Ternary Interactions and Energy Transfer between Fluorescein

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Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers Katarzyna Ratajczak, and Magdalena Stobiecka J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04295 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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The Journal of Physical Chemistry

Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers

Katarzyna Ratajczak and Magdalena Stobiecka*

Department of Biophysics, Warsaw University of Life Sciences (SGGW), 159 Nowoursynowska Street, 02776 Warsaw, Poland

ACS Paragon Plus Environment

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ABSTRACT

The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs–FITC–ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant KSV = 113±22 M-1). The temperature coefficient of KSV is positive (αT = 4.15 M-1deg-1). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC–ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (KSV = 598±29 M-1) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.


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INTRODUCTION Currently observed expansion of nanotechnology into biological systems has created a range of new research areas, such as nanobiosensing

1-5

, bioimaging

6,7

, targeted drug delivery

8,9

, and

others, resulting in deeper understanding of biological processes and providing new ways for designing highly effective medical therapies. Extensive studies have recently been conducted regarding theranostic methods in nanomedicine, based on the high promise of targeted drug delivery to cancer cells using nanocarriers. Among the nanocarriers considered, graphene oxide nanosheets (GONs) offer biocompatibility 8 and richness of molecular binding capabilities

10-12

.

The interactions of GONs with fluorophores lead to strong fluorescence quenching by GONs, which on one hand provide opportunities for development of new nanosensing platforms and on the other hand may interfere with other fluorescence biodetection methods and bioimaging. Hence, in this work, we have investigated the interactions and energy transfer in the ternary system: GONs – FITC – ATP, which has not been investigated before. Here, FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'triphosphate). We have found unusual interactions between FITC and ATP, with complex temperature dependence and with unexpectedly high contribution of internal self-quenching modes. Moreover, the quenching of FITC by GONs appeared to be moderated by ATP. The elucidation of these new observations is relevant for theranostic nanocarrier studies since these interactions may interfere with or modify the drug delivery. FITC is a xanthene dye, utilized for its high quantum yield and stability in biological environments. It has been broadly employed for marking antibodies, enzymes, and other proteins, as well as nucleic acid molecular beacons and aptamers. To our knowledge, direct interactions of ATP with FITC have not been explored. Earlier studies 13 have indicated that ATP


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takes part in energy-dependent quenching of excited chlorophyll, but the process involved is very complex and consists of the reactions of photosystem I and II (multiple-protein scaffolds with cofactors absorbing light at peak absorption of 700 and 680 nm, respectively), enzymatic reactions, energy production with acidification, etc. As a result, there is no direct quenching of excited chlorophyll by ATP. ATP is an energy-providing molecule and, thus, it interacts with all biomolecules that require energy to drive the life processes they are involved in. ATP participates in mitochondrial respiration

14

, hypoxia-reperfusion

15-17

, apoptosis

18,19

, glycolysis, and a variety of other life

processes. The cancer growth requires vast amounts of energy, and so, the ATP production is greatly increased in cancer cells

20

. Therefore, the interactions of ATP with anti-cancer drug

nanocarriers are of special interest. The detection of ATP has been studied by a variety of sensing methods, including fluorescent and electrochemical aptasensors 21-24, enzyme biosensors 25-27

, fluorescence resonance energy transfer (FRET) 28, and surface-enhanced Raman scattering

(SERS) 29. Graphene and its oxidized form, graphene oxide (GO), with their close-packed 2D honeycomb structure, offer excellent electrical, optical, mechanical and thermal properties. Thus, the exfoliated graphene and GO have potential applications in nanoelectronics cells

34

and biomedical research

35,36

30

, sensors31-33, fuel

. Recently, many studies have been carried out to develop

GONs for nanoparticle nanocarriers. We have developed GON nanocarriers for cancer cell transfection and delivery of nucleotide molecular beacons encoded for detection of survivin mRNA and its deactivation 8. Other studies concerned gene therapy 11,37, drug delivery 10,12 and cancer therapy 38.


