Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin

Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin

Accepted Manuscript Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin Kiyoshi Matsuyama, Ihsan Neil...

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Accepted Manuscript Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin Kiyoshi Matsuyama, Ihsan Neil, Keiichi Irie, Kenichi Mishima, Tetsuya Okuyama, Hiroyuki Muto PII: DOI: Reference:

S0021-9797(13)00209-9 http://dx.doi.org/10.1016/j.jcis.2013.02.047 YJCIS 18652

To appear in:

Journal of Colloid and Interface Science

Received Date: Accepted Date:

22 November 2012 28 February 2013

Please cite this article as: K. Matsuyama, I. Neil, K. Irie, K. Mishima, T. Okuyama, H. Muto, Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin, Journal of Colloid and Interface Science (2013), doi: http://dx.doi.org/10.1016/j.jcis.2013.02.047

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Bioimaging application of highly luminescent silica-coated ZnO-nanoparticle quantum dots with biotin

Kiyoshi Matsuyamaa,*, Ihsan Neila, Keiichi Irieb, Kenichi Mishimab, Tetsuya Okuyamac, Hiroyuki Mutod a

Department of Biochemistry and Applied Chemistry,

Kurume National College of Technology, 1-1-1 Komorino, Kurume, Fukuoka 830-8555, Japan Email: [email protected] b

Department of Neuropharmacology, Faculty of Pharmaceutical Sciences,

Fukuoka University, 8-19-1 Nanakuma Jonan-ku, Fukuoka 814-0180, Japan c

Department of Materials Science and Engineering,

Kurume National College of Technology, 1-1-1 Komorino, Kurume, Fukuoka 830-8555, Japan d

Department of Electrical and Electronic Information Engineering,

Toyohashi University of Technology, 1-1 Hibarigaoka Tenpaku-cho, Toyohashi, Aichi 441-8580, Japan

* Corresponding author Kiyoshi Matsuyama E-mail address: [email protected] Telephone +81-942-35-9403 Fax +81-942-35-9400

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Abstract We synthesized ZnO-nanoparticle quantum dots (QDs) as a fluorescent probe for biological applications. Highly luminescent silica-coated ZnO-nanoparticle QDs dispersed in an aqueous medium were synthesized using the sol-gel process. The ZnO-nanoparticle QDs were coated with silica to improve the water stability of the ZnO nanoparticles. NH2 groups were introduced on the surface of the silica-coated ZnO-nanoparticle QDs first by the addition of 3-aminopropyltrimethoxysilane and then by biotinylation with sulfosuccinimidyl-6-(biotin-amido) hexanoate (sulfo-NHS-LC-bioton). We demonstrated that avidin-immobilized agarose beads were tagged by the silica-coated ZnO-nanoparticle QDs with biotin by the selective avidin–biotin interaction, furnishing a fluorescent image upon excitation with UV light. Furthermore, use of the silica-coated ZnO-nanoparticle QDs with biotin in cell-labeling applications was attempted, and attachment of the silica-coated ZnO-nanoparticle QDs with biotin to nerve cells and actin filaments was achieved.

Keywords: ZnO quantum dot, ZnO colloid, Fluorescent image, Photoluminescence, Bioimaging, Avidin–biotin interaction

