Glutathione-functionalized Mn:ZnS/ZnO core/shell quantum dots as potential time-resolved FRET bioprobes

Abstract

High-quality glutathione-capped Mn:ZnS/ZnO core/shell quantum dots were prepared in aqueous solution through the nucleation-doping method by using low-cost inorganic salts as precursors. Remarkable improvements of photoluminescence (PL) were achieved by introduction of a ZnO shell around the Mn:ZnS QDs through basic hydrolysis of Zn(NO₃)₂. The Mn:ZnS/ZnO quantum dots have been demonstrated to be sensitive time-resolved Förster resonance energy transfer (TR-FRET) bioprobes for the detection of a trace amount of biomolecules such as avidin at a concentration of 3 nM with a linear response ranging from 10 to 100 nM. We anticipate that the QDs may have great potential for versatile applications in biodetection and bioimaging.

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Introduction

Recently, wide bang gap semiconductors quantum dots (QDs) like ZnS, ZnSe or CdSe doped with Manganese (Mn²⁺) have come to the forefront of functional nanomaterials for a variety of biological applications.^1–12 Doping with atomic impurities (Mn²⁺) is an efficient way to manipulate the fluorescence emission spectra of the QDs. Particularly, Mn-doped ZnS (Mn:ZnS) QDs, which are free of Cd and Se, are greener materials and have attracted considerable attention for their application in biomedicine. In Mn:ZnS dots, a small amount of Mn²⁺ is incorporated into the host ZnS nanocrystalline lattice, which results in visible fluorescence at 585 nm attributed to a ⁴T₁(⁴G) → ⁶A₁(⁶S) transition of Mn²⁺ ions.^1–3 Compared to traditional organic fluorophores and Cd-containing quantum dots commonly used in bioimaging and biodetection, Mn-doped QDs show superior features such as low toxicity, high chemical stability, high resistance to photobleaching, long photoluminescence (PL) lifetimes (a few milliseconds), and large Stokes shifts.¹ Moreover, doping with Mn²⁺ can also impart remarkable magneto-optical properties to the QDs.⁴ In particular, when combined with the technique of time-resolved (TR) luminescence that utilizes the long-lived emission of Mn²⁺ by setting appropriate delay time and gate time, the short-lived background luminescence or interferences such as scattered lights and autofluorescence from cells and tissues can be effectively suppressed, which thus results in high signal-to-noise ratio.^5–13

In the past decade, various approaches were reported to synthesize Mn:ZnS QDs, mostly focused on organometallic routes and aqueous synthesis.^14–19 However, some drawbacks still existed in the prepared Mn:ZnS QDs such as the dual emissions from the band edge emission and dopant emission, low quantum yield and photoluminescence instability, which would limit their applications in certain fields.²⁰ Therefore, development of a safe synthesis of Mn:ZnS QDs with pure dopant emission, stronger luminescence and better photostability is critical for the further application. Peng's group demonstrated a doping strategy based on decoupling the doping process from the nucleation and/or growth, and designed nucleation-doping to prepare Mn-doped ZnSe QDs in phosphine-free 1-octadecene solution.^3,21,22 The nucleation-doping was realized by a mixed dopant and host precursor during the nucleation. After nucleation, the growth of the ZnSe host overcoated the dopants. The Mn:ZnSe QDs synthesized by this strategy had a diffuse interface between the MnSe core and ZnSe shell, which had high PL quantum yield and thermal stability up to the boiling point of the solvent.^3,21,22

In this work, we describe the preparation of Mn:ZnS QDs by a alternative nucleation-doping strategy with glutathione (GSH) as the capping reagent in aqueous solution. The resulting QDs were found to be with high optical quality. Moreover, capping QDs with inorganic shells is an efficient strategy to optimize their PL properties and photochemical stability. Herein, we take advantage of the GSH-capped Mn:ZnS QDs as template cores to introduce a ZnO shell. This passivation allows a marked improvement of the emission properties of the QDs. Only pure dopant emission at ca. 585 nm without trap state emission was observed. The core/shell Mn:ZnS/ZnO QDs have a PL QY of 21% in water at neutral pH. By employing the technique of time-resolved fluorescence resonance energy transfer, we demonstrate for the first time the use of Mn:ZnS/ZnO as luminescent bioprobes in a TR-FRET assay of avidin protein with a detection limit down to nanomolar range.

