**3. Materials and methods**

ensemble averaging that is always present in experiments performed on layers, as the one described above. In addition, when layers of emitters are studied, there is always a certain thickness of this layer, across which the strength of the interaction, in this case the energy transfer, varies, sometimes considerably. As a result, an average effect is measured in fluorescence microscopy experiment, similarly as in the case of plasmonic interactions associated with metallic nanoparticles [37]. In this context, the value of single nanocrystal experiment manifests itself in the observation of suppression of fluctuations of fluorescence intensity of single nanocrystals upon deposition on graphene. This indicates the effect of graphene-induced fluorescence quenching also on the photophysics of the nanocrystals.

158 Recent Advances in Graphene Research

Very recently, both concepts were combined in a single experiment, where distance depend‐ ence of the energy transfer rate to a monolayer graphene was studied for individual emitters, both zero-dimensional CdSe/CdS nanocrystals and two-dimensional CdSe/CdS/ZnS nanopla‐ telets [38]. Both types of energy donors were separated from graphene with ultrasmooth dielectric film of magnesium oxide with a thickness varied from just a few Å up to several tens of nanometers. In terms ofradiative energy transfer efficiency (>95%), both structures exhibited similar behaviour upon direct coupling to graphene. Important differences appear when emitters are separated from graphene with a spacer. While for zero-dimensional nanocrys‐ tals the energy transfer rate scales with a distance according to d-4 law, the energy transfer rate of the two-dimensional platelet decays is affected to a lesser degree. This is explained by a theoretical model, which includes the contribution of thermal distribution of free excitons in a two-dimensional quantum well at finite temperatures. The results confirm that graphenenanocrystal hybrid structure, governed by both charge transfer and Förster-type resonant energy transfer, is a suitable system to explore the influence of exciton dimensionality and

**Figure 5.** (a) Selected luminescence decays of individual CdSe/CdS nanocrystals separated from graphene by an MgO spacer with increasing thickness. The thin black lines are fits based on biexponential decays convoluted with the in‐ strument response function. (b) Statistically averaged measured decay rate *γ* as a function of the thickness of the MgO spacer. (c) Statistically averaged product of the number of emitted photons per exciting laser pulse *N*em and the decay

Very efficient energy transfer from organic dyes to graphene-based materials has also been used to visualize the structure and determine the morphology of graphene-dye hybrids.

localization, as well as distance, on the energy transfer rate (**Figure 5**).

rate *γ*. From Federspiel et al. [38]. Reprinted with permission from ACS.

In this section, we describe the structure and properties of peridinin-chlorophyll-protein (PCP), a light-harvesting pigment-protein complex, as well as graphene-based materials that are energy acceptors in our hybrid assemblies. Next, we present experimental techniques employed for investigating the energy transfer, which include—in addition to standard absorption and fluorescence spectroscopy—high-resolution confocal fluorescence microsco‐ py coupled with time-resolved capability and spectrally resolved detection.

#### **3.1. Peridinin-chlorophyll-protein**

Peridinin-chlorophyllprotein complex from algae Dinoflagellates *Amphidinium carterae* belongs to photosynthetic complexes that are responsible for light harvesting and transfer‐ ring the energy to reaction centres [2]. We focus here on aspects relevant for understanding the energy transfer between pigments comprising the PCP complex and graphene/rGO.

PCP is a water-soluble protein functioning as an antenna external to the membrane. The structure of the PCP complex, shown in **Figure 7**, has been determined with 1.3-Å resolution using X-ray crystallography [45]. In its native form, PCP consists of two chlorophyll *a* (Chl *a*) and eight peridinin (Per) molecules embedded in a protein matrix. The pigments are ar‐ ranged in two almost similar clusters, with the distance between Mg atoms of the two Chl *a* in one monomer being 17.4 Å. The ratio of Per to Chl *a* of 4:1 indicates that PCP utilizes the carotenoids as its main light-harvesting pigments.

The absorption spectrum of the PCP complex, displayed in **Figure 7**, features an intense, broadband from 400 to 550 nm that is mainly due to Per absorption. The contribution of Chl *a* appears at 440 (Soret band) and 660 nm (*Q*<sup>Y</sup> band). The fluorescence emission of the PCP complex originates from weakly coupled Chl molecules and it appears at 673 nm with a 30% quantum yield and a decay time constant of 4 ns, as shown by red line in **Figure 7**. Upon absorption of light, peridinins in PCP transfer their electronic excitation to Chl *a* molecules. The efficiency of this excitation energy transferis higherthan 90% as evidenced by almost ideal correspondence between absorption and fluorescence excitation spectrum. Clearly, the absorption spectrum of PCP enables the photosynthetic apparatus to harness the sunlight not only in the red spectral range but extends into the blue-green spectral region. From the point of view of the experiments described in this chapter, it is important to consider the PCP complex as a donor that can be excited at essentially any energy from 350 to 650 nm, with this excitation yielding emission at the same wavelength of 673 nm. This property distinguishes PCP, and many other photosynthetic complexes, from frequently used emitters, such as organic molecules or semiconductor quantum dots, that are much more selective in their optical characteristics.