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1. Introduction Since Alivisatos et al. [1] and Nie et al. [2] first labeled biological cells with luminescent semiconductor nanocrystals [i.e., quantum dots (QDs)], QDs with highly fluorescent and photostable properties have been found to be excellent tools in imaging cellular structures and events [3-5]. The most commonly used QDs are Cd-based compounds (e.g., CdS, CdSe, and CdTe) because such compounds produce bright fluorophores with controllable surface properties and they are highly resistant to photodegradation, etc. However, Cd-based QD probes have limited use because of the high cytotoxicity of cadmium. This cytotoxicity is attributed to the release of highly toxic free Cd2+ ions, which kills cells [6, 7]. However, ZnO-nanoparticle QDs have been shown to be potentially applicable in biological cell-labeling applications [8-17]. These materials have also been shown to be potentially applicable in the manufacture of light-emitting diodes (LEDs) and optical devices [18-23]. ZnO is relatively nontoxic, whereas other semiconductor materials, including Cd-related compounds, are harmful to cells and human health, and they eventually damage the environment [7]. Although ZnO-nanoparticle QDs can be synthesized by various processes, including chemical vapor deposition (CVD) [24], hydrothermal synthesis [25], the polyol process [11], and the sonochemical method [26], the sol-gel process in an alcoholic medium is the conventional method, with a high production rate and relatively low energy cost [27-30]. To stabilize ZnO photoluminescence, surface modification can be achieved by using organic ligands [11, 14, 15], SiO2 [10, 21], and polymers [12, 13, 16, 17]. In previous work, we investigated a method of surface modification of ZnO QDs using organic ligands to obtain polymeric hybrid films of ZnO-nanoparticle QDs and poly(methyl methacrylate) (PMMA) with high luminescence and tunable emission color [23]. The surface modification techniques are needed in order to use ZnO-nanoparticle QDs in biological cell-labeling applications because the photoluminescence of ZnO-nanoparticle QDs produced in an alcoholic medium using the sol-gel process have poor stability against water. However, the use of ZnO-nanoparticle QDs for bioimaging applications is affected by such surface modification and by the conjugation of biomolecules onto the QDs. One of the most effective methods for improving the surface functionalization of nanoparticles is silica coating. Silica was selected as the coating material because of its biocompatibility, water stability, and rich surface chemistry [5, 31]. Although silica-coated ZnO-nanoparticle QDs have been used in cell-labeling applications [8, 10], there are few reports on the bioconjugation technique. After conjugation with the appropriate target ligands, antibodies, or proteins, silica-coated ZnO-nanoparticle QDs may exhibit highly selective binding, making them useful for fluorescence imaging. In this study, we report the preparation of biotinylated ZnO-nanoparticle QDs and the labeling of avidin-immobilized beads with these QDs by use of the adivin–biotin interaction. To

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demonstrate the applicability of this method, we attempted to use the biotinylated ZnO-nanoparticle QDs in cell-labeling applications. Recently, SelegÅrd et al. reported biotinylation of ZnO nanoparticles using (3-mercaptopropyl)trimethoxysilane and iodoacetyl-PEG2-biotin [32]. However, the properties of the prepared ZnO nanoparticles as bioprobes were not reported. To the best of our knowledge, the present study is the first report on the preparation of biotinylated ZnO-nanoparticle QDs with good water dispersibility and high photoluminescence emissions for cell-labeling applications. Figure 1 shows the functionalization procedure of ZnO QDs nanoparticles. To achieve the conjugation of biotin molecules on the ZnO QD nanoparticle surfaces, we focused on the introduction of NH2 groups on the silica-coated ZnO-nanoparticle QDs by surface modification of 3-aminopropyltrimethoxysilane (APTMS). The silica-coated ZnO-nanoparticle QDs were completely dispersed in water and had high photoluminescence properties and stability in water. The NH2 groups of the APTMS-modified silica-coated ZnO-nanoparticle QDs were cross-linked with biotin using sulfosuccinimidyl-6-(biotin-amido) hexanoate (sulfo-NHS-LC-bioton). Biotin was used for functionalization because avidin has a high affinity for biotin molecules, making the biotin–avidin complex suitable as a model system for recognition bioimaging processes [33, 34].