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In this work, we have investigated fluorescence quenching effects of molecules in ternary system: GONs – FITC – ATP using fluorescence spectroscopy and resonance elastic light scattering (RELS). We have successfully applied RELS previously for monitoring assembly processes of core-shell gold nanoparticles

39-41

and, in combination with fluorescence resonance

energy transfer (FRET), for detecting point mutations in DNA

42

, determination of melting

characteristics and development of highly sensitive nucleotide molecular beacons for Hg2+ homocysteine

43

, and survivin mRNA

5,8

43

,

. Herein, we consider interactions which we have

encountered between FITC and ATP and their complex temperature dependence. An attempt to elucidate the predominant quenching mode is presented. Also, the evaluation of the energy transfer from FITC to GONs and the role of ATP in this transfer have been analyzed.

EXPERIMENTAL Chemicals.

Fluorescein isothiocyanate, isomer I (FITC), adenosine triphosphate (ATP),

trizma hydrochloride (Tris-HCl), and dimethyl sulfoxide (CH3)2SO (DMSO) were purchased from Sigma-Aldrich (Poznan, Poland) and used as received. Single layer graphene oxide nanosheet (GON) nanocarriers dispersed in water, with thickness 0.6-1.2 nm and single-layer ratio >80%, were purchased from ACS Materials LLC (Medford, MA, USA). All chemicals used for investigations were of analytical grade purity. Aqueous solutions were prepared with freshly deionized water with 18.2 MΩ cm resistivity (Hydrolab, Wiślina, Poland). All concentrations of added reagents cited in this paper are final concentrations obtained after mixing. Curve fitting was performed using the Simplex algorithm.


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Instrumentation. A Spectrometer model LS55 (Perkin Elmer, Waltham, MA, USA), used for fluorimetric measurements, was equipped with a 20 kW pulsed Xenon light source operating in 8 µs pulsing mode.

Separate monochromators for the incident beam and the detector beam

provided monochromatic radiation with wavelengths from 200 nm to 600 nm with 1 nm resolution. The detector system consisted of two detectors: a photomultiplier tube and an avalanche photodiode. The resonance elastic light scattering (RELS) spectra were obtained at 90o angle from the incident (excitation) light beam. The excitation wavelength was set to λex = 480 nm. Both the excitation and emission slit widths were set to 5 nm and scan speed 500 nm/min. Measurements were performed in Tris-HCl buffer (10 mM, pH 7.4). The images of GONs nanocarriers were obtained using a transmission electron microscope (TEM) Model JEM 1200 EX and scanning electron microscope (SEM) Model JSM-6390LV (Jeol, Japan).

Procedures. The FITC stock solution (20 µM) was prepared in DMSO. The stock solutions of GONs (1 mg/mL) and ATP (150 mM) were prepared in TrisHCl buffer. Freshly prepared solutions of GONs, GONs with FITC, GONs with ATP, and solutions with ternary structures GON@ATP,FITC or GON@FITC,ATP were stable for one day, as tested with RELS and fluorescence measurements. The concentration of FITC was kept in the nanomolar range (typically 1-120 nM) to avoid any inner filter effects. ATP does not absorb at the excitation or emission wavelengths of FITC and does not cause any light energy loss. The GONs concentration was kept under control, within linear scattering range 8, using RELS measurement. The measurements with single or two-component solutions were performed directly after mixing. During the experiments with ternary structures, the measurements were performed after 10 min waiting after each addition. The molecular dynamics (MD) simulations and quantum mechanical


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(QM) calculations of electronic structures of graphene oxide, ATP and FITC and their interactions were performed using density functional theory (DFT) with B3LYP functional and 6-311G* basis set, embedded in Wavefunction (Irvine, CA, USA) Spartan 14 software. The electron density ρe is expressed in atomic units, au-3, where 1 au = 0.52916 Å and 1 au-3 = 6.7491 Å-3.

RESULTS and DISCUSSION Quenching of FITC fluorescence by ATP The fluorescence of FITC in alkaline solutions is strong due to the predominance of the dianionic form of FITC and its high fluorescence quantum yield Φ2- = 0.93 44. At physiological pH of 7.4, the quantum yield is still very high since 85.8% of FITC remains in the di-anionic form (see: pKa values in the next section). There are also indications that mono-anion is fluorescent as well but with much lower yield of Φ- = 0.37 45. Hence the net Φ at pH = 7.4 can be estimated as:

