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Abstract
This work deals with the fluorescence properties of the methylene blue (MB) fluorophores loaded on metal oxide nanoparticles, such as TiO2, ZnO, and Al2O3 based on laser-induced fluorescence (LIF) spectroscopy. At first, MB is provoked by diode laser at 665 nm, then the fluorescence emissions are recorded using a Czerny-Turner spectrometer. The lucid red shift appears during the right angle LIF measurements of (MB + TiO2NPs) suspensions, where NP are well distributed among the fluorophores. Despite that, the LIF of (MB + TiO2) demonstrates notable red shift in terms of NP concentrations; however, (MB + ZnO) and (MB + Al2O3) exhibit a negligible one. The larger red shift occurs for the NPs with greater refractive indices due to the optical elongation. Furthermore, the quenching coefficients KTiO2, KZnO, and KAl2O3 are measured according to the linear Stern-Volmer formalism. The quenching effect in a (TiO2+MB) attests to be much stronger than that of other nanoparticles of interest. The discrepancy in the fluorescence emissions of MB at the attendance of different metal oxide NPs is very significant during simultaneous imaging/diagnosis and treatment of tumors.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Recently, biomedical nanomaterials have received more attention because of their prominent biological characteristics and biomedical applications. Due to the development of nanomaterials, the metal oxide nanoparticles show promising and far-ranging prospect for biomedical field, especially as anti-bacteria, anticancer drug delivery, cell imaging, bio-sensing, and so on [1,2]. Compared with other metal oxide NPs, ZnO with the comparatively inexpensive and relatively less toxic property exhibits excellent biomedical applications [3–5]. ZnO nanoparticles are among the most promising emerging fluorescent labels for cellular imaging. Furthermore, hemolysis assay was performed to evaluate the biocompatibility of these NPs in vitro and even ZnO at very high doses, ensures their potential in biomedical applications [6]. On the other hand, photocatalyzed $\textrm{Ti}{\textrm{O}_2}$ NPs are used to eradicate cancer cells [7]. Particularly, the photo catalysis technique of $\textrm{Ti}{\textrm{O}_2}$ is an emerging treatment for a variety of cancers due to its high therapeutic efficacy and less side effect for healthy tissues [8]. It is worth noting that $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ NPs benefits various applications in the field of biomedicine and biotechnology, drug delivery, bio-sensing, destruction of microbes and bio-molecular stabilization too [9]. The NPs are toxic to cancerous cells, however minimal side effect to healthy cells [7].
MB is a phenothiazine dye, a kind of photosensitizer (PS) drug, with absorbance peak at 665 nm. MB is vastly used as PS in photodynamic therapy (PDT) [10]. Despite the material safety data sheet of Vanderbilt Environmental Health & Safety (VEHS) emphasizes that MB is slightly hazardous in the case of skin and eye contact, ingestion and inhalation, however no evidence is reported to be a carcinogenic material during its vast bio-applications [11]. Therefore, it is recognized as a well-known low toxic biocompatible material. Furthermore, MB exhibits photo toxicity toward a variety of tumor cells [12,13]. In last decade, the nanoparticles have received increasing attention as a potential for delivering PDT agents [14,15]. MB coupled nanoparticles are employed as the hybrid chemo drugs in PDT to utilize the assisted photo thermal therapy [16]. The local administration of MB is successfully carried out in the intra tumoral treatment of inoperable esophagi lesions [17]. Several biocompatible fluorophores such as Indocyanine green (ICG) [18], Cyanine7 (Cy7) [19–21], dialkylcarbocyanine fluorophores [22], and MB are also examined for the purpose of fluorescence imaging. In addition, high attention is received on the fluorophores which emit at NIR spectral region over therapeutic window (600-1200 nm) [23]. The latter is based on the fact that the most biomolecules show slight absorbance over that spectral range and give minimum interference such that the detection sensitivity can be significantly enhanced [24]. MB realizes fluorescence imaging of the healthy and cancerous tissues. In particular, wide-field planar near-infrared (NIR) fluorescence imaging revolutionizes the human surgery by providing real-time image guidance to the surgeons for the purpose of minimally invasive tumoral resection, while the healthy tissues, blood vessels and nerves are strictly avoided [25]. Steady-state and time-resolved fluorescence spectroscopy were employed to assist in vivo diagnosis and the subsequent laser assisted cancer therapy [26,27]. Various NPs have been also exploited to improve the diagnostic methods based on LIF spectroscopy [28,29]; however, the emission characteristics mainly depend on the dye concentration, the NP density as well as the optical properties of the excitation beam.
