Biology

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Preparation, textural and photoluminescence characterization of green ﬂ uorescence protein-immobilised Ga-ZnO (GZO)-nanocomposites

Jothi Ramalingam Rajabathar

^a,b,

⁎ , Murugan A. Munusamy

^c

, Hamad A. Al-Lohedan

^a

aSurfactants Research Chair, Chemistry Department, College of Science, King Saud University, P.O.Box. 2455, Riyadh 11451, Saudi Arabia

bSchool of Engineering, University of Ulsan, Ulsan, South Korea

cDepartment of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

a b s t r a c t a r t i c l e i n f o

Article history:

Received 31 August 2016 Accepted 22 October 2016 Available online 24 October 2016

Nanostructured thinfilms of Gallium doped Zinc oxide (GZO) with nanodisk/nanorods and nanoflower morphologies are fabricated on a piezoelectric substrate. Pure wurtzite phase of GZO with nanostructure morphologies were prepared by a one-step spin coating process, followed by hydrothermal treatment. A non-ionic polymer (polyethylene imine) was used as a structure assisting agent to selectively form the nanodisks or nanoflowers depending on the reaction conditions. The morphology, nanostructure, and physicochemical properties of GZO were studied by X-ray diffraction, scanning electron microscopy (SEM) and Raman spectroscopy. The thicknesses and lengths of the individual GZO nanodisks were measured by FE-SEM. Thefine nanodisk and nanoflower structures are obtained. Greenfluorescent proteins were immobilised on the as-synthesised GZO nanostructured materials by dip coating. Atomic force microscopy was used to study the surface roughness of the GZO nanodisks, nanoflowers and nanorods. Photoluminescence techniques were used to study the GZO nanodisk structural defects and its optical properties. Fluorescence spectrometry analyses confirmed the binding of greenfluorescent protein on the GZO nanostructure surface. The biocompatibility study of GZO nanostructures have been studied using Human HT-29 colon cell lines. Trace levels of greenflorescent protein immobilised on Ga-doped ZnO nanodisks and nanoflowers showed good activity for UV light sensing.

Keywords:

Gallium ZnO thinﬁlm

Greenﬂuorescence protein Human HT-29

Sensors ZnO nanodisk Nanoﬂowers

1. Introduction

The synthesis of gallium doped zinc oxide nanostructured thinﬁlms, using a variety of techniques, and targeted towards applications in photo catalysis, sensors and dye sensitised solar cells has been reported [1–5]. However, there still remain many challenges in the development of optimised processes to form ordered nanostructures with controlled morphology that show high reactivity and activity for solar cell application. The biological properties of protein bound on metal ion doped ZnO nanostructures have not been studied in detail. However, ZnO based nanocomposites have been tested for potential applications as gas sensors and in photo catalytic reactions for degradation of dyes[6–7]. Zinc oxide based materials have also shown promise in the fabrication of light emitting diodes and optical devices[8]. ZnO is relatively nontoxic in contrast to other semiconductor materials such as CdS and CdSe;

Cd-based compounds are harmful to human health and can lead to toxic waste increasing environmental pollution[7]. Apart from their

attractive optoelectronic properties, nanostructures of ZnO have a num- ber of advantages in applications such as biosensing and photo catalysis due to the high aspect ratio, presence of a polar surface along the c-axis, higher electron mobility, and non-toxicity. More importantly, the isoelectric point of ZnO is around 9.5, which is perfectly suitable for the immobilisation of bio-molecules such as proteins that have a low isoelectric point(IEP); the immobilisation process is driven by electrostatic attrac- tion in suspensions when maintained at the appropriate pH[9]. Zinc oxide nanoparticles, porousfilms, nanocombs, and nanorods have been developed for biosensor applications to detect molecules such as cytochrome-c, protein, uric acid, glucose and phenolics[10–18]. Gallium doping in Zinc oxide lattice increases the electron conductivity of ZnO by a factor of three as compared to pristine ZnO. Recently, the fabrication of thinfilms of gallium doped ZnO nanodisks by polymer assisted hydrothermal reaction has been reported[19]. Nanodisk formation depends solely on the preparation methodology that involves the presence of a polymeric additive and the use of an aluminium coated substrate. Inorganic nanomaterials are important components in sensors used in monitoring environmentally polluting gas species, and also in sensors with specific properties such as piezoelectricity, which allow the development of transducers using either surface or bulk Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 202–212