Φ = 0.858Φ2- + 0.142Φ- = 0.85. Note that we do not calculate here the quantum yield for a fluorophore from the dependence between quantum yields of two fluorophores, involving their absorption values and integrated emissions, as well as the effects of different wavelengths

46-48

,

since the quantum yields for both fluorophores are known and we estimate here the effective quantum yield for a mixture of two fluorophores at a certain concentration ratio, resulting from a given pH adjustment. The addition of ATP to FITC solutions results in fluorescence quenching, as illustrated in Figure 1A,B. The analysis and elucidation of the quenching mode has been performed on the basis of the Stern-Volmer equation 49: IFL,0/IFL = 1 + KSV CATP

(1)


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where IFL,0 and IFL are the fluorescence intensities in the absence and the presence of ATP, respectively, and CATP is the concentration of the quencher, ATP [M]. Since the dependencies IFL,0/IFL = f(CATP) for all temperatures examined were non-linear, the Stern-Volmer quenching constants KSV were determined from the initial slope of the curves, at CATP = 0. For 25 °C, the obtained value of the quenching constant is: KSV = 113±22 M-1. The low value of KSV indicates that the observed quenching may either be due to static or dynamic mechanism, or both. The affinity of FITC to ATP is associated with the π-π interactions between the aromatic xanthene triple rings of FITC and adenine double rings of ATP, but hydrogen bonding between oxygens of FITC and hydroxyl and amine groups of ATP play also a role. These interactions have been corroborated using QM calculations (see: vide supra). Since quenching may proceed either via a dynamic quenching mechanism or a static quenching mechanism and the mathematical form of the quenching equation in both cases is the same, except for the meaning of the slope constant ∂(IFl,0/IFl)/∂Q, one needs to consider the concentration level of the quencher and the temperature dependence of the quenching constant to further elucidate the quenching mechanism. Looking for further experimental verification of the FITC quenching mechanism, we have analyzed the temperature dependence of the fluorescence quenching process. The temperature coefficient of quenching, αT, defined as: αT =

∂K SV ∂T

(2)

provides additional information on the quenching mechanism, as follows:

αT < 0, for static quenching mode,

(3a)

αT > 0, for dynamic quenching mode.

(3b)


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In the case of static quenching, αT is negative since in the static quenching model, KSV becomes equal to the association equilibrium constant Ka for the ground-state associate DQ formation from the excited donor D* and quencher Q: D* + Q = DQ Ka =

C DQ C D CQ

(4)

.

(5)

Ka normally decreases with increasing temperature due to the lower stability of the complex at higher temperatures. In the case of dynamic quenching, αT is greater than zero since KSV is represented by the kinetic rate constant in the dynamic quenching model: KSV = kq τ0

(6)

where kq [M-1s-1] is the bimolecular collision quenching rate constant and τ0 [s-1] is the excited state lifetime of FITC in the absence of ATP quencher. Here, kq increases with temperature while

τ0 is temperature invariant. Therefore, the result: αT > 0 follows from the increase of the diffusion rate and the number of collisional events with increasing temperature. The value of αT determined from experimental data of Figure 1C is positive: αT = 4.15 M-1deg1

, pointing to the importance of dynamic quenching contribution. If the dynamic quenching were

the dominant mode of FITC de-excitation, then the rate constant kq = KSV / τ0 = 113/(4.1×10-9) = 2.83×1010 for t = 25 °C, assuming that the lifetime of an excited FITC in the absence of ATP is:

τ0 = 4.1 ns 45.


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Figure 1. Quenching of FITC fluorescence by ATP: (A) FITC emission spectra for increasing ATP concentration CATP [mM]: (1) 0, (2) 0.5, (3) 1.0, (4) 1.5, (5) 2.0, (6) 2.5; t = 23 °C; (B) Dependence of IFL,0/IFL on CATP for t [°C ]: (1) 30, (2) 35, and (3) 40. (C) Temperature dependence of: (1) KSV and (2) KSQ. (D) Temperature dependence of quenching efficiencies: (1)

ηSV for CATP = 2.5 mM and (2) ηSQ for CATP = 0 mM. Conditions: 10 mM TrisHCl buffer pH 7.4, CFITC = 0.12 µM, λex = 480 nm, λem = 514 nm.

