Introduction

Quantum dots (QDs) are inorganic semiconductor nanocrystals with remarkable optoelectronic properties originated from quantum confinement of the electronic motion, otherwise unavailable in either discrete atoms or bulk materials [1–3]. QDs have several advantages over conventional organic fluorophores because of their excellent resistance to photobleaching and optical properties tunable by size and material composition [4]. Most importantly, the broad absorption with narrow emission spectra of QDs makes them potential candidate for multiplex applications in bio-imaging as well as bio-tagging [1, 3]. In recent years, the extraordinary photophysical properties of QDs have been successfully exploited in various fields such as optoelectronic devices, imaging, as well as biodiagnostics [3, 5–7]. Various sensing application has been developed based on the quenching of QDs fluorescence by metal nanoparticles or other molecules [8, 9]. QDs can also act as efficient energy transfer donors or acceptors in fluorescence resonance energy transfer (FRET)-based applications [10, 11]. Several bioanalysis formats like molecular beacons, TaqMan probes, and immunoassays have been developed based on either quenching or FRET of QDs [1, 11].

In the last few years, a great deal of research—theoretical as well as experimental—has been carried out to understand the influence of metal surface on the emission properties of fluorophore [12–14]. A fluorophore near a metal film or metal nanoparticles (NPs) has been reported to show a varied degree of fluorescence depending on the distance between the metal surface and fluorophore [15, 16]. However, the exact mechanism of differential fluorescence exhibited by a fluorophore near a metal surface is still to be understood completely. In this regard, the fluorescent enhancement of conventional fluorophore or QDs in presence of silver nanoparticles (Ag NPs) has been well studied [17, 18]. However, there are only few reports where Ag NPs have been used as an efficient quencher of the fluorophore [19, 20]. Tay et al. has showed that the photoluminescence of surface-functionalized Si was quenched by Ag NPs [21]. Recently it has been reported that Ag NPs quenched the fluorescence of 1,4-dihydroxy-2,3-dimethyl-9,10-anthraquinone depending upon the particle size [22]. Ag NPs have been reported to act as enhancer of FRET efficiency, where hybridization of the donor-labeled oligonucleotide with the acceptor-labeled complimentary strand on Ag NP surface led to larger Förster distance. Zhang et al. showed that the FRET efficiency between cy5 and cy5.5 on the surface of Ag NPs was increased with particle size [23]. The report suggested that the conventional Förster distance can be increased to 75% by varying the particle size of the Ag NPs. However, the length scale for detecting dynamic changes in FRET-based methods is limited to a distance of <10 nm by the nature of the dipole–dipole mechanism. On the other hand, Yun et al. [24] reported that the energy transfer from a dipole to a nanometal surface (nanometal surface energy transfer, NSET) follows a 1/R 4 distance dependence and the traditional Förster distance can be increased up to the range of 22 nm. The quenching property of Ag NPs in FRET or NSET applications could stem from their plasmon resonance in the visible range which makes them strong absorbers with large extinction coefficients [25]. The development of long-range FRET is, therefore, critical to investigate the biological multi-component systems such as ribosomes or various nucleoprotein complexes [24].

In this paper, we report the fluorescence quenching of zinc sulfide quantum dots (ZnS QDs) by Ag NPs. Ag NPs were observed to act as an efficient quencher of ZnS QDs, exhibiting K sv value approaching 9 × 109 M−1. The corresponding Stern–Volmer plot indicated mixed quenching mechanism to be involved in the present study. The static quenching was investigated by absorbance study as well as regaining of quenched fluorescence on salt addition. The energy transfer process involved in the present quenching was studied by time-resolved fluorescence spectroscopy.

Experimental

Materials

Zinc acetate dihydrate [Zn(CH3COO)2, 2H2O], sodium sulfide [Na2S], trisodium citrate, silver nitrate, and acetic acid (glacial, 99–100%) were purchased from Merck India Ltd, Mumbai, India. Chitosan (high MW, >75% deacetylated) was obtained from Sigma–Aldrich Chemical Pvt. Ltd., Kolkata, India. All chemicals employed were of AR grade and were used as received without further purification. Milli-Q ultra pure water (>18 MΩ cm, Millipore) was used in all experiment.