SiO2 layer

NH2

OCH3 OH

ZnO

OH

H2N(CH2)3

+

Si

Si O

OCH3

ZnO

OCH3

OH

O

O Si O O Si

APTMS

NH2 NH2

O SO3Na

NH2

ZnO

NH2

O

+

N O

NH2

NH O

S O

Sulfo-NHS-LC-Biotin

O

HN

ZnO

H3C

NH

HN

O

NH

O NH NH

S O

silica-modified ZnO QDs coupled with biotin Fig. 1 Schematic illustration showing the biofunctionalization of silica-coated ZnO-nanoparticle QDs with biotin. The ZnO-nanoparticle QDs were first modified with tetraethyl orthosilicate

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(TEOS), and the NH2 groups introduced on the surface of the silica-coated ZnO-nanoparticle QDs were conjugated with the biotin. 2. Experimental 2.1 Materials Zinc acetate dihydrate [Zn(CH3COO)2·2H2O, purity: >99.9%], tetraethyl orthosilicate (TEOS, purity: >98%), 3-aminopropyltrimethoxysilane (APTMS, purity: >98%), and avidin-immobilized agarose gel beads were purchased from Sigma-Aldrich. Lithium hydroxide monohydrate (LiOH·H2O, purity: >98%) was obtained from Wako Pure Chem. Ind., Ltd., and sulfosuccinimidyl-6-(biotin-amido) hexanoate (sulfo-NHS-LC-biotin) was purchased from Pierce. 2.2 Preparation of silica-coated ZnO-nanoparticle QDs The details of the preparation method for ZnO-nanoparticle QDs using the sol-gel process are described in a previous paper [23], with zinc acetate dihydrate and lithium hydroxide monohydrate as the raw material and the base catalyst, respectively. The typical preparation method is as follows. Zinc acetate (0.5 mmol) and lithium hydroxide (8.6 mmol) were dissolved in 100 mL of ethanol. This precursor solution was then heated at 70 °C for 2 h while stirring, resulting in ZnO-nanoparticle QDs dispersed in an ethanolic solution. Then, 25 μL of TEOS and 100 μL of pure water were separately added to 50 mL of the as-prepared solution. The resulting solution was then treated by ultrasonic vibration for 10 min before further stirring for 12 h at room temperature. The solution was then centrifuged at 10,000 rpm for two cycles. The purified particles were then dispersed in absolute ethanol for storage. 2.3 Preparation of silica-coated ZnO-nanoparticle QDs with biotin APTMS (25 μL) and pure water (100 μL) were separately added to 50 mL of the as-prepared solution containing silica-coated ZnO-nanoparticle QDs. The resulting solution was then treated by ultrasonic vibration for 10 min before further stirring for 12 h at room temperature. The solution was then centrifuged at 10,000 rpm for two cycles. After removal of free APTMS by centrifugation, sulfo-NHS-LC-biotin aqueous solution was poured into the colloidal solution and stirred for 2 h. Free biotinylation agents were removed to furnish the biotinylated ZnO colloidal aqueous solution. 2.4 Tagging of avidin beads to silica-coated ZnO-nanoparticle QDs with biotin The biotinylated ZnO-nanoparticle aqueous solution (100 μL) and avidin-immobilized agarose gel beads (100 μL) were poured into ultrapure water (5 mL) and stirred for 5 h. Silica-coated ZnO-nanoparticle QDs without biotin were also used in the abovementioned procedure as the control. Agarose gel beads tagged with biotinylated ZnO nanoparticles were observed by fluorescence microscopy (Nikon, TS100-F) with an ultraviolet fluorescence filter

