Fluorescence “Giant” Red Edge Effect
4.3 Characterization
4.3.1 Structural characterization
Structure analysis and characterization focuses on contribution of significant com- ponents such as crystal structure, structural parameters, and position of the func- tional groups as these have important effects on the characteristics and properties of the material. These are generally known to be structureactivity or structureprop- erty. In other words, these are collectively termed as structural characterization techniques—namely, XRD, SEM, TEM, Raman, XPS, FTIR, AFM, UVvis, etc.
4.3.1.1 X-ray powder diffraction
XRD is an efficient analytical approach for determining material’s crystal structure, unit cell size, crystal spacing, and phase identification. CQDs are both amorphous and crystalline in nature which can be confirmed by XRD results; this may depend on the synthesis procedure. Zhang et al. reported the XRD pattern of CQDs shows the diffraction peak at 20.5 of 2θ reflecting the crystalline graphitic structure (Fig. 4.19A)[56]. Monte et al. reported low intense diffraction peak centered at 20
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Figure 4.19 (A) XRD pattern of crystalline CQDs[56], (B) XRD pattern of amorphous CQDs [57], (C) shows high degree of C (002) plane for the partial graphitic and the amorphous nature of N-CQDs[58]. (D) XRD pattern of UCQD[45].CQDs, Carbon quantum dots.
of 2θ which corresponds to amorphous carbon phase (Fig. 4.19B) [57]. Xiao et al.
successfully synthesized N-CQD characterized by using XRD which exhibits the broad peak centered at 2θ524. For the partly graphitic and amorphous character of N-CQDs, this spectrum is attributed to disordered high degree of carbon plane (002) (Fig. 4.19C)[58]. In XRD pattern for undoped CQDs, as shown in the Fig. 4.19D, the diffraction peak is observed at 2θ522.4reflecting to amorphous nature[45].
4.3.1.2 Scanning electron microscope
An SEM gives information about surface morphology, granular orientation, crystal- lographic information, and chemical composition of CQDs. The morphology of CQDs clearly shows spherical and spongy nature and the size of about 5060 nm, as shown in Fig. 4.20A [47]. Also Fig. 4.20B shows SEM image of M-CQDs around 100 nm[51]. The surface morphology of the CQDs cannot be obtained at higher magnifications in the SEM, hence we can go for the TEM as it gives good surface morphology and further crystal information.
4.3.1.3 Transmission electron microscope
TEM is a technique where a beam of electrons is transmitted through an ultrathin specimen. These electrons are interacting with the atoms in the material which gives magnified and focused image onto an imaging device which can be detected by a sensor or a camera. The principal approach for visualizing CQDs is TEM, which provides crucial information on particle shape, size distribution, and crystal- line structure. Thambiraj et al. observed HRTEM by adding a drop of CQDs solu- tion in copper grid and coating it. After the grid dried thoroughly, it was investigated using energy-dispersive X-ray spectroscopy (EDS) with a 200 kV acceleration voltage[59]. Because TEM has a high resolution of 0.1 to 0.2 nm, it may be utilized to determine the ultrastructure of CQDs[60]. TEM indicates that Figure 4.20 SEM images of (A) CQDs[47]and (B) M-CQDs[51].CQDs, Carbon quantum dots.
the CQDs are near spherical in shape and uniformly distributed, which are in range between 2.4 and 4.7 nm in diameter [61]. In determination of crystallinity, two types of lattice fringes are used, that is, in-plane lattice spacing and interlayer spac- ing, respectively. According to Zou et al., interlayer spacing typically focused at around 0.34 nm, while in-plane lattice spacing focused at 0.24 nm [62].
Thambhiraja et al. claimed that the HRTEM image obviously indicates that the CQDs are smaller in size, approximately spherical in form, and monodispersed, with particle size distributions examined. The author observed CQDs that have a graphite-like structure and produce 87.18 wt.% high purity carbon[59].Fig. 4.21A shows the TEM image of CQDs are spherical and monodisperse, and the particle size of the CQDs is 2.560.5 nm, andFig. 4.21Bshows particle size of CQDs cor- responding to the (102) crystal planes of sp2 graphitic carbon. Fig. 4.21C shows selected area electron diffraction (SAED) pattern of CQDs.
Precisely, crystal size and crystalline nature of CQDs can be studied by using SAED pattern. As reported by Chen et al., bright spots in the SAED pattern of the TEM analysis indicate the presence of CQDs and determine the type of CQDs as amorphous or poor crystalline[63].