However, the formation of associates and a contribution of the static quenching mechanism cannot be excluded due to the nonlinear character of the dependence of IFl,0/IFl vs. CATP. To gain further insights into the nature of FITC quenching by ATP, we have also explored fitting of the equation for mixed dynamic-static quenching mode to the experimental data:


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I Fl ,0 I Fl ,

2 = (1 + k qτ 0C ATP )(1 + K a C ATP ) = 1 + aC ATP + bC ATP

(7)

where a = kqτ0 + Ka and b = kqτ0 Ka. However, reasonable fits with R2 > 0.93 could only be obtained for a ≈ 0, which prohibits distinguishing between the dynamic quenching constant kqτ0 and the static quenching constant Ka. Since ATP does not absorb light at the FITC excitation and emission wavelengths, the inner filter effects are excluded. The source of nonlinearity of the Stern-Volmer dependence is therefore still unclear. In addition to these complications, there is also a self-quenching effect of FITC which we have observed in temperature scan measurements (SI, Figure 1,2) and which should be taken into account in the mechanistic elucidations. The obtained αT value should thus be related to the temperature coefficient of self-quenching rather than to the level αT = 0, indicated in conditions (3) above. The analysis and definition of self-quenching modes are discussed in more detail in the next section.

FITC self-quenching and temperature coefficient correction Self-quenching of a fluorophore is usually referred to as a collisional quenching of an excited fluorophore with other molecules of the same fluorophore. To be effective, the concentration of the fluorophore must be high, and so it is limited to low quantum yield species. Here, with FITC, the concentrations are in the nanomolar range, so this type of self-quenching is negligible. However, we have observed an evident self-quenching of FITC manifested by steady fluorescence decrease and quenching constant KSV increase with increasing temperature (Figure 1C,D curves 1), despite of the low FITC concentration (120 nM) and in the absence of other quenchers. The reason is likely due to the excitation of rotational mode of the phenyl ring and


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some vibrational modes with increasing temperatures. This type of self-quenching, which could be called internal self-quenching to distinguish it from the collisional self-quenching, can be formally treated in the following way: Let's consider a reaction in which an active form of FITC, Dact, is converted to an inactive form Dinact: → ←

Dact

Dinact

(8)

(d0 – x) x where d0 is the total concentration of D species and x is the concentration of the inactive form. The self-quenching equilibrium constant KSQ is then given by: K SQ =

x ( d 0 − x)

(9)

It is apparent that KSQ is temperature dependent. It increases with increasing temperature as more fluorescence-active molecules Dact become deactivated by rotational-vibrational excitations. The concentration of Dinact is given by:

x=

d 0 K SQ

(1 + K SQ )

(10)

The fluorescence emission intensity IFl as a function of KSQ can be described by:

 K SQ I Fl = εd = ε (d 0 − x) = I Fl , 0 1 −  1 + K SQ 

(

)

 1  = I Fl , 0  1 + K SQ 

(

)

(11)

which yields an equation for the internal self-quenching in the form similar to that of the SternVolmer equation but lacking the concentration dependence:

I Fl ,0 I Fl

= 1 + K SQ

(12)


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since the entire quenching is induced by internal excitation rather than bimolecular collisions. The dependence of KSQ on temperature for FITC alone (i.e. without ATP quencher) is presented in Figure 1C, curve 2. The obtained temperature coefficient of KSQ is: αT = 8.80×10-3 deg-1. The temperature dependence of quenching efficiency η defined by the equation: 

η = 1 − 

I Fl  I Fl ,0 

(13)

was also investigated. In Figure 1D, a plot of ηSV and ηSQ vs. t is presented. Unlike the case of KSV and KSQ, the quenching efficiencies can be directly compared and so their temperature coefficients. It is seen in Figure 1D that the lines for ηSV and ηSQ run almost in parallel, but with slightly higher slope for ηSV than that for ηSQ. The temperature coefficients of quenching efficiencies:  ∂η    ∂T 

(14)

βT = 

were determined from the slope of these lines: βT,SV = 11.1×10-3 deg-1 and βT,SQ = 8.8×10-3 deg-1 (with R2 of 0.98 and 0.97, respectively). These results indicate that 79% of the increase in quenching efficiency observed with increasing temperature is due to FITC internal selfquenching. The remaining 21% is the net value coming from the positive contributions from a dynamic quenching (collisions with an ATP quencher) and negative contribution from supramolecular ensemble of FITC with ATP and counterions.

