conducting polymers facilitate an electron transfer from biomolecules22 and the immobilization usually occurs with such polymers as polypyrrole23, polyaniline 6, polythiophene24, polyacetylene23, polyphenylenevinylene23,25. Among these conducting polymers Ppy is of particular interest because of its relatively low oxidation potential, which enables the formation of Ppy films and particles electrochemically or chemically, or even enzymatically from aqueous solution18,19. A number of experimental techniques have been applied for the characterization of the GOx performance.26 Due to conformational instability of GOx, the investigation of this dimeric flavoprotein is to some extent restricted. Fluorescence-based methods have received great attention because of a few important factors, namely: (i) high sensitivity combined with minimal background signal;27,28 (ii) non-invasive auto-fluorescence monitoring;29,30 (iii) autofluorescence of both oxidized/reduced forms of flavin-adenine dinucleotide (FAD), which are 3 ACS Paragon Plus Environment

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forms of natural GOx cofactor and are involved in the active redox-center of this enzyme;31-33 (iv) in some cases, the fluorescence quenching correlates with changes of the fluorescence wavelength, therefore, the environmental surrounding of fluorescent moiety (e.g. the structure of the protein) can be identified.34,35 Enzymes can be regulated and affected by multiple mechanisms and the enzyme entrapment or immobilization to a polymer matrix is one of the options to change kinetic parameters of enzymes12,15. Studies to understand the fluorescence-related behavior and the interaction of some enzymes, namely of GOx and horseradish peroxidase with Ppy, have been performed in our previous work.36,37. We demonstrated that GOx coated with Ppy is more stable compared to free GOx dissolved in solution, because non-encapsulated GOx dissociates to FAD and forms apo-enzyme33. Therefore, the fluorescence attributed to dissolved FAD significantly increases. It was also demonstrated that the combination of autofluorescent proteins and fluorescence quenching polymers could increase the selectivity and sensitivity of immunosensors.36 Extending our previous work, a more detailed study concerning the fluorescence quenching technique is provided.37 The estimation whether the fluorescence variation results by changes of intracellular processes or changes in the polymer matrix environment has been described.15 In the present study, we investigated the effect of the Ppy-based coating on the absorption and fluorescence of glucose oxidase and its cofactor FAD in a sodium acetate buffer solution. The nanosecond fluorescence quenching was observed for the GOx/Ppy composite system by the steady-state absorption and time-resolved fluorescence spectroscopy technique. That allowed us to compare the influence of Ppy on the fluorescence quenching of glucose oxidase composite with polymer. The obtained results lead to a better understanding of the enzyme/polymer system performance and reveal the mechanism of conformation changes of FAD and related variations of the fluorescence intensity. The study allows

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underpinning of the interpretation of the enzyme microenvironment behavior under varying surrounding conditions using the fluorescence technique.

2. EXPERIMENTAL Glucose oxidase (GOx, from Aspergillus Niger, 201 U/mg, M ~186 000 (Lit.)) and pyrrole monomer (Py, purity better than 97.6%) were purchased from Fluka Chemical Co. (Buchs, Switzerland). Flavin adenine dinucleotide (FAD), D-(+)-glucose (Glu) (purity ≥ 99.5%, anhydrous, M 180.16 g/mol, Art.-Nr. HN06.3) was purchased from Carl Roth GmbH + Co. (Karlsruhe, Germany). Before investigations glucose solutions were stored at 4 °C and before the first investigations glucose in solution was allowed to mutarotate overnight. All solutions were prepared using deionized water purified with the water purification system Millipore S.A. (Molsheim, France). All experiments were conducted in 0.05 M sodium acetate buffer, pH 6.0, containing 0.1 M of KCl. This buffer solution was prepared from CH3COONa●3H2O (purity better than 99.5%, M 136.08 g/mol) and KCl (purity better than 99.5%, M 74.55 g/mol), both of which were obtained from Riedel-de Haen (Seelze, Germany) and Merck KGaA (Darmstadt, Germany). All other chemicals used in the present study were of an analytical grade of the highest quality. The final concentration of GOx in all solutions was 1.1 mg/ml. The concentration of FAD solutions was about 16 mM. In pyrrole polymerization solutions, the concentration of pyrrole was 236 mM. Polymerization solutions consisted of four main components: (i) pyrrole (polymerizable monomer); (ii) GOx (an enzyme that produces hydrogen peroxide, which is initiator of polymerization reaction); (iii) Glucose –substrate of GOx and (iv) 250 µM of dissolved oxygen (oxidizing substrate, standard concentration in acetate buffer, pH 6.0, at 298 K).