Experimental

Chemicals

Glutathione (GSH, 99%), avidin–sulforhodamine 101, biotin, N-hydroxy succinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was from Sigma (St. Louis, MO, USA). Zn(NO₃)₂ (99%), MnCl₂ (99%) and Na₂S (99%) were obtained from Shanghai Reagent Company. Ethanol (CH₃CH₂OH, anhydrous) was of analytical grade and used without further purification. Other chemicals were of analytical grade. Phosphate buffer solution (PBS, 25 mM, pH = 7.4) was prepared by mixing the solutions of K₂HPO₄ and NaH₂PO₄. The ultra pure water with 18.2 MΩ (Millipore Simplicity, USA) was used in the experiments.

Synthesis of the GSH-capped Mn:ZnS quantum dots

A nucleation-doping strategy was adopted for the preparation of the core/shell Mn:ZnS/ZnO QDs. In the first step, GSH-capped Mn:ZnS core QDs were prepared using a mixture of the dopant and the host precursor. Subsequently, in alkaline conditions, Zn(NO₃)₂ is hydrolyzed into Zn(OH)₂, which dehydrates to produce ZnO clusters that grow at the surface of Mn:ZnS nanocrystals to form the ZnO shell (Fig. 1).

Fig. 1 Schematic representation of the two-step synthesis of the Mn:ZnS/ZnO core/shell QDs.

In a typical experiment, 0.5 mL MnCl₂ solution (0.04 M) was loaded into a 250 mL three-necked flask and degassed for 30 min by bubbling with nitrogen. 100 mL fresh NaS solution was added to the N₂-saturated MnCl₂ solution at pH 9 in the presence of GSH as the stabilizing reagent. The reaction was then switched from nitrogen bubbling to nitrogen flow and subjected to a reflux at 30 °C for 20 min. 10 mL of Zn(NO₃)₂ stock solution (0.08 M) was injected into the reaction solution. The Mn–Zn–S and Zn-to-GSH precursor ratios were 1 : 40 : 20 and 1 : 3, respectively. The mixture was kept stirring for another 40 min with 60 °C, and then the solution was aged at 40 °C under air for 2 h to form GSH-capped Mn:ZnS quantum dots. Finally, the reaction mixture was allowed to cool down to room temperature and the as-prepared Mn:ZnS QDs were used directly without any post-treatments. The Mn:ZnS QDs could be precipitated by ethanol, and the precipitate was centrifuged, washed with ethanol, and dried in vacuum at room temperature.

Deposition of ZnO shell around the Mn:ZnS QDs

For the ZnO shell growth, a simple procedure based on hydrolysis and condensation of Zn (NO₃)₂ in basic medium was performed according to previous report with some adaptations.³⁰ Typically, 100 mg of prepared Mn:ZnS QDs were dispersed in 150 mL of water. The pH was adjusted to 11.5 with 0.1 M NaOH. Then, 5 mL of 0.1 M Zn(NO₃)₂ aqueous solution was added dropwise to the GSH-Capped Mn:ZnS suspension under vigorous stirring with 40 °C. The ZnO/Mn:ZnS molar ratio was varied by changing the volume of 0.1 M Zn(NO₃)₂ aqueous solution. The solution was aged at 40 °C under air for 2 h to form the Mn:ZnS/ZnO core/shell QDs. The obtained Mn:ZnS/ZnO core/shell QDs were purified by centrifugation in ethanol, washed 3 times with ethanol, and then dried in vacuum at room temperature.