Bavali et al. reported red/blue shifts of LIF using $\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$, $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$, and $\textrm{Si}{\textrm{O}_2}$ in the host Rd6G, RdB, Coumarin 4, and Coumarin 7 ethanolic solutions [30]. However, Rd6G, RdB, Coumarin 4, and Coumarin are not biocompatible fluorophore. Moreover, Pahang et al. have examined the fluorescence quenching effects of carbon nano-structures (Graphene Oxide and Nano Diamond) coupled with MB. This attests that the chemical bonding formations mainly affect the quenching coefficient and spectral shift [31].
The fluorescence properties are useful for steady monitoring during fluorescence imaging in nano-onchology accompanying the selective cancer therapy. On the other hand, metal oxide NPs have received much attention recently due to their use in cancer therapy. Studies have shown that different metal oxide NPs induce cytotoxicity in cancer cells, but not in normal cells [7]. Thus, the fluorescence properties have been extensively investigated here based on the red spectral shifts in terms of the fluorophore concentrations and the additive metal oxide NP densities in (MB+ NP) suspensions. Particularly, these fluorescence properties resemble to be very helpful for simultaneous imaging and cancer therapy in nano oncology and drug release.
At first, the LIF and PL of the different concentrations of pure MB have compared to double check the results. We have shown that LIF is a better than PL, because PL fails detecting the signals for dye suspensions at dense concentrations. Then, the influence of $\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$, $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ nanocarriers/quenchers on the spectral properties of the LIF emission of MB aqueous suspensions is investigated. It is shown that the red shift depends on the refractive indices of NPs. Then the quenching coefficients ${\textrm{K}_{\textrm{Ti}{\textrm{O}_2}}}$, ${\textrm{K}_{\textrm{ZnO}}}$, ${\textrm{K}_{\textrm{A}{\textrm{l}_2}{\textrm{O}_3}}}$ are measured according to linear Stern-Volmer formalism.
2. Material and methods
The $Ti{O_2}$ nanoparticles are purchased from Sigma-Aldrich Co. with purity 99/7% and density 3.9 gr/mol and mean particle size of 25 nm. The $ZnO$ nanoparticles are supplied from Plasma Chem Co. with purity 99/5% and density 5.6 gr/mol and average particle size of 25 nm. The $A{l_2}{O_3}$ nanoparticles are purchased from Nanostructured and Amorphous Materials Inc. Co. with purity 99/97% and density 3.97 gr/mol and mean particle size of 25 nm. Various NPs in MB (${\textrm{C}_{16}}{\textrm{H}_{18}}{\textrm{N}_3}\textrm{SCl}$) suspension having the molecular weight of 319.86 gr∕mol, are prepared using the deionized water as the solvent.
LIF measurements are performed by making use of the diode laser at 665 nm with 100 mW power to excite the fluorophore molecules. Moreover, the Ava Spec 2048 fiber optic spectrometer is exploited with diffracting grating over a wide spectral range of 200-1100 nm having 0.4 nm resolution. The Avabench-75 symmetrical Czerny-Turner design with 2048 pixels CCD detector array is used to record the fluorescence emissions. A cubic quartz cuvette (1cm × 1cm × 4cm) is selected as the irradiation cell and the detection angle is set to be perpendicular to the laser beam (right angle arrangement). Figure 1 illustrates the schematic of experimental set-up for LIF spectroscopy.
Fig. 1. Schematic setup for right angle LIF experiment of (MB + NPs) suspensions and schematic of photon trajectory due to scattering, absorption, emission and reabsorption events.
Moreover, the UV-VIS absorption spectra are examined via a Perkin Elmer spectro-photometer with 0.1 nm accuracy over 200-1100 nm. Also, photoluminescence (PL) measurements are carried out using Perkin Elmer LS 55 photo Luminescence Spectrometer with photomultiplier (PMT) array detector and pulsed Xenon flash lamp at line frequency 50 Hz over 200-1100 nm spectral range with spectral accuracy of 1 nm.
Eventually, TEM images were collected using PHILIPS CM 300, 200 kV transmission electron microscope.