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Steady state and fluorescence decay measurements of all solutions were periodically performed in several series. The parameters of all spectroscopic instruments remained stable during the measurement in order to standardize the measurement conditions. Absorption spectra were measured using the Jasco V-670 spectrophotometer. All optical measurements were performed in PMMA cuvettes with the 10-mm optical path length. To maintain uniform measurement conditions of the investigation the solutions were kept in the same closed cuvettes during one-month lasting experiment series. Fluorescence spectra in the 400–750 nm range were measured with the EdinburghF900 fluorescence spectrometer (Edinburgh Instruments, United Kingdom). A picosecond pulsed diode laser EPL-375 that emits about 70–ps duration pulses at 375 nm was used to excite fluorescence. The average pulse power was 0.15 mW/mm2. The laser radiation intensity was maximum throughout all the measurements during one month and the aperture gap was 5 nm. All fluorescence spectra were corrected for the instrument sensitivity. The optical density at 375 nm in the cuvette with 10 mm optical path of all samples was in the range of 0.1. Fluorescence decay kinetics in the nanosecond time range was measured using the Time-Correlated Single Photon Counting (TCSPC) method by utilizing the same Edinburgh F900 spectrometer. The pulse (375 nm) repetition rate was 2 MHz and the time resolution of the setup was about 100 ps. The laser radiation intensity, the aperture gap, and the measurement time for the all kinetics measurements were the same. For the time-resolved fluorescence measurements in the picosecond time range a femtosecond Yb:KGW oscillator (Light Conversion Ltd., Lithuania) was applied. The oscillator produced 80 fs 1030 nm light pulses at the 76 MHz repetition rate, which were frequency tripled to 343 nm with the HIRO harmonics generator (Light Conversion Ltd., Lithuania), attenuated, and focused into the ∼100 µm spot on the sample, resulting in the average excitation power of about 1 mW mm–2.

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The maximum time resolution of the whole system was about 2.5 ps. Fluorescence signals were taken with a streak camera (Hamamatsu, Japan) under the excitation wavelength of 343 nm. During the fluorescence measurement samples were mixed all the time.

3. RESULTS AND DISCUSSION 3.1. Absorption and Fluorescence of FAD. Visible and near UV absorption spectra, as well as fluorescence properties of GOx, are determined by its cofactor FAD. Therefore, for a better understanding of variations of spectroscopic properties of GOx, together with GOx solutions we have investigated solutions of FAD under the same experimental conditions. The photophysical properties of FAD quoted are well studied38-40, but its changes and dynamics over a 4-week period are still unknown. Absorption spectra of FAD have two maxima at 380 nm and 450 nm (Figure 1a). Both peaks are attributed to FAD, which hosts a π-π* jump over three FAD isoalloxazine rings. Initially, the absorption band was observed at 375 nm, while on the 29th day the peak slightly shifted to shorter wavelengths and it was observed at 373 nm. During this period the intensity of this peak slightly increased. During the first day of measurement the second absorption peak was registered at 450 nm, while on the 29th day the position of the peak shifted by 5 nm to shorter wavelengths. During the 29days period, the intensity of the second peak slightly increased. Moreover, the second absorption peak registered on the 22nd day was more intense than that on the 29th day. These changes are associated with the reduced forms of FAD and changes of its geometrical configuration. The FAD fluorescence spectra had a single peak, which intensified significantly over the time of FAD incubation. On the first measurement day, the fluorescence band was registered at 532 nm, while after 29 days the position of the peak shifted to 527 nm and the fluorescence intensity increased about 8 times. 7 ACS Paragon Plus Environment