4.3.1.4 Raman spectroscopy
Raman scattering is used as very basic analytical tool for detecting CQD formation. A standard Raman spectrum has two peaks, one for the D band and the other for the G band. The D band is about 1350/cm and is attributed to disordered sp2 carbons, whereas the G band is about 1600/cm and is attributed to crystalline graphite carbons in-plane E2gstretching vibration mode[15]. The ratio of the D and G intensities of the characteristic Raman bands can be utilized to examine structural characteristics such as crystallinity and the relative concentration of core over surface carbon atoms [15].
Wang et al. demonstrated two predominant peaks at 1340 and 1590/cm attributed to the disordered D and crystalline G bands as shown inFig. 4.22Aand intensity ratio of Figure 4.21 (A) TEM image of CQDs, (B) histogram of size distribution[56], and (C) SAED pattern CQDs.CQDs, Carbon quantum dots.
Source: Adapted with permission from B. De, B. Voit, N. Karak, Carbon dot reduced Cu2O nanohybrid/hyperbranched epoxy nanocomposite: mechanical, thermal and photocatalytic activity, RSC Advance 4 (102) (2014) 5845358459, doi:10.1039/c4ra11120f.
the D-band and G-band (ID/IG) of the CQDs was about 1.3, indicating a large amount of amorphous nature in CQDs[3]. Kumar et al. analyzed that the occurrence of pristine CQDs is indicated by the strong and intense Raman peak of the G-band observed at 1578/cm in comparison to the weak peak of the D-band at 1331/cm, and the ratio of intensities of ID/IG was determined to be 0.59, confirming the CQDs purity [64].
Yongsheng and his coworkers explained CQDs through Raman peak G-band at 1590/
cm and weaker D-band peaks around 1357 and 1418/cm shown inFig. 4.22Band con- cluded CQDs are predominantly graphitic based on G peak strength[65].
Ramar and co-authors proved that Raman spectroscopy is a nondestructive proce- dure for detecting and evaluating nanostructures that is rapid, precise, and powerful.
The authors recorded Raman spectra for UCQDs and N-CQDs using a Lorentzian fit, and computed an ID/IG intensity ratio of 0.86, which indicates the cleanliness of the UCQDs and is inversely related to the common size of the sp2crystalline region. At the same time, N-CQDs are extremely sensitive to strain effects in the sp2system, with an ID/IG intensity ratio of 0.854, which is nearly identical to that of UCQDs (see Fig. 4.22), D and G band of UCQDs and N-CQDs[45].
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Figure 4.22 ( A) Raman spectroscopy of CQDs peak at 1340 and 1590/cm. (B) CQDs show graphitic nature. (C) UCQDs and N-CQDs.CQDs, Carbon quantum dots.
Source: Adapted with permission from Refs. (A)[3]; (B)[65]; (C)[45].
4.3.1.5 X-ray photoelectron spectroscopy
XPS gives information of particular atomic units on surface of CQDs. With mono- chromatized Al Ka radiation, the spectral analysis reveals unique nitrogen-, oxy- gen-, and carbon-bonded units shown on the CQDs surface[66]. XPS is a technique for analyzing primarily C and O components, with minimal N and S element XPS can be used to determine the chemical structure and content of CQDs, according to Wang et al. The XPS survey spectra of CQDs showed three typical strong peaks at 284.7, 399.4, and 531.9 eV, which were commonly assigned to C1s, N1s, and O1s, respectively, showing that CQDs largely comprised C (73.18%), N (7.23%), and O (19.59%) elements, as shown inFig. 4.23A. Four peaks occurred at 284.3, 285.1, 286.4, and 288.4 eV in the C1s spectra (Fig. 4.23B), originating from the sp2CC/
C5C, CN, CO, C5O groups, respectively [67]. The N1s band of CQDs (Fig. 4.23C) may be deconvoluted into two peaks at 399.4 and 400.9 eV, which cor- respond to the pyrrolic N and amine groups, respectively. The primary component of N-CQDs was pyrrolic N, which produced through the dehydrolysis reaction
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Figure 4.23 XPS spectrum of CQDs, (A) XPS full-survey spectrum of CQDs, (B) high- resolution XPS spectrum for C1s, (C) N1s and (D) O1s.CQDs, Carbon quantum dots.
