Introduction

In the past decade, iron oxide magnetic nanopaticles (MNs) and semiconduductor quantum dots (QDs) have attracted great research interest. MNs have unique surperparamagnetic properties and exhibit magnetization only in the presence of a magnetic field, which make it have potential applications in data storage technology, separation of biomolecules, and nucleic aicd detachment [1, 2]. QDs are nanoscale spherical particles that have two main advantages compared to organic fluorophores: its unique optical properties, such as excellent photostability and narrow symmetric emission profile with a broad excitation range [3], the other one is its emission wavelength can be tuned by changing the size of QDs and simultaneously allowing many-color QDs to work by a single narrow-band excitation source [4]. Because of excellent advantages, QDs are currently of great interest as emitting materials for biolabeling applications [5, 6]. Many of these applications require the nanoparticles to be chemically stable, uniform in size and well dispersed in liquid medium. As the chemical stability of MNs is pretty poor and QDs are still facing some unsolved problems as follows: ultra-sensitivity of their fluorescence to the surface states, cytotoxicity of heavy metal ions used in the process of synthesis which are released upon photo-oxidation, and chemical and colloidal instabilities in harsh chemical environments [7, 8]. So, they are usually coated with a protection layer, such as dextran polymer [9] or SiO2 [10]. Surface modification of QDs and MNs with silica layer has led to improved stability, low toxicity, and higher biocompatibility, and protects the QDs against corrosion of the biological buffers. In addition, the rich and well-known surface chemistry of silica makes bioconjugation more convenient [11]. SiO2 has been considered as one of the most ideal material for protection of MNs and QDs.

In fact, coating inorganic nanoparticles with silica has been widely investigated. The well-known stöber method has succeeded in obtaining core-shell structured semiconductor nanocrystals/SiO2 particles [12], iron oxide nanocrystals/ SiO2 [10], and the multi-functional microspheres [13]. Apart from the stöber method, the microemulsion approach can also be used to coat II-VI nanocrystals with silica [14]. Most recently, reverse microemulsion method has started to be addressed. In comparison with the stöber method, the reverse microemulsion method can yield more uniform spheres in the size range of 30–50 nm, the reaction condition of the reverse microemulsion method is relatively robust, and the resulting silica spheres have smoother surfaces [15]. The reverse microemulsion method has been used to fabricate the CdTe/SiO2 [16] and Fe3O4/SiO2 nanoparticles [17].

Because MNs have excellent magnetic properties and QDs have excellent optical properties, so the nanoparticles embedded with QDs and MNs will have wider application in biomedicine and biology including magnetic separation and detection of cancer cells, bacteria and viruses. Up to the present, very few studies have been carried to synthesize bi-functional magnetic and luminescent nanoparticles [13, 18–23]. The simultaneous encapsulation of both QDs/SiO2 microbeads and MNs/SiO2 microbeads in silica shell has been achieved by stöber method [13], reverse microemlusion [18] and inverse suspension method [19]. More recently, magnetic and florescent silica microspheres were prepared by silica-coated MNs as core, followed by layer-by-layer (LBL) assembly of poly electrolytes and QDs onto the core surfaces, which were then coated with a final silica shell [20]. Numpon Insin and co-workers used silica microspheres as ligand system. QDs and MNs were incorporated onto the ligands, which were then coated with a silica shell [21]. Nie and co-workers demonstrated the simultaneous or sequential doping of both oil-soluble Fe3O4 nanocrystals and QDs in silica microcapsules, the microbeads were then changed from oil-soluble to water-soluble by the addition of surfactants [22]. Reverse microemulsion method was employed by Rong He et al. to prepare the bi-functional nanoparticles embedded with MNs and QDs [23]. To our knowledge, it is the first time that some characteristics of bi-functional nanoparticles and the effect of MNs on PL intensity were studied in detail for this method.