3. Result and discussion
Initially Fig. 2(a) illustrates the UV-VIS absorbance spectra of pure MB. The maximum absorbance takes place over red spectral range with a couple of certain characteristic peaks at 613 and 663 nm. Furthermore, a couple of UV characteristic emissions appear at 260 nm and 291 nm too. The peak centered at around 663 nm is due to π-π* transition associated with the resonance of the π electrons from the Sulphur resonating with those from the Cs in thiazinic center. The higher energy peak at 613 nm corresponds to π-π* transition of benzene rings [24].
Fig. 2. (a) UV-VIS spectral absorbance of MB and overlap of normalized absorption-emission spectra at a typical 50 µM concentration, (b) Spectral absorbance in terms of MB concentrations, Inset: absorbance versus concentration at typical peak 663 nm.
Furthermore, Fig. 2(a) demonstrates the corresponding overlapping area of the normalized absorption and fluorescence emission spectra. The Stokes shift of 27 nm is obtained for MB at 50 µM which shows a significant overlapping leading to the lucid re-absorption events.
Moreover, Fig. 2(b) displays the spectral absorbance in terms of MB concentration, which lucidly demonstrates a linear function at 663 nm as shown in inset. When the concentration increases, then the absorbance elevates, however no spectral shift appears.
The LIF of the various MB concentrations are examined. The fluorophore molecules are excited by means of a diode laser emission at 665 nm. Figure 3(a) depicts the peak emission wavelength versus MB concentration. This obviously emphasizes that the higher re-absorption events take place at dense solutions while the rate of red shift becomes smaller [30,31]. Note that the lucid red shift is mainly due to high re-absorption events in large overlapping according to Fig. 2(a). The inset of Fig. 3(a) illustrates the fluorescence emission spectra against different concentrations of pure MB over a wide range of concentration (10–500µM) which initially elucidates the enhancement of the fluorescence signal and rise of red shift rate at dilute MB concentrations and then a notable reduction of fluorescence intensity corresponding to slowing down rate of the red shift at denser concentrations.
Fig. 3. (a) Emission wavelengths in terms of MB concentrations that indicates a notable red shift, inset: Fluorescence emissions due to various concentrations of MB solution (10–500µM), excited by diode laser line at 665 nm, (b) PL emission wavelengths in terms of MB concentration (10–200 µM), inset: PL spectra for various MB concentrations.
Similarly, PL data verifies those of LIF. Figure 3(b) illustrates the peak emission wavelength versus MB concentration to attest the degree of red shift. The inset of Fig. 3(b) highlights the PL spectra for different MB concentrations ranging (10–200 µM). The findings confirm LIF spectra up to 200 µM because of the technical limitation of PL instrument. However, the LIF spectrometer enables us to collect signals from dense solution up to 1000 µM. On the other hand, PL fails to detecting the signals for dye suspensions at dense concentrations above 200 µM. Here, LIF and PL spectrometers give us complementary data to double check the findings particularly by scanning of the excitation wavelengths. In comparison, the use of coherent source and high resolution spectrometer are considered as the advantage of LIF, whereas PL utilizes PMT to detect very faint emissions.
Subsequently, varieties of the guest NP densities are examined for the host fluorophore (MB) in certain concentrations during the systematic measurements in favor of the fluorescence spectral shift. The suspensions are treated by making use of the ultrasonic bath in order to assure NPs homogeneously diffuse throughout the dye solutions.
At first, $\textrm{Ti}{\textrm{O}_2}$ NPs are added to the dye solution. Figure 4(a) illustrates the corresponding LIF spectra due to a typical concentration (100 µM) of aqueous solutions for various $\textrm{Ti}{\textrm{O}_2}$ NPs densities 5, 25, 50, 150, 250, 500, 1000, 1500, 2000, 2500 (µg/cc), respectively. This indicates that a sensible red shift takes place, particularly in low densities and even at more dense $\textrm{Ti}{\textrm{O}_2}$ NPs for a certain MB concentration as shown in Fig. 4(b). The fact, this mainly arises from the elongation of optical path (nd) due to the excessive NPs in suspension that gives rise to further re-absorption events leading to the larger red shift. The intensity of fluorescence decreases due to the elevation of quenching events at higher NP densities as shown in the inset Fig. 4(a).
Fig. 4. (a) LIF spectra of fluorescence intensity versus $\textrm{Ti}{\textrm{O}_2}$ NP density at a certain MB concentration of 100 µM, inset: max intensity versus $\textrm{Ti}{\textrm{O}_2}$ density at the same MB concentration, and (b) Peak fluorescence wavelength in terms of $\textrm{Ti}{\textrm{O}_2}$ density.