Bioconjugation of the Mn:ZnS/ZnO QDs with biotin

The free carboxyl groups on the surface of the QDs allow further conjugation with biomolecules such as biotin, by following the well-established EDC/NHS protocol,²⁶ in which biotin is covalently bound to the GSH-functionalized QDs through its carboxy group by using the cross-linking reagent EDC and NHS in phosphate-buffered saline (PBS) solution at room temperature. In this work, 20 mg as-prepared QDs was dissolved in 0.1 M phosphate buffered saline (PBS) solution (pH 7.4) followed by the addition of 0.2 mmol EDC and 0.5 mmol NHS. The mixture was then stirred for 15 min to activate the carboxylic group of QDs. Subsequently, 0.2 mmol biotin dispersed in 0.1 M PBS solution (pH 7.4) was added to the above solution, and the mixture was stirred for 20 h at room temperature. To reduce nonspecific interaction, the rest active sites were blocked with 1% BSA solution. After reaction overnight, the free nonconjugated biotin as well as the isourea byproduct of the conjugation reaction were removed by ultrafiltration. The above mixture was subjected to ultrafiltration using a 50 000 MW filter; after the lower phase was removed, the upper phase containing Mn:ZnS/ZnO–biotin conjugations was decanted, diluted with PBS, and the solution was stored at 4 °C. The estimate of the coupling yield of biotin–QDs was shown in ESI.†

Homogeneous TR-FRET detection of avidin based on biotinylated Mn:ZnS/ZnO QDs

200 μL of sulforhodamine 101-labled avidin solution with different concentration was added to a 1 mL calibrated quartz cuvette. To each cuvette, 200 μL of biotinylated QDs dispersed in PBS solution (pH 7.4) was subsequently added. The cuvette was then incubated for 30 min at room temperature. Finally the quartz cuvette was subjected to the measurement of TR-FRET spectra a LS-55 fluorometer (Perkin Elmer) fluorescence spectrometer, upon excitation at 350 nm, delay time of 0.2 ms, gate time of 0.4 ms, and cycle time of 20 ms. For comparison, nonbinding control experiments were performed by employing the as-prepared QDs instead of the biotinylated QDs as bioprobes under otherwise identical conditions.

Characterization

X-ray diffraction (XRD) measurements were performed on a Shimadzu XRD-6000 powder X-ray diffractometer, using Cu Kα (λ = 1.5405 Å) as the incident radiation. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was performed on a Perkin-Elmer Optima 3000 DV after dissolving the aqueous Mn:ZnS QDs in 5% hydrochloric acid. Transmission electron microscopy (TEM and HRTEM) samples were prepared by dropping the samples dispersed in water onto carbon coated copper grids with excess solvent evaporated. TEM images were recorded on a Shimadzu JEM-2010 CX with an accelerating voltage of 100 kV. HRTEM images were recorded on a JEM-2010 F with an accelerating voltage of 200 kV. Fluorescence measurements were performed using a Shimadzu RF-5301 PC fluorescence spectrometer. The room-temperature photoluminescence quantum yield of the Mn:ZnS/ZnO QDs was estimated following the ref. 23 by using quinine sulfate as a reference standard (see ESI†). The time-resolved fluorescence spectrum was performed on a LS-55 fluorometer (Perkin Elmer). UV-Vis absorption spectra were obtained by a UV-3600 spectrophotometer (Shimadzu). The measurement of the infrared spectroscopy was performed using a Nicolet IR100 infrared spectrometer. The X-ray photoelectron spectra (XPS) were taken on a Thermo Scientific K-alpha electron energy spectrometer using Al Kα (1486.6 eV) as the X-ray excitation source. XPS sample was purified and washed carefully in order to remove any impurity phase. All of the measurements were performed at room temperature.