In this paper, we described the preparation of bi-functional composite nanoparticles by directly using water-soluble Fe3O4 nanoparticles and QDs nanoparticles as cores, followed by encapsulation with a silica shell via reverse microemulsion method. QDs were obtained by hydrothermal method using mercaptosuccinic acid (MSA) as stabilizer [24]. The preparation of Fe3O4 nanoparticles was based on co-precipitation of ferrous and ferric ion solutions. With the aim of embedding more CdTe QDs in silica composite nanoparticles, we used poly (dimethyldiallyl ammonium chloride) (PDDA) to balance the electrostatic repulsion between CdTe QDs and silica intermediates. The MNs-QDs/SiO2 composite nanoparticles show good stabilities toward pH and ionic strength. Subsequently, the surface of silica composite nanoparticles was modified with 3-aminopropyltrimethoxysilane (APS) and 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP), and the nanoparticles modified with –NH2 were conjugated with goat anti-rabbit IgG, which showed it potential application in both separation and biolabeling.

Experimental

Reagents and chemicals

All chemicals used were of analytical reagent grade and used without further purification. n-hexane, tritonX-100, acetone, ethanol were purchased from Tianjin NO. 1 Chemical Reagent Factory. Mercaptosuccinic acid (MSA) (99+ %), poly (dimethyldiallyl ammonium chloride) (PDDA, Mw = 70000 g/mol) were obtained from J&K Chemical Corric. Chloride (FeCl3·6H2O) and ferrous chloride (FeCl2·4H2O) were purchased from ACROS. Cyclohexane. Tellurium powder (200 mesh, 99.8%), tetramethylammonium hydroxide (TMA, 25%), CdCl2 (99+ %) and NaBH4 (99%), Tetraethoxysilane (TEOS), 3-aminopropyltrimethoxysilane (APS), and 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP) were purchased from Sigma-Aldrich Chemical Co. FITC labeled goat anti-rabbit IgG (FITC-IgG ) obtained from Beijing Ding Guo Biotechnology Co. FITC-IgG and glutaraldehyde were diluted with PBS to the concentrations used only immediately prior to use. The water used in all experiments had a resistivity higher than 18 MΩ/cm.

Instrument

Fluorescence spectra were obtained at room temperature using a ShimadzuRF-5301 PC spectro fluorophotometer. UV-Vis absorption spectra were obtained using a Varian GBC Cintra10e UV-Visible spectrometer. To disperse the nanoparticles, a bath ultrasonic cleaner (Autoscience AS 3120, Tianjin, China) was used. In both experiments, a 1 cm path-length quartz cuvette was used to measure the absorption or fluorescence spectrum. X-ray photoelectron spectroscopy was performed on a PHI5000C ESCA X spectrometer with Cu KR excitation (1686.6 eV). The samples for TEM measurements were obtained by placing one drop of the samples on copper grids coated with carbon. JEOL-1230 transmission electron microscope was then employed for Transmission electron microscopy (TEM) images. and. Scanning electron microscopy (SEM) was carried out on a Philips XL30 microscope, and Hysteresis loops of the samples were obtained from JDM-B vibrating sample magnetometer (VSM).

Preparation of magenertic iron oxide nanoparticlels

Monodisperse iron oxide dispersion were prepared using the method already described [25]. Briefly, based on the co-precipitation of Fe2+ and Fe3+ by adding 25% NH3·H2O into the mixture of iron salts with a molar ratio (Fe3+ : Fe2+) of 2:1 under vigorous mechanical stirring and N2 protection. After 15 min, the precipitates were isolated from the solution by magnetic decantation and washed three times by water. TMA was added to peptize the magnetic nanoparticles, and then the alkaline ferrofluid was diluted with water. The concentration of the MNs was 5.4 mg/mL