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that includes a medium-width band-pass excitation filter (330–380 nm) coupled to a long-pass barrier filter (420 nm). 2.5 Labeling of nerve cells of mouse spinal cord with silica-coated ZnO-nanoparticle QDs with biotin A mouse spinal cord was fixed in 4% paraformaldehyde and then embedded in paraffin. The spinal cord sections were incubated in a 1:200 dilution of rabbit polyclonal anti-TRPA primary antibody (Abnova Inc., Taipei City, Taiwan) overnight at 4 °C. The sections were then incubated with a 1:1000 dilution of biotin-conjugated anti-rabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 2 h at room temperature. The nerve cells in the mouse spinal cord were washed twice with a phosphate-buffered solution (PBS). For avidin attachment to the nerve cells of the mouse spinal cord, the nerve cells with antibody were filled with 10 μg/L avidin in PBS. For biotinylated ZnO-nanoparticle QD attachment to the nerve cells, the nerve cells with the attached avidin were filled with biotinylated ZnO-nanoparticle QDs in PBS. Silica-coated ZnO-nanoparticle QDs without biotin were also used in the abovementioned procedure as the control. The nerve cells of the mouse spinal cord with biotinylated ZnO-nanoparticle QDs were observed by fluorescence microscopy (TS100-F, Nikon) with an ultraviolet fluorescence filter combination that included a medium-width band-pass excitation filter (330–380 nm) coupled to a long-pass barrier filter (520 nm). 2.6 Characterization The chemical species of the products on the ZnO-nanoparticle QD surfaces were examined by Fourier transform infrared (FT-IR) spectroscopy (FT-IR470, Jasco Co.) using the KBr method, and the photoluminescence (PL) properties of the ZnO colloid were measured by PL spectroscopy (FP-6200, Jasco Co.). The PL quantum yields (QYsample) of the ZnO nanoparticles were referenced against standard rhodamine 6G ethanol solution quantum yields (QYref = 95%) [35]. The QY values of the ZnO QD samples were calculated by comparing the integrated PL intensities (excited at 350 nm) and absorbance values (at 350 nm) of ZnO QDs with those of the reference rhodamine 6G according to the following equation [12, 36]:

2 2 QYsample  QYref (msample / mref )(nsample / nref )

(1)

where m is the gradient from the plot of integrated PL intensity as a function of absorbance and n is the refractive index of the solvent. The structure of the silica-coated ZnO-nanoparticle QDs was analyzed by transmission electron microscopy (TEM) (JEOL, Hitachi H-7100FA). We prepared the sample by dipping

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carbon-coated copper grids into the sample solution and then drying the grids at room temperature. The sections were observed using a TEM operating at 200 kV. The particle size and particle-size distribution (PSD) of the silica-coated ZnO nanoparticles were determined by a particle size analyzer (Microtrac UPA-150, Nikkiso Co. Ltd.). The experimental results for the particle size measurement are a mean value of different experiments.

3. Results and discussion 3.1 Silica-coated ZnO-nanoparticle QDs The FT-IR spectra of the unmodified ZnO QDs and the silica-coated ZnO QDs are shown in Figs. 2(a) and (b), respectively. Details of the FT-IR spectra of the unmodified ZnO QDs were reported in our previous paper [23]. For the unmodified ZnO QDs, the peak at 499 cm−1 corresponds to the stretching modes of Zn–O. The large broad bands from 1405 to 1598 cm−1 are coincident with those typically observed for acetate groups complexed with metallic zinc and correspond to C=O stretching (1598 cm−1) and C–O stretching (from 1405 to 1459 cm−1). The broad peak between 3400 and 3500 cm−1 is the stretching vibration of

the –OH group on the surface of the ZnO QDs. Figure 1(b) shows the FT-IR spectra of the silica-coated ZnO QDs; the silica layer of the ZnO QD surface is characterized by Si–O–Si stretching at 1040–1140 cm−1 [37]. The presence of Si–O–Si stretching on the FT-IR spectra indicates the successful formation of a silica layer on the surface of the ZnO QDs. The colloid dispersibility and PL properties of unmodified ZnO and silica-coated ZnO QDs in water were also considered. After the redispersion of unmodified ZnO QDs (prepared by the hydrolysis of zinc acetate with LiOH in ethanol) in water, a turbid colloidal solution was obtained, as shown in Fig. 3(a-1). Conversely, the silica-coated ZnO QDs were completely dispersed in the water, giving a transparent colloidal solution, as shown in Fig. 3(b-1). The silica-coated ZnO nanoparticles remained transparent for more than ten days. A better colloid dispersibility was obtained for the silica-coated ZnO QDs because the affinity of the silica-modified ZnO QDs for water was higher than that of the unmodified ZnO QDs. The silica layer acts as a capping agent to prevent the agglomeration of ZnO-nanoparticle QDs. The unmodified ZnO QD colloidal solutions exhibited no emissions under UV irradiation (350 nm), as shown in Fig. 3(a-2). On the other hand, the silica-coated ZnO-nanoparticle QD colloidal solution exhibited a significant blue emission, as shown in Fig. 3(b-2). Figure 4 shows the PL emission and excitation spectra of the silica-coated ZnO colloidal solution in water. The excitation spectra were measured at an emission wavelength of 491 nm, whereas the emission spectra were measured under excitation at a wavelength of 350 nm. Similar PL spectra were obtained in both cases [10]. The quantum yield of the silica-coated ZnO QDs was 58%, which is higher than that of ZnO QDs prepared by the classical sol-gel method

