Kenryabc,
Kian Ping Lohbd and
Chwee Teck Lim*bce
aNUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456, Singapore
bCentre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore, Singapore 117543, Singapore. E-mail: ctlim@nus.edu.sg
cDepartment of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
dDepartment of Chemistry, National University of Singapore, Singapore 117543, Singapore
eMechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
First published on 28th April 2016
We investigate the molecular interactions between graphene oxide (GO) and blood plasma proteins, in particular, the influence of GO on the intrinsic fluorescence of these proteins. We observe that GO acts as an efficient quencher of the intrinsic fluorescence of albumin, globulin, and fibrinogen. Interestingly, we also note for the first time that, in addition to the robust fluorescence quenching, GO is capable of selectively amplifying the fluorescence emission of fibrinogen up to approximately 30% or 1.3 fold under certain concentrations but not those of albumin and globulin. We suggest that GO may possibly play a dual role in controlling the intrinsic fluorescence emission of the plasma proteins. Furthermore, this role switching may be influenced by the competition between the aggregation and encapsulation effects. We propose that the GO-induced intrinsic fluorescence quenching is driven by the physical encapsulation of the plasma proteins by GO nanosheets. Contrastingly, the GO-mediated fluorescence amplification is promoted by an aggregation of fibrinogen.
With a molecular weight of 65 kDa, albumin is one of the smallest and most abundant proteins in the circulatory system. The globular albumin is generally known as a multifunctional transporter molecule and it constitutes about 55% of the plasma proteins in blood. Structurally, albumin comprises three homologous domains in which each domain may be further classified into two sub-domains.15 Globulin is another vital globular protein responsible for numerous physiological functions, such as molecular transport and maintenance of immune system. With a molecular weight of approximately 92 to 120 kDa and constituting roughly 38% of the plasma proteins, globulin is the second most abundant plasma protein. Fibrinogen, on the other hand, is a large protein with a molecular weight of approximately 340 kDa. It is a macromolecular plasma protein which plays an active and essential role in the blood coagulation process. The native structure of fibrinogen is of triglobular configuration which consists of a small central domain connected to two terminal globular regions by the α–helical coiled-coil domains.16 In addition, a single fibrinogen molecule possesses a length of about 45 to 50 nm.16,17
Generally, blood plasma proteins possess three aromatic residues, i.e., tryptophan, tyrosine, and phenylalanine, that contribute differently to their intrinsic fluorescence emissions.18,19 Out of these amino acid residues, tryptophan is recognized as the dominant source of UV absorption and intrinsic fluorescence emission of proteins at around 280 nm and 350 nm, respectively. The tryptophan fluorescence is much stronger than those displayed by the other two aromatic amino acids. In fact, the fluorescence intensity of tryptophan is approximately eight and 140 times higher than those of tyrosine and phenylalanine, respectively. Although the quantum yields of tryptophan and tyrosine are similar, the fluorescence of tyrosine is frequently quenched in the native proteins probably as a result of its energy transfer to tryptophan or because of the tyrosine–peptide chain interactions.20 On the other hand, the intrinsic fluorescence of phenylalanine is negligible as it possesses low absorptivity and low quantum yield. The intrinsic tryptophan fluorescence emission of a protein, interestingly, is strongly influenced and highly sensitive to its local microenvironment. For example, when tryptophan is buried within the hydrophobic core of the protein, it possesses a high quantum yield and displays strong fluorescence intensity. Conversely, its quantum yield decreases and tryptophan exhibits weak fluorescence emission when it is exposed to a hydrophilic solvent.
Here, we examine the GO–plasma protein interaction, in particular, the effect of GO on the intrinsic fluorescence of the three plasma proteins of albumin, globulin, and fibrinogen. We observe that GO quenches the intrinsic tryptophan fluorescence of all plasma proteins efficiently. Remarkably, we also demonstrate that, besides the robust fluorescence quenching, GO is capable of selectively amplifying the intrinsic tryptophan fluorescence of fibrinogen up to about 1.3 fold under specific low concentrations but not those of albumin and globulin. To the best of our knowledge, this is the first study reporting on the novel GO-induced enhancement of the intrinsic fluorescence of fibrinogen. In light of our experimental observations, we propose that GO can possibly play a dual role in controlling the intrinsic fluorescence of the plasma proteins. This fluorescence manipulation may be dictated by the direct competition between the aggregation and encapsulation effects.
Subsequent to the optical characterizations of both GO and plasma proteins, we started investigating the GO–plasma protein interactions by evaluating the absorbance characteristic of these proteins in the presence of GO nanosheets (Fig. 2). We observed that with the plasma proteins fixed at 1.1 μM, the addition of GO nanosheets in the system induced changes in the characteristic absorption spectra of all plasma proteins. Specifically, we noted diminished 280 nm absorption bands coupled with a corresponding increase in the absorption bands between 230 nm and 260 nm (Fig. 2a–c). In fact, the absorption spectra of the three proteins gradually adopted the shape of that of pure GO nanosheets as their concentration in the system increased. This demonstrates the strong molecular interactions between GO nanosheets and blood plasma proteins as well as possibly, a subtle change in the structures of the amino acid chains of the plasma protein molecules.