Synthesis of Chitosan-Stabilized ZnS QDs

The synthesis of chitosan-stabilized ZnS QDs was carried out using a modified inorganic wet-chemical synthesis method reported by Warad et al. [26]. Briefly, 21 mg zinc acetate (0.01 M) was added to 9 ml of 0.05% (w/v) chitosan solution under constant stirring and heated at 80 °C for 15 min. After cooling to room temperature, 1 mL of freshly prepared 16 mg mL−1 sodium sulfide was added dropwise to the mixture in an ice bath with continuous stirring. This resulted in the formation of a milky white suspension without any sedimentation for more than 24 h, indicating the synthesis of ZnS nanocrystals. The as-prepared colloidal solution of ZnS QDs was ultrasonicated to remove the dissolved gases, if any. Next, ZnS QDs were characterized by transmission electron microscopy (TEM) by a JEOL 2100 UHR-TEM instrument. Five microliters of ZnS QDs solution was drop-cast on carbon-coated copper grids and subsequently air-dried before TEM analysis. X-ray diffraction (XRD) pattern were recorded with Brucker D8 Advanced X-ray diffraction measurement system, with Cu Kα source (λ = 1.54 Å). UV-visible absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Steady-state fluorescence intensity was measured using Jobin-Yvon Fluoromax-3 spectrofluorimeter at an excitation wavelength of 315 nm. Quantum yield of ZnS QDs were calculated according to Williams' method [27] using Rhodamine 101 as a reference (Q.Y. = 100%) [28].

Synthesis of Citrate-Capped Ag NPs

Citrate-capped Ag NPs were prepared by a modified Turkevich method [29]. Briefly, 1 mM aqueous solution of AgNO3 was heated until it started to boil. As the boiling started, sodium citrate (5 mM) was added to it. The color of the solution slowly turned yellow and then grayish yellow after 30 min of boiling, indicating formation of Ag NPs. The reaction was performed under refluxing condition to avoid loss of water by evaporation during boiling. The solution was cooled to room temperature and kept for 1 day. In order to remove excess Ag+ ions, if any, the Ag NPs solution was centrifuged at 13,000×g for 15 min and the pellet was washed for several times. Finally, the washed Ag NPs were resuspended in Milli-Q water and subsequently characterized by using UV-visible absorption spectroscopy (Varian Cary 100 spectrophotometer) and TEM (JEOL 2100 UHR-TEM instrument). The particle size determined by TEM analysis was used to calculate the concentration of Ag NPs.

Fluorescence Quenching of ZnS QDs by Ag NPs

The chitosan-stabilized ZnS QDs were diluted to 1.4 μM in Milli-Q water and different concentrations of citrate-capped Ag NPs were added to it. The pH of all the samples was 5.5. After 1 h of incubation, each sample was studied by steady state fluorescence as well as absorption spectroscopy. For all steady state fluorescence, measurement excitation was set at 315 nm and the fluorescence emission spectra were recorded in the wavelength range of 360–560 nm. For quenching experiments, the integrated fluorescence intensity in the abovementioned range was used to determine F o/F since it gave a flat baseline free from Raman scatter. Time-resolved intensity decays of the ZnS QDs both in absence and presence of quencher were recorded using a Life Spec II spectrofluorimeter (Edinburgh Instrument). The sample was excited by PicoQuant 308 nm LED source, and the decay was measured in a time scale of 0.0488 ns/channel. The decay curves were analyzed by FAST software, provided by Edinburgh Instrument along with the fluorescence instrument.