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(where the quantum yield is usually below 10%). The silica layer caused the water stability for the PL properties of the ZnO QDs. Significant improvements have been made to ZnO QDs in the past few years, and their quantum yields have been improved [10, 38]. The quantum yields of ZnO QDs could reach 60% with SiO2 surface modification [21]. A similar quantum yield was obtained for the silica-coated ZnO QDs in our experiment.

The particle morphology of silica-coated ZnO QDs was characterized by TEM and dynamic light scattering (DLS) analysis. Figure 5 shows the TEM image of the silica-coated ZnO QDs. It can be seen that they are uniform, monodispersed in the silica matrix, and about 3 nm in diameter. However, the mean particle size of silica-coated ZnO QDs measured by DLS is 115 nm (standard deviation 55 nm), as shown in Fig. 6. A possible reason for this difference is that the silica layer surface on the ZnO QDs might have caused the agglomeration of the prepared composite particles. This process was carried out under excess LiOH for zinc acetate, which indicates that the hydroxyl groups coated the surfaces of the ZnO nanoparticles, which probably resulted in silica condensation on the ZnO-nanoparticle surfaces. The alkoxy groups of TEOS accelerate the aggregation of silica-coated ZnO QDs [39].

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Abs.[−]

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(c)silica-modified ZnO QDs coupled with biotin

2 (b)silica-modified ZnO QDs

1 (a)unmodified ZnO QDs

0 4000

3000

2000

1000

400

Wavenumber [cm-1] Fig. 2 FT-IR spectra of (a) unmodified ZnO-nanoparticle QDs, (b) silica-coated ZnO-nanoparticle QDs, and (c) silica-coated ZnO-nanoparticle QDs coupled with biotin.

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(a-1)

(b-1)

(c-1)

(a-2)

(b-2)

(c-2)

Fig. 3 Photographs of ZnO-nanoparticle QDs in water under ambient light (left-hand side) and UV (350 nm) irradiation (right-hand side). (a) Unmodified ZnO, (b) silica-coated ZnO, and (c) silica-coated ZnO coupled with biotin.

Silica-coated ZnO QDs Silica-coated ZnO QDs coupled with biotin 1.2

1.2

1

Normalized PL emission intensity[a.u.]

(a)

Normalized PL exitation intensity[a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(b)

1

0.8

0.8

0.6

Ex_ZnO_SiO2

Em_ZnO_SiO2

0.6

0.4

Ex_ZnO_biot

Em_ZnO_biot

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0.2

0.2

0

0

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Wavelenght[nm]

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Wavelenght[nm]

Fig. 4 PL spectra of silica-coated ZnO-nanoparticle QDs and silica-coated ZnO-nanoparticle QDs in water: (a) PL excitation spectra and (b) PL emission spectra. The PL excitation spectra of silica-coated ZnO QDs with and without biotin were detected at 486 nm and 482 nm, respectively.

The PL emission spectra of silica-coated ZnO QDs with and without biotin were

excited at 355 nm and 287 nm, respectively.