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The fluorescence decay kinetics of the FAD solutions was measured at 530 nm throughout the 4-weeks period (Figure 1b). Traces were approximated by several exponents that were used to calculate the average fluorescence lifetime. On the first measurement day, the fluorescence decay rate of FAD was about 2.9 ns. During the first two weeks, it increased up to 4.5 ns. The main photophysical parameters of FAD, GOx, FAD/Ppy, GOx/Ppy and Ppy are presented in Table 1. An edge-to-edge distance between the electron donor and acceptor groups in different flavoproteins could change from 3 Å to 16 Å41-43. In the ‘closed’ FAD conformation, in which flavin (acceptor) and adenine (donor) moieties are close to each other, and intramolecular photoinduced electron transfer occurs in several picoseconds44. The distance between the electron donor and acceptor groups of FAD increases and this can be evaluated by the characteristic ultrafast intramolecular electron transfer process observed by fluorescence measurements45. In order to ascertain the electron transfer process in FAD solutions, the samples were further characterized by time-resolved fluorescence with the streak camera under excitation at 343 nm (Figure 1c). From these measurements we can clearly identify the fastest fluorescence decay component with a lifetime of about 4.4 ps. The similar partial ultrafast fluorescence quenching is reported in other studies,46, 47 and attributed to intramolecular electron transfer between adenine and isoalloxazine moiety of ‘closed’ FAD conformation, also called “stacked” conformation. Molecular dynamics simulations and time-resolved fluorescence spectroscopy revealed that FAD

might have several

conformations: a ‘closed’ conformation, which is characterized by ultrafast fluorescence quenching (about 7 ps), several intermediate conformations, and an ‘open’ conformation, which is characterized by almost 3 orders of magnitude longer fluorescence lifetime (4.7 ns in water)48. After 22 days we did not observe any fast fluorescence decay component (Figure

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1c). During the 3-week period FAD changed its conformation from the ‘stacked’ to ‘open’ form. We also measured fluorescence spectra dynamics on a picosecond timescale (data are not shown): (i) during the first 5 ps after the excitation the fluorescence maximum of FAD solution was at around 515 nm; (ii) during 100 ps, the fluorescence maximum slightly redshifted (up to 520 nm), but FWHM did not change. It suggests that during the excited state lifetime FAD did not change its redox state and remained in the oxidized state. It was determined that the steady-state fluorescence spectra of FAD did not experience changes during the measurement time of 29 days. These results show that initially ‘stacked’ FAD conformation dominated in solution. About 50% of the FAD fluorescence was quenched during first 10 ps and this part of the signal did not contribute to the steady-state fluorescence, while fluorescent molecules were in two different open conformations characterized by about 1.7 and 4.6 ns decay times. After two weeks, FAD fully unfolded and almost only the ‘open’ FAD conformation with the 4.6 ns decay time remained. Associated changes in time of the absorption spectra indicate that different conformational states also have slightly different absorption spectra. The long wavelength band is apparently composed of two components and the intensity of the long wavelength component decreases with time when conformational changes take place. However, it is difficult to predict which one, ‘closed’ or short-lived ‘open’ component, or both of them are responsible for the gradually disappearing long wavelength absorption component. 3.2 Quenching of FAD Fluorescence in Gox. Absorption and fluorescence spectra of GOx are shown in Figure 2a. Absorption of GOx is identical to that of FAD in the 320– 550 nm spectral range, but in our samples, it was twice lower. The absorption spectra of GOx