Next, we sought to investigate the effect GO nanosheets would induce on the intrinsic fluorescence emission of the three plasma proteins (Fig. 3). The intrinsic fluorescent probes of the three plasma proteins were excited at 280 nm and their fluorescence emissions were recorded from 310 to 420 nm. Fig. 3a–d show the evolutions of the intrinsic fluorescence of 1.1 μM albumin and globulin in the presence of GO nanosheets with increasing concentration, respectively. As anticipated, the emission intensity of both albumin and globulin decreased progressively with a corresponding increase in the GO concentrations from 0.5 to 25 μg mL−1. Strong interactions between GO nanosheets and albumin and globulin were noted according to the efficient quenching of the protein fluorescence emission. Importantly, this experimental data coincided with conventional observations in which GO serves as an effective fluorescent quencher for amino acids, proteins, and other fluorescent materials.8,9
In addition to those of albumin and globulin, we examined the evolution of the fluorescence emission of 1.1 μM fibrinogen in the presence of GO nanosheets with various concentrations (Fig. 3e–g). Intriguingly, in our system, besides the commonly recognized fluorescence quenching (Fig. 3f and g), we observed an amplification of the intrinsic fluorescence of fibrinogen in the presence of GO nanosheets with low concentrations, ranging from 1 μg mL−1 to approximately 3 μg mL−1 (Fig. 3e–g). Particularly, the intrinsic fluorescence of fibrinogen increased up to around 1.3 fold or 30% with the addition of 2 to 2.5 μg mL−1 GO (inset of Fig. 3g). Nevertheless, the low GO concentration-induced intrinsic fluorescence amplification was not detected from the fluorescence spectra of albumin and globulin. Beyond these GO concentrations, the fluorescence intensity of fibrinogen was gradually quenched and the emission reduced with no shift in its peak wavelength (Fig. 3f).
To shed light on the possible physical mechanisms underlying the manipulation of the intrinsic fluorescence of albumin, globulin, and fibrinogen by GO nanosheets, we sought to characterize the structural morphologies of the plasma protein–GO complexes upon the addition of GO with a range of concentrations (Fig. 4 and 5). As control, the surface morphologies of freshly cleaved mica (i.e., substrate on which the plasma proteins and GO–protein complexes were deposited and imaged) (Fig. 4a) and 1.1 μM albumin (Fig. 4b) and globulin (Fig. 4d) were examined using the tapping mode of an AFM. As observed, the albumin and globulin molecules possessed a regular circular shape and an approximate size of a few to tens of nm. Sectional profiles revealed that albumin and globulin displayed similar heights of about a few nm (Fig. 4b(ii and iii)) to 10 nm (Fig. 4d(ii and iii)), respectively. Contrastingly, the GO–albumin and GO–globulin complexes adopted irregular shapes (Fig. 4c and e). Furthermore, the topographical sectional features of the GO–protein complexes demonstrated that they had a height of approximately 20 nm (Fig. 4c(ii and iii) and e(ii and iii)) which was significantly larger than those of the albumin and globulin molecules. Importantly, upon a closer examination of the structural morphologies of the complexes, it is interesting to note that apparently, the GO–albumin and GO–globulin complexes were formed from the encapsulation or wrapping of individual protein molecules or aggregates within GO nanosheets. This, in fact, provides an important clue to the physical mechanisms driving the GO-induced fluorescence quenching.
Subsequent to characterizing the structural morphologies of the GO–albumin and GO–globulin complexes, we performed similar morphology characterization on the GO–fibrinogen complexes, especially, when the fluorescence emissions were amplified and quenched at GO concentrations of 2.5 μg mL−1 and 25 μg mL−1, respectively (Fig. 5). As control, 1.1 μM fibrinogen was deposited on mica and surface-characterized using AFM (Fig. 5a). As depicted in the AFM topographical images, the fibrinogen molecules adopted an elongated spheroid shape and possessed height of roughly five to 10 nm. When GO of low concentration of 2.5 μg mL−1 was mixed with fibrinogen, unlike albumin and globulin which were directly encapsulated or wrapped by GO nanosheets, it was highly likely that fibrinogen adsorbed onto the GO surface and aggregated into spherical-shaped structures, forming the GO–fibrinogen complex (Fig. 5b). Sectional profiles revealed that the fibrinogen aggregates possessed height of up to approximately 50 to 60 nm, depending on the size of the aggregates (Fig. 5bii and iii). However, when a higher concentration of GO of 25 μg mL−1 was added, fibrinogen was observed to form large spherical- and ellipsoid-shaped aggregates with heights of up to around 160 nm (Fig. 5c). Moreover, it was clearly evident that some fibrinogen aggregates were covered, encapsulated, or fully wrapped by multiple layers of large GO nanosheets, leading to the formation of large GO–fibrinogen complexes. In light of all the obtained data, we proposed the possible mechanisms driving the observed manipulation of the intrinsic fluorescence of the three plasma proteins (Fig. 6). In fact, the requirement for a particular low GO concentration for the fluorescence amplification of fibrinogen suggests that GO might play a dual role in controlling the fluorescence emission of fibrinogen.