⁎ Corresponding author at: Surfactants Research Chair, Chemistry Department, College of Science, King Saud University, P.O.Box. 2455, Riyadh 11451, Saudi Arabia.

E-mail addresses:[email protected],[email protected](J.R. Rajabathar).

Contents lists available atScienceDirect

Journal of Photochemistry & Photobiology, B: Biology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p h o t o b i o l

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acoustic waves to measure perturbations in fundamental frequencies due to the added mass on their surfaces. Recent developments in biosensor research indicate that the crystalline form of SiO2, a piezoelectric crystal with an inert surface is a potential candidate for the immobilisation of proteins for bio-detection[20]. Zinc oxide has a higher piezoelectric coefﬁcient (0.43 C/cm²) and also has a reactive surface.

Nanostructured ZnO with high piezoelectricity can enhance the sensi- tivity of the sensor system and also improves bulk acoustic wave reso- nators with high resonance frequencies (2.0 GHz)[21]. The reactive ZnO surface provides the opportunity to obtain effective bio friendly– ZnO interfaces for the development of UV and gas sensors to enable de- vice fabrication directly on silicon coated substrates thus paving the way for full integration with read-out and signal processing circuitry

compatible with silicon technology[22]. The present study describes the detailed methodology for the synthesis of Gallium doped ZnO nanostructures with nanodisk and nanoflower morphology and the characterization of these structures using a variety of techniques such as XRD, FE-SEM, AFM and Photoluminescence spectroscopy. The biocompatibility study and stability of cell lines with GZO were carried out using Human HT-29 cell lines by MTT assay method. The as-synthesised protein immobilised GZO thin films with nanodisk and nanoflower morphologies are further tested for UV-light sensing activity targeted towards possible application in UV sensing. GZO materials have been studied both under dark and under light irradiation to char- acterise the greenfluorescent protein bound on the GZO nanomaterial surface.

Fig. 1.Schematic representation of GZO nanostructure formation.

Fig. 2.X-ray diffraction patterns of (A) 1%GZO nanodisk and nanoﬂower and (B) 2% and 3% GZO nanodisk.

J.R. Rajabathar et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 202–212 203

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2. Experimental

2.1. Preparation of Gallium doped ZnO thinﬁlms

Ga-doped ZnO sol was prepared from zinc acetate dihydrate and gallium nitrate hydrate (Sigma-Aldrich). Methoxyethanol and monoethanolamine were used without further puriﬁcation.

Polyethylenimine (PEI) solution (30% in water) was purchased from TCI chemicals, Japan. Gallium (1, 2, and 3 mol%) doped ZnO (designated as 1% GZO, 2% GZO, and 3% GZO) solutions used for the coating of a seed layer on the substrate, were prepared by adding appropriate amounts of gallium nitrate to 0.1 M zinc acetate solution. A few drops of the GZO (1% GZO, 2%GZO and 3% GZO) solution were deposited on the AlN/Si substrate followed by spin coating at 1000–2500 rpm (10–30 s). After each deposition, theﬁlms were heated on a hot plate at 300 °C for 10 min. Four to six layers of the GZO thinﬁlms were deposited to obtain a thickness of 300–350 nm after which the samples were annealed at 500 °C.