Eventually, Fig. 5 depicts the peak fluorescence wavelength emission of various suspensions (MB + $\textrm{Ti}{\textrm{O}_2}$), (MB + $\textrm{ZnO}$) and (MB + $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) versus NPs densities at certain MB (50, 100, 200 µM) concentration respectively. Figure 5(a) obviously represents that notable red shifts appear in terms of $\textrm{Ti}{\textrm{O}_2}$ densities at certain MB concentrations (50, 100, 200µM). The larger wavelengths at peak emission appear versus $\textrm{Ti}{\textrm{O}_2}$ densities mainly due to the excessive re-absorption and the optical path elongation. In the case of MB concentration ranging 50 to 200 µM, a red shift of ∼11 nm undergoes for $\textrm{Ti}{\textrm{O}_2}$ densities (0.5 - 2500 µg/cc).
Fig. 5. Fluorescence peak of (NP guests + MB) suspensions at certain MB concentrations (50, 100 and 200 µM) as a function of (a) $\textrm{Ti}{\textrm{O}_2}$, (b) $\textrm{ZnO}$, (c) $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ NP densities ranging 0.5 - 2500 µg/cc. Note that the plateau does not appear in (MB + $\textrm{Ti}{\textrm{O}_2}$ NPs) suspension, whereas the lucid plateau is seen for the other suspensions of interest.
Similarly, Fig. 5(b) displays the peak emission wavelengths versus $\textrm{ZnO}$ NP densities in various MB concentrations and Fig. 5(c) shows the emission wavelengths in term of $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ NP densities in various MB concentrations. Note that the spectral shift does not take place in case of (MB + ZnO) and (MB + $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) over wide range of NP densities. Indeed (MB + ZnO) and (MB + $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) suspensions undergo a different behavior that give rise to the plateau indicating the balance of re-absorption and quenching events leading to complex formations. In fact, a max red shift of 6.9 and 7.2 nm are measured for (MB + $\textrm{ZnO}$) and (MB+$\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) suspensions respectively versus 50-200 µM MB concentrations.
Finally, Fig. 6 plots the peak emission wavelength in terms of NP densities (0.5 - 2500 µg/cc) at a certain MB concentration (100 µM) for different NP densities of $\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$ and $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$. Similarly, the notable red shift appears in the case of $\textrm{Ti}{\textrm{O}_2}$ NP additives. Conversely, the other nanoscatterers ($\textrm{ZnO}$, $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) demonstrate negligible spectral shifts. This arises from the small refractive indices of $\textrm{ZnO}$ and $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ NPs leading to the negligible red shift which enhances the competitive mechanism to balance the induced blue shift due to the complex formations at dense NP densities.
Fig. 6. Emission wavelength in terms of NP density in the case of various nanoscatterers 0.5 - 2500 µg/cc which benefit ∼ 25nm mean diameter suspended in typical 100 µM MB solution.
The refractive indices of the various types of nanoscatterers of interest ($\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$ and $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) in the dye solutions are measured to be 2.8, 2.1 and 1.8 respectively. This elucidates that for a given NP density, larger red shift takes place particularly where the higher refractive index is used due to the optical path elongation which arises mainly from the Rayleigh elastic scattering cross section (${{\sigma }_\textrm{s}}$), in accordance with longer random walk [31,32]. The latter gives rise to the higher re-absorption rates. The graphical representation of the dominant mechanism of the multiple scattering, the fluorescence emission and the re-absorption events are given in Fig. 1.
In fact, the plateau arises from competitive effects of red and blue shift mechanisms. The former originates from the re-absorption rate and larger random walk at dilute suspensions, whereas the latter attests the depopulation of fluorophores and the formation of complexes and conjugate molecules at dense suspensions. The result gives rise to the balance of competitive effects to form a wide plateau. In the case of $\textrm{Ti}{\textrm{O}_2}$ due to the higher optical path and large NP refraction indices, then the red shift overcomes the blue shift.