**Figure 7.** Pigment structure of the PCP complex together with absorption (black line) and fluorescence (red line) meas‐ ured in aqueous solution at room temperature.

Previous studies of PCP complexes have been carried out on the ensemble [46, 47] and singlemolecule levels [48, 49]. Transient absorption in femtosecond timescale revealed main energy transfer pathways between pigments comprising the complex, and it also was demonstrated that the interaction between the two Chl *a* molecules is relatively weak with transfer times of the order of 10 ps [47]. These findings were also corroborated with fluorescence studies of individual PCP complexes: it has been shown that it is possible to distinguish emission originating from each of the two Chl *a* molecules and using the property of sequential photobleaching of the Chl, the energy splitting between the two molecules in the monomer was determined [49].

The simplicity of the PCP complex, its water solubility that facilitates easy sample fabrica‐ tion, its small size (~4 nm) and unique spectral properties have rendered this complex as a model system for fabricating hybrid nanostructures for studying interactions at the nano‐ scale [3]. These include in particular extensive work focused on plasmon-induced effects associated with interactions between pigments comprising the PCP complex and metallic nanostructures [37].

#### **3.2. Graphene-based materials**

ring the energy to reaction centres [2]. We focus here on aspects relevant for understanding the energy transfer between pigments comprising the PCP complex and graphene/rGO.

PCP is a water-soluble protein functioning as an antenna external to the membrane. The structure of the PCP complex, shown in **Figure 7**, has been determined with 1.3-Å resolution using X-ray crystallography [45]. In its native form, PCP consists of two chlorophyll *a* (Chl *a*) and eight peridinin (Per) molecules embedded in a protein matrix. The pigments are ar‐ ranged in two almost similar clusters, with the distance between Mg atoms of the two Chl *a* in one monomer being 17.4 Å. The ratio of Per to Chl *a* of 4:1 indicates that PCP utilizes the

The absorption spectrum of the PCP complex, displayed in **Figure 7**, features an intense, broadband from 400 to 550 nm that is mainly due to Per absorption. The contribution of Chl *a* appears at 440 (Soret band) and 660 nm (*Q*<sup>Y</sup> band). The fluorescence emission of the PCP complex originates from weakly coupled Chl molecules and it appears at 673 nm with a 30% quantum yield and a decay time constant of 4 ns, as shown by red line in **Figure 7**. Upon absorption of light, peridinins in PCP transfer their electronic excitation to Chl *a* molecules. The efficiency of this excitation energy transferis higherthan 90% as evidenced by almost ideal correspondence between absorption and fluorescence excitation spectrum. Clearly, the absorption spectrum of PCP enables the photosynthetic apparatus to harness the sunlight not only in the red spectral range but extends into the blue-green spectral region. From the point of view of the experiments described in this chapter, it is important to consider the PCP complex as a donor that can be excited at essentially any energy from 350 to 650 nm, with this excitation yielding emission at the same wavelength of 673 nm. This property distinguishes PCP, and many other photosynthetic complexes, from frequently used emitters, such as organic molecules or semiconductor quantum dots, that are much more selective in their

**Figure 7.** Pigment structure of the PCP complex together with absorption (black line) and fluorescence (red line) meas‐

Previous studies of PCP complexes have been carried out on the ensemble [46, 47] and singlemolecule levels [48, 49]. Transient absorption in femtosecond timescale revealed main energy transfer pathways between pigments comprising the complex, and it also was demonstrated that the interaction between the two Chl *a* molecules is relatively weak with transfer times of the order of 10 ps [47]. These findings were also corroborated with fluorescence studies of

carotenoids as its main light-harvesting pigments.

160 Recent Advances in Graphene Research

optical characteristics.

ured in aqueous solution at room temperature.

Graphene oxide was synthesized from graphite powder using the modified Hummers and Offeman method described elsewhere [50, 51]. Reduced graphene oxide was prepared from graphene oxide by reduction with hydrazine. In our procedure, graphene oxide powder (2.5 mg) was dispersed in 5 ml of distilled water and placed in an ultrasound bath for 30 min. In a separate vial, 1.55 μl of 65% hydrazine monohydrate solution was added to 1 ml of distilled water. Then, 0.5 ml of the prepared hydrazine solution was added to 0.5 mg/ml-graphene oxide solution. Finally, the mixture was transferred into a round-bot‐ tomed flask, put in an oil bath, heated up and maintained at 100°C for 24 h. After this time, a clear brown solution of graphene oxide turned into black precipitate of reduced graphene oxide flakes. The final solution was washed five times with water and ethanol, and then filtered. The remaining reduced graphene oxide flakes were dried, dissolved in distilled water and left in an ultrasound bath for 1 h before further use. As estimated from XPS measurements, *C*/*O* = 7–10 and 1.7–2 ratios were measured for rGO and GO, respec‐ tively, pointing towards substantial reduction efficiency of the synthesis procedure [52]. Afterwards, rGO flakes were dispersed in distilled water, in an ultrasound bath.