Results and discussion

Crystal structure, size and shape of GSH-capped Mn:ZnS core

The powder X-ray diffraction (XRD) patterns recorded from the Mn:ZnS core QDs are shown in Fig. 2. The intense and wide peaks, characteristic for nanoparticles, are positioned at 2θ = 28.7°, 48.1°, and 56.4°, which oriented along the (111), (220), and (311) directions, and are in agreement with the JCPDS no. 77-2100 for cubic zinc blende ZnS. No diffraction peaks from impurities were detected in the samples. This result shows that Mn²⁺ doping into the ZnS host nanocrystals does not disturb the crystal structure. The incorporated Mn²⁺ concentrations in the Mn:ZnS core QDs was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). And the doped Mn concentration (Mn/Zn molar ratios) in the Mn:ZnS core QDs was 0.94%.

Fig. 2 XRD patterns of the Mn:ZnS core QDs and the Mn:ZnS/ZnO core/shell QDs. Diffraction lines for cubic phases of bulk ZnS are shown for the guidance.

Fig. 3 (A) shows wide-field TEM image together with a representative high-resolution TEM (HRTEM) image of the Mn:ZnS QDs. Nearly monodispersed QDs were obtained with an average diameter of 3.2 nm. Clear lattice fringes were observed in the high-resolution TEM (HRTEM) image reveal high crystallinity of the QDs. The distance of 0.33 nm between adjacent lattice fringes is consistent with the literature value for the (111) d spacing, 0.324 nm (JCPDS 77-2100).

Fig. 3 TEM image of the Mn:ZnS cores (A) and the Mn:ZnS/ZnO core/shell QDs (B). A representative HRTEM is given as the inset.

Epitaxial growth of the ZnO shell around the Mn:ZnS QDs core

In alkaline conditions, Zn(NO₃)₂ is hydrolyzed into Zn(OH)₂, which dehydrates to produce ZnO at the surface of Mn:ZnS nanocrystals to form the ZnO shell. A marked decrease of pH from 11.5 to 8 was observed during the addition of Zn(NO₃)₂ with using a Zn(NO₃)₂/Mn:ZnS molar ratio of 0.12, thus confirming the consumption of NaOH in the hydrolysis reaction. As shown in Fig. 2, the narrower diffraction peaks comparing with Mn:ZnS core were due to the formation of a bigger particle size with the growth of the shell of ZnO around the Mn:ZnS core. No XRD peaks from the crystalline ZnO was shown in Fig. 2, indicating that the amount of ZnO is very low.

The average diameter of Mn:ZnS/ZnO core/shell QDs was 4.8 nm, larger than that of the core QDs. The difference means that the shell thickness is around 0.8 nm. From the HRTEM image, the lattice spacing (0.33 nm) agrees well with that of the Mn:ZnS core indicating that the introduction of the ZnO shell keeps unchanged the lattice spacing of ZnS nanocrystals. Because the ZnO layer introduced at the periphery of the Mn:ZnS QDs is very thin, and the core and the shell have similar electron densities and lattice parameters, it is difficult to recognize the interface by image contrast.

The X-ray photoelectron spectra (XPS) results further confirmed the proposed Mn:ZnS/ZnO core/shell structure. As shown in Fig. 4, the peaks located at 1021.6, 532.1, and 162.2 eV correspond to Zn 2p, O 1S and S 2p, respectively. The molar ratio of oxygen (O) to sulfur (S) can be calculated by dividing the area of the XPS peaks by their respective sensitivity factors (2.93 for O and 1.68 for S).^29,30 The molar ratio of O : S increased remarkably from 0.53 : 1 in the Mn:ZnS core nanocrystals to 5.2 : 1 in the Mn:ZnS/ZnO core/shell nanocrystals, which confirmed the growth of the ZnO shell.

Fig. 4 XPS spectra of the Mn:ZnS core and the Mn:ZnS/ZnO core/shell QDs.