2.2. Ga-ZnO nanostructures formation by polymer assisted hydrothermal process

The ZnOﬁlms on AlN/Si substrates with different Ga doping levels were treated hydrothermally in the presence of 3% polymer (PEI)

solution for the formation of GZO nanodisk/nanoflowers. To prepare GZO nanorods, a similar procedure was employed but without the addition of the polymer solution. In the hydrothermal process, 40 mL of zinc nitrate and 40 mL hexamethylene tetramine solutions (equimolar concentrations of 0.1 M) were mixed with 3% polymer solution (10 mL) and the contents were poured into an autoclave with a Teflon lining. 1% GZO coated AlN/Si substrate wasfixed vertically inside the Teflon container and heated at 90 °C for 20 h to grow nanodisks. For growing nanoflowers, the GZO coated substrate was kept in a horizontal position inside the Teflon autoclave container. The same procedure was repeated for the different compositions, namely, 2% and 3% Ga-doped ZnO nanodisk/nanoflower. The schematic diagram of formation of the different GZO nanostructures is given inFig. 1.

2.3. Protein immobilisation on GZO nanostructures

A 1 mL (5μM) solution of GFP in Tris-HCl buffer (pH 7.0) was incubated at 37 °C with GZO coated substrates for different time intervals ranging from 15 to 30 min. After dipping the substrates in the protein solution, theﬁlms were dried at room temperature before characterization. The phase and morphology of the prepared samples were characterised by XRD and FE-SEM, respectively. The crystal structures of the samples were analyzed by X-ray diffraction analysis using a Rigaku ultra-X diffractometer (Cu Kαradiation, 40 kV, 120 mA). FE- Fig. 3.(a–d) FE-SEM images of hydrothermally synthesised of GZO nanodisks at different conditions.

204 J.R. Rajabathar et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 202–212

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SEM connected with energy dispersive X-ray (EDX) facility (model- JSM-6500F) and atomic force microscopy (AFM) using a Park system Xe 100 E microscope were used for surface characterization.

Photoluminescence properties of synthesised sample were analyzed by Jobin Yvon HR 800 UV (HORIBA Jobin Yvon, Inc., Edison, NJ) using Ar laser as the excitation source. Fluorescence images of GFP-GZO bound substrate recorded by Fluorescence spectrometer connected to microscopy to record the images under dark and light emission conditions (model RF-5301PC, Shimadzu, Japan). A Keithley 4200-SCS pa- rameter analyser was used to study the UV sensing activity of the protein immobilised GZO nanostructures.

Human HT-29 colon cancer cell lines were obtained from the Amer- ican Type Culture Collection (ATCC). Cell viability was assessed by MTT assay. Briefly,HT-29 cells, 5 × 10³cells/well were seeded in a 96-well plate for 24 h. GZO nanostructures in ethanol (1 mg/mL) at varying concentrations (0, 2.5μM, 5μM, 7.5μM, and 10μM) was added to the cells and incubated for 24 h. Thefinal concentration of ethanol 0.5% in the cell culture medium. We did (0, 2.5μM, 5μM, 7.5μM and 10μM) only for 24 h and no sensitive effect observed in the cell lines using ethanol [23]. After incubation, 10μL of MTT (5 mg/mL) was added into each well for 2–4 h. The insoluble formazan crystals formed were dissolved in dimethylsulfoxide (DMSO) and quantified with a microplate reader (ELx800, BioTek, USA) at 540 nm. Survival rate percentage was

measured using the formula: (Absorbance of treated sample)/(Absor- bance of control) × 100.

3. Results and discussion

The XRD patterns of GZO with different nanostructured morphologies are shown inFig. 2. Major peaks were seen at 2Ɵvalues of 31.8(100), 34.5(002), 36.3(101), 47.6(102), 56.6(110) for GZO nanoﬂowers and 31.7(100), 34.3(002), 36.1(101), 47.4(102), 56.5(110) for GZO nanodisks. For GZO, the most intense peak has a 2θvalue of 43.4 (002). The crystallite size corresponding to each peak was calculated from the FWHM values using Scherer's formula.