Predominant species in solution In the evaluation of ATP-FITC interactions, the electrostatic interactions have also been taken into account. At a neutral pH, the ion charge of predominant FITC species can be determined from the respective pKa values which are: pKa1 = 2.05 (for H3R+/H2R), pKa2 = 4.35 (for H2R/HR-


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), pKa3 = 6.62 (for HR-/R2-)

50,51

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, where the structures of the completely protonated form H3R+

and deprotonated form R2- of FITC are presented in Figure 2. On the other hand, the acid-base equilibria for ATP are characterized by the following pKa values: pKa1 = 4.3 (for H2R2-/HR3-), pKa2 = 6.5 (for HR3-/R4-). The structures of ATP forms, H2R2-, HR3-, and R4-, are shown in Figure 2. Thus, the deprotonated anionic forms of ATP (R4-) and FITC (R2-) are predominant in solutions at a neutral pH. The strong repulsions between these anionic forms considerably hinder the binding interactions between ATP and FITC. Thus, the analysis of the predominant forms of ATP and FITC at pH 7.4 supports the dynamic quenching mechanism. In order to reduce the effect of electrostatic repulsions between ATP and FITC in neutral solutions, a high ionic strength medium to shield negative charges of the anionic molecules and an addition of divalent cations for bridging purposes have to be considered.

Figure 2. Acid-base equilibria and the ionic forms of ATP and FITC.


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Quenching of FITC fluorescence by GONs The interactions of dye molecules with nanoparticles have recently been explored due to the observed pronounced quenching of the dye fluorescence by plasmonic nanoparticles

3-5,52

. The

graphene nanosheets and exfoliated graphene oxide-based drug nanocarriers (GONs) show also strong quenching ability toward fluorescent dyes 8. Here, we consider the quenching of FITC molecules by GONs as one component of the ternary interactions FITC-ATP-GONs. For the purpose of comparisons, we represent the GONs nanocarrier concentration in terms of the concentration of graphene rings rather than the number of individual particles, since the molar concentration of rings can be directly correlated with the number of active centers on a GON for the π-π interactions with rings of FITC or ATP. To convert the concentration of the GONs stock solution of 1 mg/mL, we assume the molar mass of a graphene ring as: M(graphene ring) = nCαCMC = 6*(1/3)*12 = 24 g/mol, where nC is the number of carbon atoms in the graphene ring,

αC is the fraction of each carbon atom belonging to the given ring, and MC =12 g/mol is the atomic mass of carbon. The content of oxygen in GONs has been determined to be p = 11 weight %, so the molar mass of an average GON ring is approximately: MGON,rings = 24/(1 - p/100) = 26.97 g/mol. Thus, CGONs = 1 mg/mL = (1/26.97) M = 37.08 mM (of GONs rings). With this definition, the Stern-Volmer quenching constant for the FITC-GONs system can be expressed with the same units as that for a bimolecular quenching of FITC with ATP, so these constants can be directly compared.


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Figure 3. Quenching of FITC fluorescence by GONs: (A) FITC emission spectra for increasing GONs concentration C

GON,rings

[µM]: (1) 0, (2) 122, (3) 245, (4) 371, (5) 493, (6) 616; t = 23 °C;

(B) Dependence of emission peak intensity IFL on C C

GON,rings

GON,rings

; (C) Dependence of IFL,0/IFL on

; (D) RELS spectra for increasing GON concentration CGON,rings [µM]: (1) 0, (2) 122, (3)

245, (4) 371, (5) 493, (6) 616; (E) Dependence of RELS intensity on CGON,rings in the presence of 0.12 µM FITC; (F) electronic structure of an exemplary model GON, which includes all major oxygen-containing functional groups, with electrostatic potential mapping on a fixed electron density surface (d = 0.08 au-3); (G) SEM image of GONs; (H) TEM image of GONs; (I) Scheme


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of interactions of FITC with GONs. Conditions: 10 mM TrisHCl buffer pH 7.4, C