Source: Adapted with permission from X. Wang, et al., Green preparation of fluorescent carbon quantum dots from cyanobacteria for biological imaging, Polymers (Basel) 11 (2019) 4, doi:10.3390/polym11040616.
between carboxyl and amine groups. Previous reports have proposed that the pyrro- lic N is likely to improve electronic cloud density of CQD surfaces, resulting in an enhancement in luminescence efficiency[68]. In the O1s spectra (Fig. 4.23D), the peaks at 531.5 and 532.9 eV are assigned to the binding energies of C5O and CO, respectively [69]. Dong et al. experimented with NSCQDs and observed the high-resolution scan of the C1s region gives information that C was present in vari- ous chemical environments. By using their experiments, concluding s2p XPS peaks occurred at 163.4 and 164.6 eV, nearer to the s2p2/3 and s2p1/3 positions for their spin-orbit couplings[24]. Thus, the creation of doping N and S in NSCQDs as well as amide N on the surface of NSCQDs were clearly indicated by C1s, O1s, N1s, and s2p XPS spectra, which both considerably contributed to the fluorescence increase of NSCQDs. As a result of these findings, carboxyl and hydroxyl groups were most likely generated on the surface of NSCQDs[70]. Overall, the resultant CQDs contained multiple O- and N-related functional groups that paves the way for their excellent water solubility and fluorescence emission[3].
4.3.1.6 Fourier-transform Infrared
FTIR spectroscopy is used to investigate the chemical bonding, surface functional groups, and structure of CQDs. The spectra of the CQDs were measured in the wavenumber ranging from 400 to 4000/cm. Meiqin et al. investigated CQDs and found oxygen functionalities peaks at 3450 (OH stretching vibrations), 2927/cm, 1407/cm (CH stretching vibrations), 1726/cm (C5O stretching vibrations), and 1639/cm (C5C stretching vibrations) as shown in Fig. 4.24A [71]. Fig. 4.24B
Figure 4.24 (A and B) FTIR spectrum of CQDs[72,73].CQDs, Carbon quantum dots.
Source: Adapted with permission from M. He, J. Zhang, H. Wang, Y. Kong, Y. Xiao, W.
Xu, Material and optical properties of fluorescent carbon quantum dots fabricated from lemon juice via hydrothermal reaction, Nanoscale Research Letters 13 (2018), doi:10.1186/
s11671-018-2581-7; A. F. Shaikh, M. S. Tamboli, R. H. Patil, A. Bhan, J. D. Ambekar, B. B.
Kale, Bioinspired carbon quantum dots: an antibiofilm agents, Journal of Nanoscience and Nanotechnology 19(4) (2018) 23392345, doi:10.1166/jnn.2019.16537.
shows that broad peak around 3435/cm is due to hydrogen bonding that can be con- cluded that there is existence of hydroxyl groups on the surface of the CQDs. Also, the peaks at 1085 and 1046/cm contribute to the asymmetric and symmetric stretch- ing vibration of COC, and the peak around 669900/cm attributed to the aro- matic out of plane of CH bending[29].
4.3.1.7 Atomic force microscopy
AFM used to identify topographic height in 2D and 3D structures[29]. Thambiraj et al. studied the surface topography of the CQDs which are decorated on the UV- treated silicon wafer. CQDs have average sizes of 35 nm and a surface roughness of less than 5 nm, in a counting scale. The CQDs’ histogram exhibited an average roughness of 4.2 nm [61]. The height profiles display that the typical topographic height difference of the N-CQDs approached 2 nm, as illustrated in Fig. 4.25, reveals the small spherical morphology of the N-CQDs[19].
4.3.1.8 UVvis spectra
UVvisible spectra frequently show apparent optical absorption in the UVvisible region; the CQDs exhibits absorption spectra of around 260323 nm. Surface pas- sivation of CQDs with various compounds leads to a shift in absorbance to longer wavelength according to the findings. The optimal emission wavelength red shifts as particle size increases[52].
Fig. 4.26A shows UV absorption spectra of N-CQDs. As shown in the figure, the absorption peak around 249 nm due to theπ!π electronic transition of aro- matic C2C bonds and another peak occur around 355 nm from the n!π elec- tronic transitions of the C5C and C2N bonds, ascribed due to the N heteroatoms induced resulted in the formation of excited defect surface states[34,58]. Four typi- cal sizes of CQDs by white and UV light are shown inFig. 4.26B. CQDs’ radiated colors are bright and vivid enough to be seen with the naked eye[68]. At 360 nm, Figure 4.25 AFM images of N-CQDs.CQDs, Carbon quantum dots.