Preparation of CdTe QD nanoparticlels

The preparation of mercaptosuccinic acids capped water-compatible CdTe nanoparticlels by hydrothermal route has been described in detail in our previous paper [24]. Briefly, fresh NaHTe solution was added to a N2-saturated CdCl2 solution in the presence of mercaptosuccinic acids at pH 11.2 in an ice-water bath. The ratio of [CdCl2]:[MSA]:[NaHTe] was fixed at 1:2.4:0.5. Then the CdTe precursor solution was put into a teflon-lined stainless steel autoclave with a volume of 50 mL. The temperature of autoclave was maintained at 160°C for 50 min. Then it was cooled to room temperature by a hydrocooling process. Stable water compatible mercaptosuccinic acids capped CdTe QDs with emission maximum at 585 nm were obtained and was used in the present experiments. Compared with the fluorescence emission of Rhodamine 6G [26], the luminescence quantum yield of the CdTe QDs used in this study is 75.2 %. The concentration of the CdTe QDs (1.03 × 10-5 M) was calculated according to the method reported by Peng et al [27].

Preparation of MNs-QDs/SiO2 composite nanoparticles

MNs-QDs/SiO2 composite nanoparticles were prepared by reverse microemulsion method at room temperature. In this method, cyclohexane was used as a continuous phase, TritonX-100 and n-hexanol were used as surfactant and co-surfactant respectively. First of all, 15 ml cyclohexane, 3.6 ml Triton X-100 and 3.6 ml n-hexanol were added to a flask, after stirring for 20 min, 200 μl MNs and 800 μl QDs were added and mixed with continuous stirring. After 30 min, 120 μl NH4OH and 120 μl PDDA solution (0.075% v/v) were added. To initiate the hydrolysis, 80 μl TEOS was added to the microemulsion system after 20 min, the reaction progressed in the dark for 24 h of stirring. The microemulsion was broken by adding 20 mL of acetone to the reaction system and the resultant precipitate was MNs-QDs/SiO2 composite nanoparticles which were washed in sequence with iso-propyl alcohol, ethanol and water. During each washing procedure, the dispersion of nanoparticles was subjected to precipitate by external magnetic field, followed by decantation of the supernatant and redispersion of the precipitate in the next solvent with the aid of supersonication. Ultimately, aqueous dispersion of MNs-QDs/SiO2 was obtained for further use.

The PL intensity of MNs-QDs/SiO2 composite nanoparticles is determined by the quantity of CdTe QDs embedded in the MNs-QDs/SiO2 composite nanoparticles. During the hydrolysis step of TEOS, the intermediate silica species can carry negative charges, the same with CdTe QDs stabilized by MSA. The electrostatic forces of repulsion between the negatively charges are not negligible, which limit the quantity of CdTe QDs embedded in the MNs-QDs/SiO2 composite nanoparticles. Therefore, PDDA was used to keep the electrostatic balance during the embedded process.

Surface functionalization of MNs-QDs/SiO2 composite nanoparticles

Surface functionalization of MNs-QDs/SiO2 composite nanoparticles is important for further application as fluorescent biomarkers. To obtain well biocompatibility and dispersibility, the surface of silica-shell was modified with –NH2. 30 μl of aminopropyltris (methyloxy) silane (APS) was injected to graft amine groups on the surface of MNs-QDs/SiO2 composite nanoparticles. In neutral solution, the amino group had a positive charge, and silica groups had negative charges. The amine-modified silica nanoparticles could back bond to the surface silanol groups, which resulted in very low total charges on the silica nanoparticle surface and no driving force existed on the nanoparticle surface to keep them separated. The excessive APS will cause aggregation of silica nanoparticles, and will consequently reduce their aqueous dispersibility. Therefore, 80 μl THPMP was added to solve this problem [28]. THPMP contains an inert stabilizing group -methylphosphonate-, which was introduced onto the surface of silica composite nanoparticles. The majority of amino groups electrostatic interact with methylphosphonate groups, which block the aggregation of nanoparticles effectively.