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20 nm Fig. 5 TEM photograph of silica-coated ZnO-nanoparticle QDs.

(a)silica-coated ZnO QDs (b)silica-coated ZnO QDs coupled with biotin

40 ZnO-SiO2

30

Number[%]

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ZnO-biotin

20

10

0 10

100 Particle diameter[nm]

1000

Fig. 6 Particle-size distribution determined by DLS for (a) silica-coated ZnO-nanoparticle QDs and (b) silica-coated ZnO-nanoparticle QDs with biotin diluted in water.

3.2 Silica-coated ZnO-nanoparticle QDs with biotin The biotin molecules were attached to the surface of the silica-coated ZnO-nanoparticle QDs using sulfo-NHS-LC-biotin. Prior to loading the biotin molecules on the silica-coated ZnO-nanoparticle surfaces, the surfaces of the nanoparticles were modified with

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APTMS to introduce an NH2 group. The FT-IR spectra of the silica-coated ZnO-nanoparticle QDs with biotin are shown in Fig. 2(c). Biotin on the ZnO QD surfaces is characterized by the vibrational mode of a cyclic urea at 1240–1250 cm−1 [37]. The presence of biotin on the FT-IR spectra indicates the successful bonding of biotin to the surface of the silica-coated ZnO QDs. The colloid dispersibility and PL properties of silica-coated ZnO QDs with biotin in water were also considered. Although silica-coated ZnO-nanoparticle QDs were completely dispersed in water [Fig. 3(b-1)], a slightly turbid colloidal solution was obtained for the biotinylated ZnO-nanoparticle QDs, as shown in Fig. 3(c-2). This is caused by the hydrophilic properties of the biotin molecules on the silica-coated ZnO-nanoparticle QD surfaces. The mean particle size of silica-coated ZnO QDs with biotin measured by DLS is 125 nm (standard deviation 62 nm), as shown in Fig. 6(b). The attachment of biotin molecules does not appreciably change the particle size. The biotinylated ZnO-nanoparticle QD colloidal solution and the silica-coated ZnO-nanoparticle QDs without biotin both exhibited a blue emission under UV irradiation (350 nm), as shown in Figs. 3(b-2) and 3(c-2), respectively. Figure 4 shows the PL emission and excitation spectra of the silica-coated ZnO with biotin colloidal solution in water. Similar emission peak wavelengths of around 490 nm were also found for the silica-coated ZnO-nanoparticle QDs without biotin. 3.3 Biological imaging using silica-coated ZnO-nanoparticle QDs with biotin Figure 7(a) shows the differential interference contrast photograph and fluorescent image of avidin-immobilized agarose gel beads stained by silica-coated ZnO-nanoparticle QDs with biotin. The blue emission from the agarose gel beads was clearly observed under the fluorescent microscope. On the other hand, no emission was observed for silica-coated ZnO-nanoparticle QDs without biotin, as shown in Fig. 7(b). These results suggest that biotinylated ZnO-nanoparticle QDs are successfully bound to the surface of the avidin-immobilized agarose gel beads through the selective avidin–biotin interaction. Attachment of silica-coated ZnO-nanoparticle QDs with biotin to the nerve cells of a mouse spinal cord by utilizing antibodies and avidin-recognition molecules has been discussed. Figure 8(a) shows the differential interference contrast photograph and fluorescent image of nerve cells of a mouse spinal cord stained by silica-coated ZnO-nanoparticle QDs with biotin. Prior to attaching the QDs with biotin, avidin was attached to the nerve cells utilizing a biotin-conjugated antibody. The green emission from the nerve cells was clearly observed under fluorescent microscope, indicating that the ZnO-nanoparticle QDs were attached by antibodies and avidin binding to the nerve cells. The interaction between nanoparticles and biomolecules (e.g. proteins) results in the formation of a biological corona on the nanoparticle surface [40]. The biological corona may reduce the photoluminescence of nanoparticles, because of the