The crystallite sizes and hkl plane values for GZO nanoflowers and nanodisks are shown in Tables S1 and S2, respectively. In the present study, the X-ray diffraction patterns clearly show differences in the intensities of the major peaks between the nanodisk and nanoflower morphologies. The (100) reflection, which is the most intense peak for GZO nanoflowers, has a crystallite size of ~ 77.8 nm and the (101) reflection, which is the highest intensity peak for nanodisks, corresponds to a crystallize size of ~ 56.1 nm (Tables S1 & S2). The XRD results clearly reflect the differences in crystallography and nanostructure between the two GZO morphologies. We have previously reported the role of the concentration of the GZO sol Fig. 4.(a–d) FE-SEM images of GZO with hybrid nanodisk/nanorods formation at different conditions.

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(that was used as the precursor for the formation of the ZnO seed layer) in the formation of the nanodisk morphology[19]. For an optimised GZO sol concentration (0.5 M sol-gel solution), fine nanodisks with the desiredfilm thickness were produced with a good control of crystallite sizes in annealedfilmsFig. 3(a–d). A loss of selectivity resulting in random formation of nanorods and nanodisks is observed for GZO (0.2 M) sol-gel solution. Therefore, in the present study, 0.5 M ZnO sol was used as the precursor for the seed layer formation to selectively obtain pure nanodisk or nanoflower morphology instead of hybrid morphology. In the hydrothermal process, the formation of the nanodisk-like morphology depends on the interaction of the polymer with the surface seed layer of ZnO particles. The effect of gallium addition on the nanostructure of ZnO has also been studied by comparing 1%, 2% and 3%

gallium doped ZnO samples.Fig. 3showsﬁne nanodisks with different thicknesses of ~ 96 nm and ~ 102 nm for polymer concentrations of 3% (Fig. 3a–b) and 4%, (Fig. 3c–d) respectively. An optimised polymer concentration in the 3–4% range results in the formation of thinner nanodisks. Increase in polymer concentration to above 4% in the hydrothermal process increases the nanodisk thickness (Fig. 6). Hence, it is concluded that doping with gallium above 2 mol% into ZnO matrix results in unfavourable morphology. The

good quality of GZO nanodisks prepared under the optimised conditions such as 1 mol% Gallium and 3% polymer solution in the hydrothermal process. Different strategies were adopted to selectively obtain nanodisk or nanoflower morphology (shown in the graphical abstract). We obtained nanodisk morphology by starting from a 0.5 M GZO sol-gel precursor in the spin coating process to make a GZO seed layer on the substrate (instead of GZO sol-gel precursor of a lower concentration, namely, 0.2 M).Figs. 4(a–d), show the results obtained when the GZO sol with a lower concentration (0.2 M) was used in the hydrothermal process. A hybrid material consisting of nanodisks and nanorod are formed on the surface of the GZO seed layer on substrate. The detailed X-ray photoelectron spectroscopy study on the influence of Ga content (1%, 2% and 3%) in the formation of nanodisks of gallium doped ZnO has been described in our previous report[19]. In the present study, we studied morphology changes in details, including FE-SEM images of GZO nanodisks with different Gallium contents, and the effect of polymer addition on the formation of different morphologies.Fig. 4(a-b) indicates that nanorods are first formed, which later transform into nanodisks. In the hydrothermal crystallization process, the fast growth of nanorods occurs with assistance from the polymer struc- turing agent after which, the presence of aluminium species on the Fig. 5.(a–d) FE-SEM images of GZO with nanoflower morphology.