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have two maxima at 380 nm and 450 nm both attributed to FAD. The absorption spectra of GOx remained stable during the experiment period of 29 days. Fluorescence spectra of GOx show two peaks. The intensity of the first peak (at 430 nm) remained stable during the one month period. This peak is attributed to tryptophan and tyrosine, which both are present in GOx27,49. The second peak, attributed to FAD fluorescence, showed identical spectral and nanoseconds-lasting decay properties. Similar changes were registered in pure FAD solution, only its intensity in pure FAD solution was higher by approximately two orders of magnitude. Taking into account the 2 times lower absorption intensity, the fluorescence efficiency of FAD in GOx solution was approximately 20 times lower than that in FAD solution. Picosecond time-resolved fluorescence measurements of GOx (Figure 2c) show that initially after the sample preparation a major fraction of FAD fluorescence (more than 80%) was quenched on a picosecond timescale. Probably this fraction was even larger; just the peak intensity was reduced due to limited time-resolution of the streak camera. Due to a very short lifetime, this fast component only slightly contributed to the steady-state fluorescence, the latter being determined by the slow unquenched fluorescence component. Most probably, the slow decay component, which has similar properties as fluorescence of pure FAD solution, originates from FAD, which has dissociated form GOx. Thus, taking into account that the FAD fluorescence efficiency in GOx solution is about 20 times lower than that of pure FAD solution, we conclude that initially after the sample preparation, about 95% of FAD is located inside of GOx and about 5% of FAD is dissociated form GOx and is located in solution. The fast FAD fluorescence quenching in GOx may be attributed to the stacked conformation. However, according to the literature data FAD in GOx is unfolded42. It suggests that the FAD fluorescence quenching mechanism in GOx is different. We predict

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that fluorescence may be quenched by the electron transfer between FAD and charged GOx fragments42. Subsequently, the evolution of FAD fluorescence in GOx solution during 29 days is very similar to that in pure FAD solution. The fluorescence intensity of both solutions gradually increases about 8 times due to unfolding of FAD in solution, remaining about 20 times weaker for the GOx solution. According to the proposed quenching mechanism, we did not observe a release of FAD from GOx. It was earlier suggested that FAD could be easily released from GOx and other proteins

50-52

. Of course, we cannot completely rule out this

process. However, our data show that the release is insignificant during a one month observation period. In the next section, we are going to demonstrate the evaluation of FAD dissociation from GOx when the GOx composite is in the complex with polypyrrole. 3.3 Properties of FAD/Ppy and GOx/Ppy Complexes Revealed by Fluorescence Quenching. Figure 3a,b, and c show evolutions of the absorption and fluorescence spectra of the FAD and GOx solutions with Ppy composites. All absorption spectra of FAD/Ppy and GOx/Ppy composites are different from those of FAD and GOx without Ppy due to the growing Ppy aggregates and formation of Ppy complexes with FAD or GOx, respectively. For better understanding of spectroscopic properties of GOx/Ppy and FAD/Ppy composites a pure Ppy solution in the same buffer was also evaluated (Figure 3c). It was shown that pyrrole underwent the polymerization in acidic solutions in the absence of a chemical oxidant or electrochemical oxidation.53 Polymerization process lasted for several weeks. We also observed some changes of spectroscopic properties of the Ppy solution within the 4-week period. Absorption spectra of pyrrole oligomers have several bands at 380, 462 and 550 nm. During the period of one month, the intensity of absorption spectra increased more than 7 times, whereas fluorescence spectra of Ppy also increased during the first days, but after one week it started to decrease. It suggests that low molecular weight pyrrole oligomers formed