The reaction system was then kept under stirring for one more day. The resultant amine-funcationalized CdTe/SiO2 composite nanoparticles were washed by the same purification procedures as those mentioned above.

Preparation of fluorescent-magnetic-biotargeting tri-functional nanoparticles

To form the IgG antibody conjugated fluorescent-magnetic-biotargeting tri-functional nanoparticles, the amine-functionalized MNs-QDs/SiO2 nanoparticles were made to react with FITC-IgG by using glutaraldehyde as a coupling reagent [29]. First of all, 100 μl amine-functionalized MNs-QDs/SiO2 composite nanoparticles were added to 100 μl of 1% glutaraldehyde solution. The mixture was vibrated for 30 min at room temperature. After that, the nanoparticles were precipitated by a magnet and washed thrice by PBS (pH = 7.4). Then 120 μl FITC-IgG solution was added and incubated with constant shaking for about 2 h. It was then washed three times with PBS to remove excess antibodies. The fluorescent-magnetic-biotargeting tri-functional nanoparticles were obtained.

Results and discussion

MNs and QDs nanoparticlels

The MNs which were embedded in the bi-functional nanoparticles should have surperparamagnetic properties. From X-ray diffraction pattern of the MNs prepared in this study (Shown in Fig. 1), it is evident that the peaks at 30.22, 35.54, 43.24, 53.5, 57.36, 62.82, and 74.16 are the characteristic peaks of the Fe3O4 nanoparticlels, and by using the XRD patterns, the approximate average size of nanoparticles can be estimated by applying the Scherrer formula as follows:

$$ {\text{D}} = {\text{K }}\lambda ~/~\beta { \cos }\theta $$

Where λ is the X-ray wavelength (nm), θ is the Bragg angle, β is the excess line broadening, and K is the constant. Thus the MNs size was estimated to be 10 nm, which is consistent with the result obtained from TEM image of the MNs (shown in Fig. 4c), so the magnetic nanoparticlels have surperparamagnetic properties [30].

Fig. 1
figure 1

Powder XRD patterns of the magnetic Fe3O4 nanoparticles

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Fig. 2
figure 2

Normalized absorption and emission spectra of the solution of CdTe QDs (dashed line) and bi-functional MNs-QDs/SiO2 bi-functional nanoparticles (solid line)

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The fluorescence emission spectra of the mercaptosuccinic acids-capped water-compatible CdTe nanoparticlels have emission maximum at about 585 nm as can be seen by the results shown in Fig. 2. The relatively narrow PL peak indicates the narrow size distribution of the CdTe QDs and few electronic defect sites.

Optical property, magnetic property, size characterization of bi-functional nanoparticles

The UV-Vis and PL emission spectra of bi-functional nanoparticles are shown in Fig. 2. Compared with the free QDs in aqueous solution, we can see that after silica coating, the maximum emission peak became blue shift and broader obviously, It had been demonstrated that the blue shift was due to the corrosion of QDs during silica deposition. Since thiol ligands must be completely removed from their surfaces, leaving the QDs unprotected, thus resulting in this blue-shift in the maximum of emission spectra [31].

The magnetic properties of the MNs and bi-functional nanoparticles were studied by using a vibrating sample magnetometer (VSM) with fields up to 10 T. Hysteresis loops of the samples were registered at temperatures of 300 K (Fig. 3). From Fig.3, it can be seen that both of MNs and bi-functional nanoparticles exhibit negligible coercivity (Hc) and remanence, typical of superparamagnetic materials. The bi-functional nanoparticles are superparamagnetic at room temperature, reaching a saturation moment of 13.1 emu per gram of material. This low saturation magnetization value, far less than the saturation magnetization of the MNs used for the preparation of these nanoparticles (55 emu per gram), can be explained by taking into account the diamagnetic contribution of the thick silica shell surrounding the magnetic cores [20].