In general, albumin and globulin are globular proteins with small physical sizes and molecular weights as compared to the large fibrinogen macromolecules. Upon interactions with GO nanosheets, it was highly likely that the GO–albumin and GO–globulin complexes formed immediately through the physical encapsulation or wrapping of the individual protein molecules by GO nanosheets (Fig. 4c and e). As these molecular interactions occurred, there would be energy transfer between GO nanosheets and plasma proteins as a result of the π–π stacking and interaction. This would eventually promote the quenching of the intrinsic fluorescence of both albumin and globulin. Also, as the physical distance separating GO nanosheets and individual plasma protein molecules reduced due to the formation of the GO–protein complexes and a progressive increase in the GO concentration in the system, a resultant increase in the π–π interaction and energy transfer would follow, leading to a corresponding increase in the GO-induced fluorescence quenching efficiency.
Fibrinogen, on the other hand, is a large elongated macromolecular structure.24 At low GO concentrations, fibrinogen and its hydrophobic tryptophan residue might bind non-specifically to GO nanosheets through hydrophobic interactions. Subsequently, the molecular motion and structural flexibility of fibrinogen decreased, leading to a corresponding decrease in the collision-induced non-radiative energy loss and a slight increase in the intrinsic fluorescence of fibrinogen. Recent study has reported that GO itself is capable of promoting and accelerating protein aggregation.25 In addition, it was possible that fibrinogen adsorbed on both sides of GO nanosheets, increasing the likelihood of the formation of more fibrinogen aggregates. Also, several studies have recently demonstrated the enhancement of the aggregation-induced fluorescence emission of active fluorophores by GO under certain concentrations.26,27
Here, it is noteworthy that under a similar low GO concentration range, the intrinsic fluorescence emissions of albumin and globulin were not amplified. Irrespective of the GO concentration in the system, the fluorescence emissions of both proteins were continuously quenched. As such, we suggest that the absence of the amplification of the fluorescence of albumin and globulin might be attributed to the synergistic effect brought about by the lower likelihood of both proteins to form aggregates as well as their rapid physical encapsulations by GO nanosheets. It was highly possible that the aggregates of the large macromolecular fibrinogen could be formed more easily as compared to those of the small albumin and globulin. This is because during the folding and/or unfolding processes, there was a higher probability for the hydrophobic backbone of fibrinogen to be exposed to external microenvironments as compared to those of albumin and globulin, triggering intermolecular aggregation.28,29 Moreover, the fibrinogen molecules comprise more domains than the albumin and globulin molecules. As a consequence, the interdomain surface contacts of fibrinogen might occur more frequently and at a higher rate than those of the other two proteins, initiating more aggregations. Finally, fibrinogen might experience more competing aggregation reactions due to the slower rate of refolding of its unfolded conformers. Intriguingly, the lower probability of albumin and globulin to form aggregates might also be ascribed to the rapid and facile physical encapsulations of these two plasma proteins due to their small sizes and molecular weights.
When the concentration of GO nanosheets increased beyond 3 μg mL−1, an opposite effect in the fluorescence emission of fibrinogen could be observed. Instead of fluorescence amplification, the intrinsic fluorescence of fibrinogen was quenched steadily in the presence of additional GO nanosheets. Interestingly, here, we observed that fibrinogen aggregates were also formed in the presence of increasing concentration of GO nanosheets (Fig. 5c). In fact, our data on the formation of a high number of fibrinogen aggregates in spite of the presence of hydrophilic GO corroborated with the observation of an earlier study,25 further confirming the capability of GO to induce protein aggregation despite its hydrophilic nature. Also, it is important to highlight that the intrinsic fluorescence quenching occurred although more fibrinogen aggregates were generated. Apparently, at high GO concentration, fibrinogen aggregation no longer played a dominant role in influencing fluorescence emission.
Alternatively, it was highly likely that an increase in the number of multilayer GO nanosheets led to an encapsulation or full wrapping of the fibrinogen aggregates (Fig. 5c), similar to that experienced by the albumin and globulin molecules. At the same time, with more GO nanosheets, the molecular interactions between fibrinogen and two or more overlapping and stacked GO nanosheets would increase proportionately as a result of the shortening of the physical distance separating the protein molecules from GO. With a significant increase in the GO–fibrinogen interactions, more energy transfer would occur between fibrinogen and GO due to a corresponding increase in the π–π stacking and interaction. Moreover, fibrinogen might experience a conformational change with increasing interactions with GO. Consequently, the shielded tryptophan residue which was previously hidden in the internal hydrophobic parts of fibrinogen would be exposed. This ultimately resulted in the dwarfing of the aggregation-induced effects for a robust quenching of the intrinsic fluorescence of fibrinogen. In short, we suggest that the fibrinogen aggregation and encapsulation effects contributed synergistically to the intrinsic fluorescence amplification and quenching of fibrinogen, correspondingly (Fig. 6).
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