The subtle red shift in the absorption spectra of the Mn:ZnS/ZnO core/shell QDs revealed the formation of a bigger particle size with the growth of the ZnO shell around the Mn:ZnS core as shown in Fig. 5. The inset was corresponding photoluminescence (PL) spectra of the QDs. The PL spectra exhibited a strong peak around 585 nm from an internal electronic transition of the Mn (⁴T₁ → ⁶A₁).^1–3 Because a large number of surface defects existed in nanoparticle surface, the nonradiative recombination paths could generate for the excitation energy in the Mn:ZnS core QDs, as a result, quenching for the emission of Mn²⁺ takes place to some extent. By the growth of an additional ZnO shell on the Mn:ZnS core, the surface defects could be greatly reduced; thus, emission intensity of the Mn:ZnS/ZnO core/shell QDs could be enhanced greatly, and that the defect related emission around 450 nm attributed to a donor–acceptor pair transition involving electron–hole recombination at a sulfur vacancy³¹ was greatly suppressed.

Fig. 5 UV-Vis spectra of the Mn:ZnS core (a) and Mn:ZnS/ZnO core/shell QDs (b), inset: the corresponding PL spectra (λ_ex = 350 nm).

As shown in Fig. 5, the Mn:ZnS/ZnO core/shell QDs obviously exhibited increased PL intensity compared with the Mn:ZnS core QDs, and the PL quantum yield (QY) of the Mn:ZnS/ZnO core/shell QDs was up to 21%. The fluorescence life-time of the Mn:ZnS/ZnO core/shell QDs at the emission of 585 nm are 1.5 ms (see Fig. S4 in ESI†), consistented with the values in previous reports.^24,25 The long fluorescence life time was propitious for the time-resolved fluorometry.

Application in homogeneous TR-FRET detection of avidin

The surface functional groups of the Mn:ZnS/ZnO QDs can be identified by FTIR spectroscopy (Fig. 6). The strong infrared bands centered at 1652 cm⁻¹ and 3390 cm⁻¹ were observed for the as-prepared QDs, which are attributed to the C=O stretching mode and the O–H stretching mode of the carboxyl groups, respectively. In contrast to that of the as-prepared QDs, the FTIR spectrum of biotinylated QDs (Fig. 6 A) displays a new peak at 3432 cm⁻¹, ascribed to the N–H stretching vibration of biotin. Moreover, a vibrational band centered at 1671 cm⁻¹ corresponding to the amide carbonyl of biotin was observed in biotinylated QDs as shown in Fig. 6 B, which was similar with previous report.³² These findings show successful binding of biotin to the surface of the QDs, and the yield of the biotin–QDs coupling was about 64%, which enables specific binding with proteins such as avidin for versatile bioapplications.

Fig. 6 (A) FTIR spectra of the as-prepared Mn:ZnS/ZnO QDs and the corresponding biotinylated QDs. (B) Expanded the area around 2000–1000 cm⁻¹ in the above FTIR spectra.

To demonstrate the use of biotinylated Mn:ZnS/ZnO QDs as biosensors, they were used for detection of traces of avidin based on TR-FRET technique, in view of the high affinity between avidin and biotin.^27,28 The principle of TR-FRET detection of avidin by employing the biotinylated QDs as energy donor and fluorophore sulforhodamine 101 (SUF 101)-labeled avidin as energy acceptor is illustrated in Fig. 7. Excitation of biotinylated Mn:ZnS/ZnO triggers energy transfer to SUF101 within a given proximity through specific binding between biotin and avidin, and results in emission from SUF 101 at its characteristic wavelength.

Fig. 7 TR-FRET detection of avidin by employing biotinylated Mn:ZnS/ZnO as donor and sulforhodamine 101 as acceptor.