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substrate causes a morphology transformation by bending the rods to form disks.Figs. 5(a–d) show GZO nanoflowers obtained by mounting the substrate vertically during the polymer assisted hydrothermal process.Figs. 5(a–b) show individual nanorods that combine to form aflower-like arrangement. SEM analysis with higher magnification inFig. 5a (1μm scale) shows the formation offlower like GZO that is made from a hexagonal arrangement of smaller nanorods. Hence, it is clear that the nanorod formation is the preliminary step after which, depending on the reaction conditions, the transformation into nanostructures of different morphologies takes place.Fig. 6shows a comparison of the FESEM images of nanodisks of GZO on AlN/Si substrate prepared with different concentrations of added polymer.Figs. 6(a–b) show the morphology of GZO nanodisks prepared with the optimised concentration of 1% GZO and 3% polymer additive.Figs. 6(c–d) show the morphology of GZO nanodisks prepared with 5% polymer that shows thicker nanodisks formed by the coalescence of the individual disks due to the presence of an excess polymer molecules. Atomic force microscopy is used to study the surface structure of the as-synthesised GZO nanostructures and our results show significant differences in AFM images for GZO with nanodisk, nanoflower and nanorod

morphology.Figs. 7–9show the AFM images of nanostructured GZO thinfilms.Figs. 7(a-d) show GZO nanorods prepared using the conventional synthesis route (images were recorded at different magnifications, namely, in the 2.15μm and 5.98μm range, surface height = 200 nm). The nanorods appeared to form a cone-like array which is clearly visible inFig. 7a–d. AFM images of GZO with nanodisk morphology are shown inFigs.8(a–b and c–d); these are somewhat different in appearance from the AFM images of nanorods. The surface of GZO nanodisk is made of larger cones with differences in the sizes of the individual nanodisks. The porosity and space between the nanodisks are different from the nanorods as well as nanoflowers GZO.Figs. 9(a–d) show the AFM images of GZO nanoflowers with images recorded at different magnifications (1.17μm and 2.25μm magnification ranges) with a surface height of 200 nm. AFM images of GZO are further differentiated in terms of surface morphology from nanodisk and nanorod structures.

Nanostructured samples with rough surface topographies are used in different applications, namely, in gas sensors and in dye sensitised solar cells. The pores found on the doped ZnO surface are probably formed by the coalescence of little voids created during the decomposition of the precursor and the subsequent vaporization Fig. 6.(a–b) FE-SEM images of GZO with thin nanodisk and (c–d) thick nanodisk morphology.

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of residual organic species during crystallization under hydrothermal process.

Fig. 10shows the room-temperature photoluminescence spectra of 1% GZO nanodisk grown by hydrothermal method. The ultraviolet emission peaks centred at about 362 nm for sample prepared at higher polymer concentration (blue line inFig. 10) and the peak at 382 nm is dominate for all other GZO sample prepared at optimised 3% and 4% polymer concentration, while the well-known broader emission situated in the blue-red part of the visible spectrum has also been observed and it is further confirmed by reported literature of bulk and doped ZnO nanostructure[24]. The above obtained PL result can be attributed to thefine crystalline quality and promising optical property simulation of as prepared GZO nanodisk. As mentioned above, the intensity can be enhanced by crystalline quality of GZO nanostructures. In addition to amending their own quality, surface plasmons or localized surface plasmons of various noble metals are also can be used to realize ZnO nanostructure photoluminescence enhancement. In the present research study, GZO nanodisk structured samples are shown enhancement in photoluminescence peak without the addition of any noble metal. Xiao et al. have also found enhancement of near-band-emission of ZnO by coating a layer of Ag implanted silicafilm[25–26]. You's group sputtered ZnOfilm on Si (001) substrate which had already been coated with 100 nm Ag nanoparticle previously, and the ultraviolet emission of the compos- ite is found to be greatly enhanced[24].