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larger Ppy units during 3-5 days aging causing an increase in the fluorescence intensity, while after one week, Ppy oligomers started to aggregate. Meanwhile, the fluorescence intensity decreased, and the fluorescence band-shape started to change: a new weak fluorescence band started to grow in the 460-550 nm region and the fluorescence average lifetime slightly increased. This fluorescence intensity decrease occurred due to the self-quenching of Ppy. Absorption spectra of FAD/Ppy, GOx/Ppy composites also showed significant changes. However, their dynamics is significantly different from that of Ppy solution. We observed only weak changes during the initial 8 days. During this time, the spectra in the 450 nm region apparently were dominated by the FAD absorption. At longer times, Ppy absorption increased and upstaged the FAD absorption. A new absorption band also appeared with time in the red spectral region, which was absent both in FAD and Ppy solutions. The fluorescence intensity of GOx/Ppy and FAD/Ppy solutions slightly increases and its decay becomes slightly slower during 29 days, because some aggregation of formed Ppy. The fluorescence decay kinetics of FAD/Ppy, GOx/Ppy and pure Ppy are shown in Figure 3d, 3e, 3f. The average fluorescence lifetimes of the FAD/Ppy composite solution at 530 nm during the four weeks period increased from 0.9 ns to 1.3 ns, while the average fluorescence lifetimes of the GOx/Ppy composite solution changed from 1.5 ns to 1.8 ns (Table 1). The presence of Ppy reduces the fluorescence decay time more than twice in comparison with that of solution without Ppy. However, the quenching effect of Ppy is much stronger; the presence of Ppy reduces the steady-state fluorescence intensity of FAD solution about 40 times, which suggests that fluorescence of a large fraction of FAD molecules is completely quenched. We have also attempted to measure fluorescence of GOx/Ppy complexes on a ps time scale (Figure 4). However, the fast decay was completely dominated by the Ppy fluorescence.

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Taking into account the twice lower absorption of GOx, fluorescence efficiencies and decays of GOx/Ppy and FAD/Ppy composites are perfectly identical. This is in a clear difference with the FAD fluorescence yield of FAD and GOx solutions without Ppy. Identical properties of FAD fluorescence in GOx/Ppy and FAD/Ppy solutions lead to the conclusion that all FADs in GOx/Ppy solution are outside GOx and form complexes with Ppy identically as in case of FAD/Ppy solution. Consequently, we conclude that Ppy oligomers cause withdrawing of FAD from GOx. Most probably, Ppy oligomers create the same stable composites with isoallosazine moiety and obstruct incorporation of FAD in GOx. A similar effect was demonstrated by M. Holzinger et al. for GOx and gold nanoparticle composite 54. Ppy oligomers make composites with isoallosazine moiety of FAD, which is stabilized by the π-π interactions between the aromatic rings. Ppy as a good p-type conducting polymer

55

can

play an electron acceptor role in the FAD/Ppy composite and efficiently quench the FAD fluorescence. We summarized our results schematically in Figure 5. During the 4-weeks incubation period of FAD in 0.05 mol L−1 sodium acetate buffer, pH 6.0, FAD changed its conformation from the ‘stacked’ to ‘open’ form, but in the case of the FAD composition with Ppy the FAD ‘opening’ process was slowed down. When GOx dissolved in the acetate buffer pH 6.0 was evaluated, the fluorescence dynamics during the first days showed that 5% of FAD molecules in ‘stacked’ conformation had been released from GOx enzyme and the other 95% of FAD molecules remained in complex with GOx. We observed the 5.4 ps fluorescence decay component, which is induced by the electron transfer from adenine to isoalloxazine moiety and from adenine to tyrosine. During the 3-weeks period, the average fluorescence lifetime and the fluorescence intensity increased about 2 and 6 times, respectively. This effect is based on the conformational change of FAD from the ‘stacked’ to ‘open’ form. It was determined that Ppy in GOx/Ppy composite slows down the dissociation of FAD from

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GOx/Ppy composite, because Ppy absorbs FAD molecules, which are dissociated from GOx. During the 4-weeks lasting period, all photoluminescence changes of the GOx/Ppy composite are folowed by the formation of FAD complex with Ppy.

CONCLUSIONS In the present paper, fluorescence spectroscopy was applied to monitor FAD dissociation from glucose oxidase in two different classes of samples: (i) native GOx and (ii) the GOx composite with the Ppy matrix. These investigations were performed to analyze stabilization of GOx by the interaction with the conducting polymer. The obtained results showed that fluorescence of FAD in solution was initially strongly quenched by the electron transfer in a closed FAD form. During one month the transformation of FAD to its open form took place resulting in the fluorescence increase by order of magnitude. In GOx solution, approximately 5% of FAD remained outside of GOx and this FAD fraction is responsible for the steadystate fluorescence, while fluorescence of FAD remaining in GOx is quenched during several picoseconds. Ppy strongly quenches the FAD fluorescence both in FAD and GOx solutions. On the other hand, Ppy aggregation causes Ppy fluorescence in the same spectral region as FAD fluorescence impedes its investigations. Identical FAD fluorescence quenching in FAD and GOx solutions suggests that Ppy causes a release of all FAD molecules from GOx.