Here SUF 101 was selected as an energy acceptor to couple with the Mn:ZnS/ZnO QDs, because it has a broad excitation peak at 585 nm that matches well with the Mn (⁴T₁ → ⁶A₁) emission band centered at 585 nm (Fig. 8A). More importantly, the short-lived broad-band emission of SUF 101 arising from direct UV excitation, can be distinguished easily from the long-lived emission of Mn²⁺, that is the apparently long lived emission of SUF 101 due to FRET could be employed for time-resolved detection. As shown in Fig. 8B, the conventional PL spectrum of a mixture of the biotinylated Mn:ZnS/ZnO QDs and SUF 101 was dominated by the emission of SUF 101, and the emission bands from Mn:ZnS/ZnO QDs were hardly observed due to interference from the SUF 101 emission band (Fig. 8B). In sharp contrast, only intense emission originating from Mn (⁴T₁ → ⁶A₁) emission were detected in the time-resolved PL spectra. These results verify that the time-resolved fluorescence technique, by utilizing the long-lived luminescence of emitters, is particularly effective in removing undesired short-lived background fluorescence. The proposed TR-FRET detection based on the Mn:ZnS/ZnO QDs brings together the advantage of very low background of the time-resolved technique and the convenience of homogeneous assay from FRET.

Fig. 8 (A) Excitation of SUF 101 (a) and emission (b) spectra of the biotinylated Mn:ZnS/ZnO; (B) conventional fluorescence spectra of the Mn:ZnS/ZnO QDs (a), sulforhodamine 101 (b), and mixture of the Mn:ZnS/ZnO QDs and sulforhodamine 101 (c). Inset: time-resolved fluorescence spectra of the mixture (c).

As shown in Fig. 9, upon excitation of the Mn:ZnS/ZnO QDs at 350 nm, the TR-FRET signal represented by a clear emission band of SUF 101 centered at 610 nm is gradually enhanced at the expense of the Mn (⁴T₁ → ⁶A₁) emission band centered at 585 nm with increasing amount of SUF 101-labeled avidin. The concentration of avidin can be quantified by determining the integrated PL intensity ratio SUF 101610/Mn585 from the observed TR-FRET spectra. For comparison, nonbinding control experiments were performed by employing the as-prepared Mn:ZnS/ZnO QDs instead of the biotinylated QDs as bioprobes under otherwise identical conditions. The specific interaction between biotin and avidin plays a key role to ensure proximity between the acceptor and the donor. Non-biotinylated NCs and SUF 101 are far apart in solution, and thus no FRET occurred in the control experiments. The calibration graph for avidin is linear over the range of 10–100 nM with a correlation coefficient of 0.996. The precision for seven repeated measurements of 36 nM avidin was 3.5% (RSD) and the detection limit (3δ) was 3 nM.

Fig. 9 TR-FRET spectra for avidin with different concentrations. Inset was calibration curve of TR-FRET detection for the integrated PL intensity ratio SUF 101₆₁₀/Mn₅₈₅ versus the concentration of avidin. The asterisk (*) symbols show the control experiments with SUF 101-labeled avidin and the no-biotinylated Mn:ZnS/ZnO QDs, for which no binding and hence no FRET occurs. The delay and gate times were set to 0.2 ms and 0.4 ms, respectively. Cycle time was 20 ms. Error bars mean the standard deviation. Each point was an average value of three independent measurements.

In summary, the low-toxic luminescent Mn:ZnS/ZnO core/shell QDs were successfully synthesized by a nucleation-doping strategy with employing glutathione as surfactant and capping agent. The QDs solution exhibited intense PL emission and long PL life-time, which thus allows one to completely eliminate the interference of short-lived background fluorescence by means of time-resolved detection. The biotinylated QDs were used as efficient TR-FRET probes to detect trace amounts of avidin in a typical avidin–biotin model system with a detection limit of about 3 nM. As a new type of TR-FRET bioprobe, the Mn:ZnS/ZnO QDs have desirable advantages such as low cost, high PL quantum yields and excellent water dispersibility and photostability, and thus may have great potential for versatile applications in time-resolved biodetection and bioimaging.

Acknowledgements

We greatly appreciate the National Natural Science Foundation of China for the financial support (21205064). This work was also supported by Fund of State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1208).

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Footnote

† Electronic supplementary information (ESI) available: PL QY determination and other experimental details. See DOI: 10.1039/c3ra45491f

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