Wang et al., 2007, reported PL spectra of visible emission band at 470, 486 and 496 of the prepared GZO nanodisk samples are due to presence of intrinsic defects, which is further confirmed by detailed reported literature data of visible emission of PL spectra of doped ZnO nanostructure[25]. They excited the prepared ZnO by a laser beam at 325 nm from a He-Cd laser, shows a strong visible emission band at about 491 nm[27]. Impurities and various intrinsic defects, such as the singly ionized oxygen vacancies and interstitial zinc atoms, were usually assigned to be the origins of this visible emission band. This indicates that the surface adsorption plays a significant role in the exciton relaxation after photo excitation. The reported PL study states that the visible luminescence of ZnO mainly originates from different defect states such as oxygen vacancies and Zn interstitials. The emission at 421 nm can be assigned to the recombination of an electron at ZnO and a hole in the VB. We also observed that the PL intensity decreases with the increase of the excitation wavelength. InFig. 9, green line indicates the sample prepared at 2% gallium addition in ZnO lattice and prepared by addition of optimised 3% polymer solution, shows the perfect PL emission near visible region without any defects peak observed. An important observation is that, GZO nanostructure has a very strong photoluminescence (PL) band at visible range accompanied by few weaker defect states emissions.Fig. 11shows the differential interfer- ence contrast photographs andfluorescent images of nanostructures GZO with different morphologies.Figs. 11(a-b) show theflorescence activity of GZO nanorods samples under dark and underfluorescence Fig. 7.(a–d) AFM images of GZO nanorods.

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irradiation. Theﬂuorescence-active proteins on the surface of ZnO emit green light when viewed in the dark. This clearly indicates the presence of the greenﬂuorescent protein immobilised on GZO nanostructures;

similarfluorescence is observed in other samples. The stability of binding nature of protein on GZO is clearly confirmed by their emitting nature upon irradiation. Before record eachfluorescence images, the GZO-bound protein washed with de-ionized water and dried in room temperature. Figs. 11 (b-c) show the presence of the protein immobilised GZO nanodisks, which display thefluorescence emitting sites in the active portion of the protein bind on GZO surface. Similar results are observed for protein-immobilised GZO nanoflower samples (Figs. 11(e-f)). Fluorescence images of GZO nanoflowers, as well as nanodisks and nanorods, show that the observedfluorescence emission is almost similar to that for protein immobilised - GZO nanoflower samples.

The green emission from the protein immobilised nanostructure of GZO is clearly observed byﬂuorescence microscopy, which indicates that the GZO-nanoparticle on substrate is strongly bound to the protein molecules. The interaction between ZnO nanoparticles and the biomol- ecule (e.g. proteins) results in the formation of a biological corona on the nanoparticle surface[28]. The biological corona could reduce the photoluminescence of the nanoparticles, because of the conformational changes that cause steric hindrance on the nanoparticle surface. In the hydrothermal process, the morphology of the product depended signif- icantly on the polymer additive and the concentration of the Zn precursor solution. However, during the hydrothermal crystallization process, the nucleation and growth of ZnO nanostructures are also affected by the nature of the chemical species present in the solution. The degree of saturation of Zn(OH)2in the ageing stage and the adsorption of

polymeric/inorganic species on the ZnO surface are two important fac- tors that inﬂuence the nucleation and growth of ZnO nanostructures.

The formation of these nanostructures generally takes place by a layer-by-layer assembly mechanism. First, individual ZnO nuclei grow along the c-axis, forming a rod-like structure that transforms into various morphologies depending on reaction conditions. ZnO surface is either positively or negatively charged and in either case, the surface attracts ions of the opposite charge from the solution and the polymer, thereby influencing the morphology of the nanostructure. The presence of aluminium nitride on the silicon substrate causes the transformation of rods into nanodisks perpendicular to the surface. Aluminium nitride acts as a catalyst for ZnO disk formation since it causes the rods to bend and the polymer molecules further assist in the formation of the fine nano-disk shape. Because of the intercalated structures, the plate- lets and disks appear to befirmly bonded to each other, providing me- chanical strength to the nanostructures.