Fig. 3
figure 3

Hysteresis loops taken at 300 K for the MNs (a) and MNs-QDs/SiO2 bi-functional nanoparticles (b)

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From SEM image of the MNs-QDs/SiO2 composite nanoparticles (Fig. 4b), it can be seen that the composite nanoparticles had good dispersibility and uniform diameter of about 50 nm, which was further verified by TEM image (Fig. 4a). The structure of core-shell of MNs-QDs/SiO2 composite nanoparticles can also been seen from TEM image, which proved the CdTe QDs and MNs were indeed embeded in silica composite nanoparticles. From the photographs of MNs, and bi-functional nanoparticles, driven by an external magnetic field (Fig. 5 a and b), we can see the brown bi-functional nanoparticles were easily manipulated with an external magnetic field. They rapidly responded to the applied magnetic field and get crowded near the point where the magnetic field existed actually. When the magnetic field was removed, the nanoparticles lost their alignments immediately and dispersed again within few seconds by virtue of ultrasonic. All the results indicate that the bi-functional nanoparticles can be obtained successfully by reverse microemulsion method.

Fig. 4
figure 4

TEM images of the MNs-QDs/SiO2 bi-functional nanoparticles (a), SEM images of the MNs-QDs/SiO2 bi-functional nanoparticles (b), TEM images of the MNs nanoparticles (c)

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Fig. 5
figure 5

Phtographs of MNs (a), MNs-QDs/SiO2 bi-functional nanoparticles (b), fluorescent-magnetic-biotargeting tri-functional nanoparticles (c) driven by an external magnetic field

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Stability of bi-functional nanoparticles

In this paper, we studied the stability of bi-functional nanoparticles in aqueous solution. The results showed that the fluorescence intensity of MNs-QDs/SiO2 composite nanoparticles is remarkably constant over time, keeping their PL intensity even after 20 days without any visible decrease. It indicates that the MNs-QDs/SiO2 composite nanoparticles resisted quenching, the silica shell has a dramatic effect on the stability of the luminescent properties and the surfaces of the nanoparticles are well passivated.

Under physiological condition, ionic strength and pH are the important parameters of the buffer solution. So the influences of pH and ionic strength on the PL properties of MNs-QDs/SiO2 composite nanoparticles and free CdTe QDs were investigated in this study (Figs. 6 and 7). From Fig. 6, it can be seen that pH of buffer largely affects the fluorescence intensity of free QDs and no fluorescence was observed at pH values below than 5, which is due to the precipitation of the QDs [32, 33]. The interaction between a ligand (MSA) and the surface cations can be regarded as a special type of coordinating bond. When hydrogen ions are added into the system, the hydrogen ions will compete for the surface ligands with the nanocrystals. Therefore, the detachment of the ligands from the QD core surface can be considered as a displacement reaction, which will destroy their dispersibility and cause aggregations [33]. As for MNs-QDs/SiO2 composite nanoparticles, it also exhibits high PL intensity when pH is 3.

Fig. 6
figure 6

Effect of pH on the fluorescent intensity of MNs-QDs/SiO2 bi-functional nanoparticles (solid line) and (dashed line)

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Fig. 7
figure 7

Effect of ionic strength on the fluorescent intensity of MNs-QDs/SiO2 bi-functional nanoparticles (solid line) and CdTe QDs (dashed line)

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From Fig. 7, it can be seen that MNs-QDs/SiO2 composite nanoparticles are less susceptible than naked CdTe QDs in different ionic strength solution. The above mentioned results indicate that the PL emission of CdTe QDs can be well protected by the silica network structure when CdTe QDs embedded in silica nanoparticles.