Source: Adapted with permission from L. Cao, et al., Competitive performance of carbon
‘quantum’ dots in optical bioimaging, Theranostics 2(3) (2012) 295301, doi:10.7150/
thno.3912.
CQD has a significant UVvisible absorption peak [62]. Two peaks at 200 and 420 nm are obtained in N-CQDs[51].
4.3.2 Photophysical analysis
Excitation-dependent PL, also known as excitation-dependent fluorescence emis- sion, is perhaps the most remarkable attribute of CQDs. Typical excitation- dependent luminescence spectra are shown, along with the accompanying colors.
The multicolor features of CQDs are highlighted by their wide spectrum range and relatively high emission peak intensities. Indeed, one of the distinctive aspects of CQDs is that the emission color can be changed according to the excitation wave- length, which has implications in a variety of applications.
4.3.2.1 Photoluminescence
PL is size-dependent optical absorption, which is one of the most interesting aspects of CQDs. It is one of the remarkable characteristics of CQDs that depends on emis- sion wavelength and intensity, as different emissive traps exist at the surface of CQD[67].
Ding et al. claimed that changing the emissive trap sites on identical particles of the same size has a greater impact on the excitation wavelength of CQDs PL. The QCE theory applicable for the PL of CQDs[12]. When the size of a material is smaller than the Bohr excitons radius and within the same order of magnitude as the de Broglie wavelength of the electron, QCE occurs, resulting in a discrete size-dependent energy bandgap between the valence and conduction bands[68]. As a result of the absorption of a photon, one electron from the valence band’s HOMO is promoted to the conduc- tion band LUMO, resulting in fluorescence, which blue shifts with decreasing particle size. Zhou et al. showed that the fluorescence of oxidized CQDs was size dependent, with both excitation and emission spectra showing a red shift when CQDs molecular weight increases[6]. Another study clarifies the visible PL of GQDs of various shapes
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Figure 4.26 (A) The UVvis absorption spectra of N-CQDs in aqueous solution are shown:
(B) white (left; ordinary lamp; hues are pale green, pale yellow, yellow, and red,
respectively) and UV light illuminate four common sizes of CQDs (right; 365 nm; from left to right the colors are blue, green, yellow, and red, respectively)[33,66].CQDs, Carbon quantum dots.
and sizes and confirms the size and shape dependence of PL. The PL emission red- shifted as the size of GQDs increases. Furthermore, maximal absorption occurred at longer wavelengths within the same size range, and intensity declined as GQD size increased. The edge states of CQDs were also governed by their shape with increasing armchair edges appearing as the size climbed above 17 nm, at which time overall shape shifted from circular to polygonal. As a result, the emission intensity as the size of the particle grew larger than 17 nm.Fig. 4.27Ashows the PL emission spectrum of CQDs stimulated from 245 to 395 nm, Fig. 4.27B shows PL decay lifetime profile of the CQDs as excitation wavelength at 337 nm and emission wavelength at 437 nm, and Fig. 4.27Cshows corresponding PL emission spectra of the eight samples, with peaks at 440, 458, 517, 553, 566, 580, 594, and 625 nm. Another potential explanation for PL is that it is caused by the presence of surface energy traps on surface imperfections
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Figure 4.27 (A) PL emission spectrum of CQDs excited between 245 and 395 nm. (B) PL decay lifetime profile of CQDs. (C) The eight samples PL emission spectra, with maxima at 440, 458, 517, 553, 566, 580, 594, and 625 nm.CQDs, Carbon quantum dots.
Source: Adapted with permission from H. Ding, S. B. Yu, J. S. Wei, H. M. Xiong, Full-color light-emitting carbon dots with a surface-state-controlled luminescence mechanism, ACS Nano, 1 0(1) (2016) 484491. doi:10.1021/acsnano.5b05406; V. A. Online, et al., Nitrogen- doped, carbon-rich, highly photoluminescent carbon dots from ammonium citrate †,
Nanoscale 6 (2014) 18901895, doi:10.1039/c3nr05380f.
caused by the passivation process. Furthermore, the optical behavior of CQDs may be determined by the dispersion of various emissive sites on the optical characteristics of the produced CQDs solution in pH 6.9 phosphate buffer as analyzed by Xu et al.