Acknowledgment Research was funded by a grant (No. SEN-21/2015) from the Research Council of Lithuania.

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Table 1. Photo-Physical Properties of Systems Consisting of Mixtures of Different Materials Dissolved in 0.05 M Sodium Acetate Buffer, pH 6.0, Containing 0.1 M of KCl1) 16 mM of FAD; 2) 1.1 mg/ml of GOx; 3) 16 mM of FAD/Ppy Composite (236 mM of Pyrrole); 4) 1.1 mg/ml of GOx/Ppy Composite (236 mM of Pyrrole); 5) Ppy (236 mM of Pyrrole) FAD GOx FAD+PPy GOx+Ppy Ppy 1st day λFl, nm 530 450, 533 434, 521 430 424 IFl,. arb.un. 1180750 61150 465160 391860 189420 τfast, ps* 4.4 ± 0.3 5.4 ± 0.3 8.6 ± 0.3 6.6 ± 0.3 τmidl, ns (%)** 1.2 (29%) 0.8 (10%) 0.55 (84%) 0.35 (52%) 0.3 (64%) τlong, ns (%)** 3.6 (71%) 2.9 (90%) 2.5 (16%) 2.8 (48%) 1.6 (36%) τave, ns 2.9 ± 0.2 2.7 ± 0.2 0.9 ± 0.2 1.5 ± 0.2 0.65 ± 0.2 22nd day λFl, nm 530 450, 533 440, 520 440, 525 425, 495 IFl,. arb.un. 7942050 336700 625200 524540 409800 τfast, ps* 10 ± 0.3 10.8 ± 0.3 6.0 ± 0.3 τmidl, ns (%)** 1.2 (2%) 1 (2%) 0.7 (71%) 0.55 (46%) 0.6 (45%) τlong, ns (%)** 4.5 (98%) 4.5 (98%) 2.8 (29%) 2.9 (54%) 2.8 (55%) τave, ns 4.4 ± 0.2 4.4 ± 0.2 1.3 ± 0.2 1.8 ± 0.2 1.8 ± 0.2 * from measurements with the streak camera ** from measurements with the spectrophotometer Edinburgh-F900

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FAD

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b

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Figure 1. (a) Absorption and fluorescence spectra of 16 mM FAD solution under excitation at 375 nm, (b) fluorescence decay kinetics at 530 nm on a long timescale measured under excitation at 375 nm, and (c) fluorescence decay kinetics at 530 nm on a short timescale under excitation at 343 nm.

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4x10

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Figure 2. (a) Absorption and fluorescence spectra of 1.1 mg/ml of GOx solution under excitation at 375 nm, (b) fluorescence decay kinetics at 530 nm on the long under excitation at 375 nm, and (c) fluorescence decay kinetics at 530 nm on a short timescale under excitation at 343 nm.

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104 1.0

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Figure 3. Absorption and fluorescence spectra (a-c) and fluorescence decay kinetics at 530 nm (d-f) of 16 mM of FAD/Ppy composite (a, d), 1.1 mg/ml of GOx/Ppy composite (b, e) and 236 mM of Ppy (c, f) solution under excitation at 375 nm.

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a at 530 nm on 1st day Ppy GOx + Ppy

0 1

b at 530 nm on 22nd day Ppy GOx + Ppy

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Figure 4. Fluorescence decay kinetics on the first (a) and 22nd (b) days after preparation of samples of Ppy (black line) and GOx/Ppy (red line) composite solution under excitation at 343 nm.

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Figure 5. Schematic representation of various structures of FAD (A), FAD/Ppy composite (B), GOx (C) and GOx/Ppy composite (D) and their dynamics.

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TOC GRAPHIC

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