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conformation change and steric hindrance of nanoparticle surface. To prevent the reduction of photoluminescence of ZnO nanoparticle QDs by biological corona formation, we used the sulfo-NHS-LC-bioton for biotinylation of silica-coated ZnO QDs. Sulfo-NHS-LC-bioton has simple alkyl spacer arms that help to minimize steric hindrance. The images of the corresponding control experiment, in which the nerve cells were incubated with the addition of ZnO-nanoparticle QDs without biotin, are shown in Fig. 8(b). No emission was observed for silica-coated ZnO-nanoparticle QDs without biotin because the selective avidin–biotin interaction was not obtained. These results show that silica-coated ZnO-nanoparticle QDs with biotin are very promising for applications in selective cell labeling.

(a)silica-coated ZnO QDs coupled with biotin

(b)silica-coated ZnO QDs

10 μm

Fig. 7 Differential interference contrast photograph and fluorescent image of avidin-immobilized agarose gel beads stained by silica-coated ZnO-nanoparticle QDs (a) with biotin and (b) without biotin.

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(a)silica-coated ZnO QDs coupled with biotin

(b)silica-coated ZnO QDs

100 μm

Fig. 8 Differential interference contrast photograph and fluorescent image of nerve cells of mouse spinal cord stained by silica-coated ZnO-nanoparticle QDs (a) with biotin and (b) without biotin.

Furthermore, attachment of silica-coated ZnO-nanoparticle QDs with biotin to the actin filament in Caco-2 cells by utilizing biotinylated phalloidin and avidin-recognition molecules has been discussed. The Caco-2 cell line is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells. Figure 9(a) shows the differential interference contrast photograph and fluorescent image of actin filament of Caco-2 cells stained by silica-coated ZnO-nanoparticle QDs with biotin. Prior to attaching the QDs with biotin, avidin was attached to the actin filament in Caco-2 cells utilizing a biotinylated phalloidin. The green emission from the actin filament was clearly observed under fluorescent microscope, indicating that the ZnO-nanoparticle QDs were attached by phalloidin and avidin binding to the actin filament. The images of the corresponding control experiment, in which the actin filament were incubated with the addition of ZnO-nanoparticle QDs without biotin, are shown in Fig. 9(b). When QDs are used as the fluorescent probe for biological applications, the cytotoxicity of QDs for living cells is noteworthy. Tang et al. discussed the cytotoxicity of silica-coated ZnO nanoparticle QDs for living cell such as NIH/3T3 cells using MTT

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assay [10]. They show that more than 85 % of the cells survived (after incubation for 24 h) when the concentration of silica-coated ZnO nanoparticle QDs was 30 μg/mL. It is suggested that silica-coated ZnO nanoparticle QDs are quite safe to living cells.

a)silica-coated ZnO QDs with biotin

b)silica-coated ZnO QDs

100μm Fig. 9 Differential interference contrast photograph and fluorescent image of actin filament of Caco-2 cells stained by silica-coated ZnO-nanoparticle QDs (a) with biotin and (b) without biotin.

4. Conclusions Synthesis techniques have been developed for the biotinylation of silica-coated ZnO-nanoparticle QDs with high luminescence and stability in aqueous solution. Silica-coated ZnO-nanoparticle QDs conjugated with biomolecules could be used as advanced fluorescence probes for biological cell-labeling applications because they have low cytotoxicity, high luminescence and stability, and high sensitivity. Acknowledgement This study was supported by MEXT KAKENHI (23760732), and by funds from Toyohashi University of Technology, Nagaoka University of Technology, and Kurume National

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College of Technology.

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16

ZnO

SiO2 layer biotin avidin biotin conjugated secondary antibody

silica-coated ZnO QDs coupled with biotin primary antibody

nerve cells

mouse spinal cord section

>Silica-coated ZnO QDs exhibited high photoluminescence emission in water. >Silica-coated ZnO QDs with biotin applied to cell-labeling applications. >ZnO QDs have low cytotoxicity.