The biocompatibility, bio-stability and the interaction of GZO with HT-29 type human cell lines have also been carried out in the present study.Fig. 12shows the GZO nanodisk and GZO nanoﬂower structures stability after incubated with HT-29 cells. MTT assay is carried out to study the activity. The various concentrations of HT- 29 cell lines in culture medium with GZO dispersed samples are tested and the respective absorbance was measured. The results indicat- ed that the prepared GZO nanostructures are showing stable interaction with HT-29 cell lines and promising biocompatibility. If the GZO is not stable with cell lines, it shows very less absorbance values, but in our experimental result showing gradual decrease in activity with respect to precursor concentration. Biocompatibility study of undoped ZnO nanowires is already reported. The ZnO Fig. 8.(a–d) AFM images of GZO nanodisks.

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nanowires are showing good biocompatibility same as prepared Ga- ZnO nanodisk and nanoﬂowers[29]. Hence, the as prepared GZO with nanodisk/nanoﬂowers are also showing similar activity and it could be a bio-safe substance for bio-imaging applications. The UV

light sensing property of the protein immobilised GZO nanodisks and nanoflowers was tested in the dark (absence) and in the presence of UV light for 1% GZO/AlN/Si nanodisk/nanoflower.Fig. 13 shows the UV sensing activity of GZO nanodisks (green line): A fast response and a short recovery time for UV light“on”for 10 s and UV light“off”(dark) for (30 s) were measured. The sensing activity of nanoflower structures of GZO is shown inFig. 13(red curve).

The recovery time for GZO nanoﬂowers after turning off the UV light was a little longer compared to that for GZO nanodisks.

4. Conclusions

Gallium doped ZnO with different morphologies such as nanodisks and nanoflowers are prepared on commercially available aluminium nitride coated silicon substrates by means of an optimised hydrothermal process with polyethylene imine as a structure assisting agent. A commercially available green fluorescent protein was successfully immobilised on the as-prepared GZO nanostructures and tested for UV sensor applications. Typical XRD patterns are observed for GZO nanodisk and nanoflower morphologies. The role of gallium doped in different concentrations into the ZnO lattice, and the effect of polymer concentration, have been studied; FE-SEM images of nanodisks were obtained. Increasing the polymer concentration in the hydrothermal process results in binded-nanodisk type morphology, as clearly identi- fied from FE-SEM images. AFM images show differences in surface structures for the various GZO nanostructures. Biocompatibility and Fig. 10.Photoluminence spectra of GZO nanodisks.

Fig. 9.(a–d) AFM images of GZO nanoﬂowers.

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interaction of as prepared GZO with HT-29 human cell line have also been showing promising activity and stability with cell interaction.

Fluorescence images and contrast photographs of GFP-bound nanostructures show goodﬂuorescence response under presence and absence ofﬂuorescence irradiation. The active sites in GFP-bound GZO nanostructures emit green light from the protein blinded sites upon ir- radiating. Hence, in the future research studies, GFP immobilised GZO

nanostructures can be used as light sensor in bioimaging applications includingﬂuorescence probes for biological cell-labelling.

Acknowledgements

This project wasﬁnancially supported by King Saud University, Vice Deanship of Scientiﬁc Research, Research Chairs.

Fig. 11.Fluorescence images of protein bound GZO (a–b) nanorods (c–d) nanodisks, and (e–f) nanoﬂowers with contrast photographs.

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Appendix A. Supplementary data

Supplementary data to this article can be found online atdoi:10.

1016/j.jphotobiol.2016.10.028.

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Fig. 12.(a) GZO nanodisk activity with human HT 29 cell (b) GZO nanoﬂower with human HT 29 cell determined by MTT Assay using HT-29 cell lines.

Fig. 13.UV-light sensing activities for protein bound GZO nanostructure with nanodisks (green line) and nanoﬂowers (redline). (For interpretation of the references to color in thisﬁgure legend, the reader is referred to the web version of this article.)