FITC

= 0.12 µM,

λex = 480 nm, λem = 514 nm. The FITC fluorescence quenching by GONs is shown in Figure 3A,B. The Stern-Volmer quenching constant KSV for the FITC-GONs system has been determined from the slope of the dependence IFL,0/IFL = f(CGONs,rings) (Figure 3C). The obtained value, KSV = 598±29 M-1, is several times higher than that for quenching of FITC by ATP. In the experiments of Figure 3, relatively low concentrations of GONs were used to avoid the inner filter effect which becomes pronounced at higher GONs concentrations due to strong absorbance of GONs in a wide wavelength range. As we have shown recently 8, the safe concentration range is defined by the linearity of the RELS signal which was measured together with the fluorescence quenching. The results of RELS intensity measurements for 0.12 µM FITC solutions with changing GONs concentrations are presented in Figure 3D,E. It is seen that scattering increases linearly with GONs concentration, indicating that there is no inner filter effect observed for the GONs concentrations used. Figure 3F shows an exemplary model of electronic structure of graphene oxide nanosheets, consisting of 52 C + 3 COOH groups + 3 CO groups + 4 OH groups, with electrostatic potential map on a fixed electron density surface, to visualize the impact of the functional groups on 3D structure of GON. Figures 3G and 3H present the SEM and TEM images of graphene oxide nanosheet carriers, respectively. Figure 3I depicts the scheme of the interactions of FITC with GONs.


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Ternary interactions of FITC, ATP, and GONs As discussed above, quenching of the excited FITC molecules by GONs nanocarriers (KSV = 598 M-1, rings) is more efficient than quenching of FITC by ATP (KSV = 113 M-1). We have found that the situation becomes more complex when all three components, ATP, FITC, and GONs, are present in a solution. It appears that quenching of FITC depends not only on the concentration of quenchers but also on the supramolecular structures that are formed which, in turn, depend on the order the components are added. Figure 4 illustrates in more detail the energy transfer mechanisms encountered in the ternary system FITC – ATP – GONs.

Figure 4. Fluorescence energy transfer (FRET) from an excited FITC molecule to GONs nanocarrier. (A-C) Mechanisms of FRET: (A) between FITC in solution and GONs; (B) between FITC bound to GON (GON@FITC) and the nanocarrier; (C) between FITC and GON covered by ATP (GON@ATP). (D,E) Emission spectra for: (1) FITC in the presence of GONs; (2)


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GON@FITC after addition of ATP, (3) GON@ATP after addition of FITC; the background curves for GONs alone and GON@ATP are also marked. Conditions: 10 mM TrisHCl buffer pH 7.4, CFITC = 0.12 µM, CATP = 2.5 mM, CGO,rings = 618 µM, λex = 480 nm, λem = 514 nm.

The efficiency of energy transfer from FITC to GONs in a binary system for the conditions of Figure 4A and D is: η = 25.6% (IFl,0 = 820 a.u., IFl = 610 a.u.). On the other hand, the efficiency of FITC quenching by ATP in a binary system at the concentration used in the experiment (CATP = 2.5 mM) is 45.1%. Hence, if the quenching of FITC by GONs and ATP were additive, one should expect a combined quenching efficiency of 70.6%. However, the experimentally obtained efficiency is only 57.8% (Figure 4B and curve 2 in Figure 4D), if ATP was added after 10 min of equilibration of FITC solution with GONs or only 48.9% (Figure 4C and curve 3 in Figure 4E), if FITC was added after 10 min of equilibration of ATP solution with GONs. Therefore, the quenching efficiencies are not additive. Also, the net efficiency is dependent on the order of component addition. This clearly indicates on a competitive binding of ATP and FITC to GONs nanocarriers. By comparing the model mechanisms 2 and 3 of Figure 4, one can reasonably assume that equilibration of GONs with solution of either ATP or FITC results in the formation of a supramolecular structure of ATP or FITC with GONs. The binding is due not only to the stacking interactions of xanthene rings of FITC or adenine rings of ATP to a graphene net of GONs but to the hydrogen bonding as well. In particular, one can see from Figure 4 that the FRET efficiency from excited FITC molecules is higher for GON@FITC in the presence of ATP (57.8%) than that for a film of GON@ATP after addition of FITC (48.9%). Since ATP is transparent in the wavelength range 400-600 nm, an ATP film on GONs should not prevent


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absorption of photons emitted by FITC and passed through the ATP film to underlying GONs nanocarrier. However, for FRET, the distance between the emission point in FITC and the absorption point in GON is larger for GON@ATP,FITC supramolecular layer structure than for GON@FITC,ATP structure. Note that direct dynamic quenching of FITC by ATP from GON@ATP is unlikely due to ATP bonding to GON.