Graphene substrates were purchased from Graphene Supermarket. We used 1 × 1-cm p-doped silicon wafers with a single-layer graphene deposited using chemical vapour deposition on a 285-nm thick silicon dioxide layer. The presence of a graphene monolayer on the sub‐ strates was confirmed using Raman spectroscopy.

#### **3.3. Sample preparation**

In order to study interactions between PCP and rGO, we prepared three solutions of rGO in water, one with the initial concentration of *C*<sup>0</sup> = 0.5 mg/ml, and two dilutions, 1:10 *C*<sup>0</sup> and 1:100 *C*0. To prepare the samples, we mixed PCP complexes in 2% polyvinyl alcohol (PVA) with these three rGO solutions in a 1:1 ratio. The final PCP concentration in each sample was 0.2 μg/ml. In order to compare the results obtained for the rGO-containing samples, we also prepared a reference sample, where PCP and PVA concentrations were the same as above and with rGO replaced by distilled water. The layers were obtained by spin-coating solutions on pure coverslips with the rotational speed of 1200 rpm for 2 min.

For optical experiments focused on studying excitation energy dependence of the energy transfer efficiency, we used highly diluted (optical density of 0.009 at 671 nm, concentration less than 10 μM) aqueous solution of PCP complexes. Such a low concentration is very important as on the one hand it strongly reduces the inner filter effect, but this also yields a thin layer of PCP complexes on a graphene surface. As a result, we minimize the fraction of PCP that is not coupled to graphene, thus takes no part in the energy transfer.

Finally, we fabricated structures for the evaluation of the effect of polymer layer (in our case PVA) on both interaction with graphene and photostability. To this end, samples were fabricated with the concentration of PVA varying between 0.2 and 0.002%. The obtained solutions were either drop-casted or spin-coated on single-layer graphene substrates. In the case of the latter approach, the concentration of PCP had to be adjusted to be slightly higher, as spin coating strongly reduces the number of PCP complexes within the focal volume of the focused laser.

#### **3.4. Experimental techniques**

The optical properties of hybrid nanostructures comprising light-harvesting complexes and graphene-based materials were studied using absorption and fluorescence spectroscopy in the visible spectral region. Absorption spectra were obtained using Cary 50 spectrophotometer, while fluorescence in solution was measured using Fluorolog 3 spectrofluorometer. A Xenon lamp with a double grating monochromator was used for excitation and the signal was detected with a thermoelectrically cooled photomultipliertube characterized by a dark current of less than 100 cps.

Fluorescence intensity maps were measured with an inverted fluorescence wide-field Nikon Eclipse Ti-U microscope equipped with an Andor iXon Du-888 EMCCD detector. For each sample, a series of 50 images were acquired in orderto allow forreliable statistics. Every image was collected for a different sample area, which allows for minimization of any photobleach‐ ing of the PCP fluorescence. Immersion objective with a magnification of 100× (Plan Apo, Nikon) and a numerical aperture of 1.4 was used, which provides a spatial resolution of about 300 nm. As a light source, we used LED illuminators (405, 480 and 530 nm) equipped with appropriate bandpass filters. Excitation power was equal to 50 μW. Fluorescence of PCP was extracted by combining a dichroic mirror (Chroma T650lxpr) and a bandpass filter (Thorlabs FB 670-10). Fluorescence intensity maps and kinetics were collected with the electron multiplying gain of 300× and acquisition times of 0.25 or 0.5 s, depending on the experimen‐ tal conditions. White-light transmission images were recorded with the same microscope, with a halogen lamp V2-A LL (12 V, 100 W) as a light source.

Spectrally and time-resolved fluorescence measurements were performed using a home-built confocal fluorescence microscope described in detail in [53]. The sample was placed on a piezoelectric translation stage. We used pulsed laser excitation at 405, 485 and 640 nm (repetition rate of 20 MHz, average power of 30 μW, power density of ~1MW/cm−2). Impor‐ tantly, PCP can be efficiently excited at 405 (Soret band), at 485 (Per) and at 640 nm (excited states of chlorophylls). The laser beam was focused on the sample by LMPlan 50× objective (Olympus) with a numerical aperture of 0.5. Fluorescence was first filtered by a longpass

filter (HQ665LP Chroma) and then the spectra were detected using Andor iDus DV 420A-BV CCD camera coupled to an Amici prism. Time-resolved measurements were performed by time-correlated single-photon-counting technique using an SPC-150 module (Becker & Hickl) with fast avalanche photodiode (idQuantique id100-20) as a detector. In order to select appropriate wavelength range, we used an additional bandpass filter (FB670/10 Thorlabs). Time resolution of the TCSPC set-up is about 100 ps.