The effect of MNs on the fluorescence of MNs-QDs/SiO2 composite nanoparticles

To examine the effect of MNs on the fluorescence of MNs-QDs/SiO2 composite nanoparticles, different proportions of MNs were used for preparing MNs-QDs/SiO2 composite nanoparticles, and the fluorescence spectra were recorded correspondingly (Fig. 8). From Fig. 8, it can be seen that with the increasing of the ratios between QDs and MNs from 8:2 (v/v) to 3:7 (v/v), the effect of MNs on the fluorescence of MNs-QDs/SiO2 composite nanoparticles became obviously. The PL intensity gradually decreased and there were a little blue shift in the emission spectra with the increase of the proportion of MNs. This phenomenon might be caused by two factors, a broad absorption spectrum of MN in the visible range (400–600 nm), perhaps due to charge-transfer transitions in this mixed valence compound, excitation light in this window will be strongly absorbed by iron oxide, thus attenuating the light intensity reaching the embedded QDs. The other factor responsible for the decreased PL intensity is that with the increasing proportion of MNs, the arrangement of MNs and QDs become more compact, the interaction between MNs and QDs would lead to energy transfer and hence reduce the PL intensity of MNs-QDs/SiO2 composite nanoparticles [34]. This kind of interaction would accelerate the corrosion of QDs during silica deposition, which will induce a blue shift in the emission spectra.

Fig. 8
figure 8

Fluorescence emission spectra of MNs-QDs/SiO2 bi-functional nanoparticles with different ratio (V/V) of QDs and MNs:, 8:2 (a), 7:3 (b), 6:4 (c), 5:5 (d), 4:6 (e), 3:7 (f)

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Bioconjugation and bioactivity

In order to prove the bioactivity of the bi-functional nanoparticles, amine-functionalized MNs-QDs/SiO2 nanoparticles were made to react with FITC-IgG by using glutaraldehyde as a coupling reagent. The fluorescence spectra of the mixture system were shown in Fig. 9. FITC is a green fluorescence dye, which has a maximum emission peak at 520 nm and the maximum emission peak of the bi-functional nanoparticles is at 573 nm. From Fig. 9, it can be seen that the fluorescence spectra of MNs-QDs/SiO2- FITC /IgG system has maximum emission peaks of 520 nm and 573 nm simultaneously, which indicates that the amine-functionalized MNs-QDs/SiO2 composite nanoparticles can be conjugated with IgG to form fluorescent-magnetic-biotargeting tri-functional nanoparticles. It can also be verified by the photographs of fluorescent-magnetic-biotargeting tri-functional nanoparticles driven by an external magnetic field (Fig. 5(c)), From Fig. 5(c), we can see the brown bi-functional nanoparticles were coated with yellow-green FITC-IgG and aggregated at the point where the external magnetic field existed. These also prove the bioactivity of the bi-functional nanoparticles and potential for various applications in separation and bioassay.

Fig. 9
figure 9

The Fluorescence emission spectra of FITC-IgG (a), MNs-QDs/SiO2 bi-functional nanoparticles (b) and fluorescent-magnetic-biotargeting tri-functional MNs-QDs/SiO2nanoparticles (c)

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Conclusion

In summary, we synthesized bi-functional nanoparticles by coating aqueous magnetic nanoparticles and CdTe quantum dots with silica shell via reverse microemulsion method. This facile method provides small size (about 50 nm), smooth morphology, high luminescent and magnetic MNs-QDs/SiO2 composite nanoparticles, which have good stability towards time, pH and ionic strength, and can be manipulated easily in the external magnetic field. PDDA was used to keep the electrostatic balance during the process of embedded process. The effect of MNs on the fluorescence of composite nanoparticles was also studied. The surface of the bi-functional nanoparticles was modified with amino group. The modified surface of bi-functional nanoparticles can conjugate with IgG to form fluorescent-magnetic-biotargeting tri-functional nanoparticles, which showed that the modified nanoparticles had good bioactivity. The fluorescent-magnetic-biotargeting tri-functional nanoparticles have potential for detection of bio-molecules such as proteins and targeting agents. The further application of the bi-functional nanoparticles is being developed in our group.