Model calculations of ATP and FITC binding to GONs The interactions of ATP and FITC with GONs and between them have been analyzed in model systems using molecular dynamics (MD) simulations and quantum mechanical (QM) calculations of electronic structures. The general methodology applied in these investigations was based on evaluating H-bonding, electrostatic and π-π interactions, followed by enabling the molecules under test to interact with each other. In Figure 5, the following interactions are illustrated: (i) FITC with GONs, (ii) ATP with FITC, and (iii) interactions in a ternary structure GON@ATP,FITC. It is seen that the GON@FITC binding is stabilized by the formation of hydrogen bonds between the oxygen of FITC and a hydrogen atom of a carboxylic group of GON and stacking configuration of FITC-GON (Figure 5A). ATP molecules formed with FITC two hydrogen bonds between deoxyribose of ATP and carboxylic group of FITC. In the ternary structure of GON@ATP, FITC, the π-π interactions between the aromatic rings of nucleobase adenine and graphene rings of graphene oxide nanosheets are observed. Also, there are hydrogen bonds formed between the phosphate group of ATP and CO group of GON. Another hydrogen bond is also observed between amine group of adenine and carboxylic group of FITC. Hence, the molecular dynamic simulations confirm that interactions do occur between all molecules under study: GONs, FITC and ATP.


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Figure 5. Interactions of (A) FITC with GON, (B) ATP with FITC and (C) ternary structure of GON@ATP, FITC; electron density surfaces ρ = 0.08 a.u. with mapped electrostatic potential, color coded: from positive (blue) to negative (red).

Conclusions We have demonstrated that the interactions between a fluorophore FITC and biomolecule ATP are relatively weak but cause considerable quenching of FITC emission (KSV = 113 M-1). They exhibit positive temperature coefficient of the quenching (αT = 4.15 M-1deg-1). The system behavior does not conform to the known models of dynamic and static quenching. The major contribution (79%) to the temperature increase of quenching efficiency is associated with internal self-quenching mode of FITC, for which a suitable model has been developed (eq. (12)). The FITC fluorescence quenching by GON nanocarriers results in much higher Stern-Volmer quenching constant KSV = 598±29 M-1, which can be ascribed to the static quenching mode and


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formation of assemblies of FITC with GON via hydrogen bonding and stacking π-π interactions. Since the temperature coefficient for the FITC quenching by GONs is near zero, it is likely that the FITC internal self-quenching modes are deactivated by supramolecular binding to GON. Molecular dynamics simulation of interaction between FITC, ATP and GON molecules indicate on the hydrogen bonds formation and π-π interactions between these molecules. The presence of counter-ions is necessary to stabilize the structures. Furthermore, in a three component system FITC-ATP-GONs, we have observed partial blocking of FITC quenching by GONs, due to the presence of ATP, which can be attributed to the formation of a stable GON@ATP film.

Supporting Information. Additional information about temperature dependence of ternary interactions are available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *Magdalena Stobiecka, E-mail: [email protected] Phone: +48.22.593.8614 Fax:

+48.22.593.8619

ACKNOWLEDGMENT This research was supported by funding provided by the Program SONATA of the National Science Center, Grant No. DEC-2012/05/D/ST4/00320.

ABBREVIATIONS GONs, graphene oxide nanosheets; FITC, fluorescein isothiocyanate; ATP, adenosine triphosphate; FRET, fluorescence resonance energy transfer; RELS, resonance elastic light scattering; MD, molecular dynamic simulations; QM, quantum mechanical calculations.


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TOC Graphic


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TOC Graphic 44x24mm (600 x 600 DPI)



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Fig 1. 101x81mm (600 x 600 DPI)


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Fig 2. 100x80mm (600 x 600 DPI)



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Fig.3 104x86mm (600 x 600 DPI)


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Fig.4 100x89mm (600 x 600 DPI)



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Fig.5 107x90mm (300 x 300 DPI)


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Ternary Interactions and Energy Transfer between Fluorescein

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