In fact, the addition of nanoparticles to MB reduces the fluorescence intensity due to extra quenching events. Figure 7 represents the fluorescence ratio (${\textrm{F}_0}/\textrm{F}$) versus the additive quencher density at a typical 100 µM of MB suspensions. It lucidly emphasizes that the ratio ${\textrm{F}_0}/\textrm{F}$ versus the nano-quencher density [Q] would be linear in static mode. This is in good agreement with the Stern-Volmer equation, ${\textrm{F}_0}/\textrm{F} = 1 + \textrm{K} [\textrm{Q} ]$, where ${\textrm{F}_0}/\textrm{F}$, $\textrm{K}$, and $[\textrm{Q} ]$ ascertain the fluorescence intensity in the absence (presence) of quencher, the quenching constant and the quencher density, respectively [31,33]. Regarding the linear regression over the experimental data, the quenching coefficients i.e., the slopes of the straight lines (${\textrm{K}_{\textrm{Ti}{\textrm{O}_2}}}$, ${\textrm{K}_{\textrm{ZnO}}}$, ${\textrm{K}_{\textrm{A}{\textrm{l}_2}{\textrm{O}_3}}}$) are determined to be 0.0049 and 0.0004 and 0.0001 (µ${{\textrm{g}/\textrm{cc}} )^{ - 1}}$, respectively. Therefore, the quenching effect in a ($\textrm{Ti}{\textrm{O}_2}$+MB) attests to be much stronger than that of other nanoparticles of interest. In fact, ($\textrm{Ti}{\textrm{O}_2}$+MB) exhibits a dynamic quenching and the suspensions undergo static quenching. Table 1 tabulates the findings including quenching coefficient and refractive index and the corresponding spectral shift of ($\textrm{NPs}$+MB) suspensions.
Fig. 7. ${\textrm{F}_0}/\textrm{F}$ ratio versus additive density according to linear Stern-Volmer quenching formalism for $\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$ and $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ in MB solution (100µM).
Table 1. Quenching coefficients, spectral shifts and refractive indices of NPs of interest in MB suspension.
TEM images of three suspensions of interest have been obtained to assure uniform distribution of NPs in suspension and evidences of some attachments. Furthermore, the size of NPs is visualized in TEM images as shown in Fig. 8 ranging 20-30 nm. $Ti{O_2}$ NPs undergoes loose or no bondings in close proximity with MB fluorophores accompanying a lucid red shift, whereas $ZnO$ and $A{l_2}{O_3}$ NPs experience a degree of blue shift, as well as evidences of some attachments with MB molecules.
Fig. 8. Typical TEM images of a) (MB+$\textrm{ZnO}$), b) (MB+$\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$).
4. Conclusion
The fluorescent hybrid scattering media is a challenging issue in bio-optics. Here, systematic experiments have been carried out to investigate MB as a biocompatible fluorophore coupled with several metal oxide NPs. Such study can be useful in simultaneous diagnosis/ imaging in the course of selective cancer therapy using (MB + metal oxide NPs).
At first, the fluorescence property of pure MB is studied at various concentrations. Then, the fluorescence properties of (MB + NP) suspensions are investigated. Three different metal oxide NPs are examined with distinct refractive indices investigating the effect of their scattering strengths on MB fluorescence emissions. The red shift against MB concentrations (and NP densities) are carefully measured. The red shifts of MB loaded in NPs with various refractive indices such as $\textrm{Ti}{\textrm{O}_2}$, $\textrm{ZnO}$, $\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$ are obtained against certain fluorophore concentrations (typically 50, 100, 200 µM) in various NPs densities (0.5 - 2500 µg/cc).
The fluorescence spectra of (MB + $\textrm{Ti}{\textrm{O}_2}$) demonstrates a notable red shift in terms of NP concentrations. In fact, further red shift occurs for the NPs with larger refractive indices mostly due to the optical elongation. On the other hand, the peak fluorescence wavelength for (MB + $ZnO$) and (MB +$\textrm{A}{\textrm{l}_2}{\textrm{O}_3}$) remains nearly invariant indicating a plateau over various nanoscatterers with low index of refraction. That is why, $\textrm{Ti}{\textrm{O}_2}$ NPs resembles to be quite suitable for bio-imaging due to its unique fluorescence features.
Eventually, the quenching coefficients of various (MB + NP) suspensions are measured using linear Stern-Volmer formalism, indicating (MB + $\textrm{Ti}{\textrm{O}_2}$) attains a larger quenching coefficient accompanying a notable red shift against the other NPs of interest.
Acknowledgment
This work is totally funded personally by Prof. Parviz Parvin.
Disclosures
The authors declare no conflicts of interest related to this article.
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