4.3.2.2 Fluorescence
As shown in Fig. 4.28A, the position of the emission peaks varies from 428 to 510 nm as the excitation wavelength increases from 330 to 470 nm, and the fluores- cence intensity increases to a maximum and then gradually falls. It means that the CQDs are affected by the wavelength of stimulation. The highest emission peak is redshifted because the CQDs have the general properties of quantum dots, such as size effects, point defects, edge effects, and so on [66]. The maximum excitation wavelength and the maximum fluorescence wavelength of N-CQDs are 350 and 445 nm, respectively. Such excitation wavelength-dependent emission character is derived from the π!n electronic transitions of the surface-attached C5C and CNH2bonds, which indicates that the fluorescence band can be tuned by adjusting the excitation wavelength without changing CQDs size. Fig. 4.28DG shows the
Figure 4.28 (A) Fluorescence spectrum of CQDs at different excitation wavelength and (B, C) represents the CQDs under normal light and under excitation at 365 nm. (DG) The CQD images captured with a fluorescent microscope at several excitation wavelengths 360, 390, 470, and 540 nm, respectively. (H) The variation in the emission peak positions by varying the excitation wavelength, and (I-A and B) fluorescence emission of CQDs observed in water and ethanol respectively.CQDs, Carbon quantum dots.
Source: Adapted with permission from Refs.[57,58,66,67].
fluorescent microscope images of CQDs under excitations at 360, 390, 470 and 540 nm, respectively.Fig. 4.28I,A and Bshows the fluorescence of CQDs under dif- ferent excitation wavelengths in water and ethanol as solvents, respectively. From the figures it can be observed that for both the solvents the peak intensity increases with increase in the excitation wavelength. For aqueous system, the peak intensity reaches the maximum at 380 nm whereas increases upto 460 nm in ethanol solvent. The peak intensity for the same CQDs in ethanol as solvent shows higher values as compared to the water-dispersed CQDs. N-CQDs have an average fluorescence lifespan of 15.0 ns, which is close to the maximum value[71]. N-CQDs have a higher QY than 80%, since N-CQDs are stable over a pH range (411), high ion strength (2 M KCl), and 4 h of continuous UV light irradiation, making them ideal for preservation and transit. Dry N-CQDs powder can be redispersed in water multiple times without aggregating in solution, and no significant changes in fluorescence spectra are detected. N-CQDs that have an extremely long fluorescence lifespan, a high QY, and outstanding stability can be used in a variety of biomedical applications.
4.3.2.3 Forster resonance energy transfer
The fluorescence phenomena of Forster resonance energy transfer (FRET) have shown in CQDs. FRET is a nonradiative energy transfer between two fluorophores that are physically near to each other (fluorescence donor and acceptor). As a result, the approach is beneficial in determining the structural characteristics of materials, particu- larly biological molecules. FRET has been used to detect changes in intramolecular dis- tances caused by protein folding processes by embedding the donor and acceptor molecules within the lipid bilayer framework. In a lipid bilayer system, containing hydrophobic CQDs that can serve as energy donors to two different acceptor mole- cules, each excited at a different wavelength. In some cases, the CQDs could also serve as a FRET acceptor for the donor molecules.Fig. 4.29demonstrates the feasibility of FRET interaction of CQDs with riboflavin for determining the concentration of the organic molecule. FromFig. 4.29Ait can be seen that there is a good overlap of the absorbance spectrum of the riboflavin with the emission spectrum of the CQDs excited at 380 nm. This overlap is essential for the FRET interaction between the two entities, where the CQD serves as the donor and the riboflavin acts as the acceptor. As shown in Fig. 4.29B when the riboflavin concentration is increased gradually, the emission intensity of CQDs at 440 nm gradually decreased, whereas the riboflavin peak at 520 nm increased gradually, demonstrating the FRET interaction between the two. As the FRET efficiency is depending on the concentration of the riboflavin molecules and the excitation wavelength of the CQDs, the FRET process efficiency can be calculated by EFRET5[(F0-F)/F0]3100 (where F and F0represent the fluorescence intensity at 440 nm of CQDs in presence and absence of the riboflavin, respectively). The calcu- lated FRET efficiency is plotted and explained inFig. 4.29C. FRET has also been used to monitor membrane remodeling. CQDs have a wide range of excitation/emission wavelengths and can act as both donors and acceptors in FRET investigations. These characteristics construct a single CQDs species that can function as an energy donor to a variety of acceptors by simply changing the excitation wavelength[71].