Results and Discussion

As evident from the TEM image (Fig. 1a), the chitosan-stabilized ZnS QDs synthesized in the present study were nearly monodisperse and spherical in shape. The average particle size was found to be 2.9 nm from the corresponding particle size distribution shown in Fig. 1b. The high-resolution TEM (HRTEM) image shown in the inset of Fig. 1a, depicts the lattice-resolved structure of ZnS QDs. The UV-visible absorption spectrum (Fig. 1c) of the ZnS QDs showed absorption edge around 300 nm, considerably blue-shifted compared to that of bulk ZnS at 340 nm [30]. This shift in band gap can be explained by the effective mass approximation model with the particle in a box approach [31]. Based on the first order approximation of Brus equation [32], the relationship between the particle radius (r) and band gap Eg in ZnS nanocrystal [33] is given by

$$ r({{\hbox{E}}_{\rm{g}}}) = \frac{{0.32 - 2.9\sqrt {{({{\text{E}}_{\rm{g}}} - 3.49)}} }}{{2(3.50 - {{\hbox{E}}_{\rm{g}}})}} $$
(1)
Fig. 1
figure 1

a TEM image of ZnS QDs with HRTEM image (inset) showing lattice fringes. b Particle size distribution, c UV-visible spectra, and d XRD pattern of the corresponding ZnS QDs

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Using Eq. (1), the diameter of the ZnS nanocrystal (considering Eg = 4.14 eV) was calculated to be 3.14 nm which is in good agreement with TEM results. The extinction coefficient of ZnS QDs at 315 nm was estimated to be 3.3 × 105 M−1 cm−1. The crystallinity of the ZnS QDs was confirmed by powder XRD analysis. The XRD pattern (Fig. 1d) consisted of characteristic peaks at 28.5°, 47.5°, and 56.5° corresponding to principal Bragg reflections in (111), (220), and (311) lattice planes, respectively, of bulk ZnS crystallizing in Wurtzite structure according to the JCPDS-ICDD database [34]. The quantum yield of ZnS QDs was calculated to be 9.5%.

Citrate-capped Ag NPs were prepared using a modified Turkevich process [29]. The TEM images revealed that the prepared Ag NPs are well-separated and mostly spheroid in shape. Selected area electron diffraction (SAED) pattern of the Ag NPs (inset of Fig. 2) indicated poly-crystalline nature of the NPs. The average particle size was determined to be 37 nm from the corresponding particle size distribution (inset Fig. 2). A strong surface plasmon resonance (SPR) band at 418 nm, characteristic of Ag NPs [35, 36], was observed in the corresponding UV-visible spectra. The extinction coefficient of citrate-capped Ag NPs at 418 nm was estimated to be 1 × 1010 M−1 cm−1. It may be mentioned here that no significant emission was observed for the prepared Ag NPs at the excitation wavelength of 315 nm.

Fig. 2
figure 2

TEM image of Ag NPs with corresponding particle size distribution and SAED pattern (inset)

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The effect of Ag NPs on the fluorescence properties of ZnS QDs was first investigated by steady state fluorescence spectroscopy. The fluorescence intensity of ZnS QDs in presence of different concentrations of Ag NPs was recorded and the results are shown in Fig. 3a. Figure 3a clearly shows that the fluorescence of ZnS QDs was efficiently quenched by Ag NPs in a concentration-dependent way. It may be mentioned here that no quenching of ZnS QDs fluorescence was observed in presence of citrate, indicating the Ag NPs to be solely responsible for quenching effect.

Fig. 3
figure 3

a Fluorescence of chitosan capped ZnS QDs (1.4 μM) in presence of (1) 0, (2) 11.5, (3) 2.30, (4) 3.45, (5) 4.60, (6) 6.90, (7) 8.05, (8) 10.35, (9) 12.65, (10) 13.8, (11) 16.1, and (12) 18.4 pM Ag NPs. b Stern–Volmer plot of the corresponding fluorescence quenching

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The fluorescence quenching was further analyzed using the well known Stern–Volmer equation [28]

$$ \frac{{{F_0}}}{F} = 1 + {K_{\rm{sv}}}[Q] $$
(2)

where, F 0 and F represents the integrated fluorescence intensity in absence and presence of quencher, respectively, K sv is the Stern–Volmer quenching constant and [Q] is the concentration of quencher. The corresponding Stern–Volmer plot (Fig. 3b) demonstrates the quenching in the present QD–NP pair is linear in the lower quencher concentration range (0–100 pM) with a K sv value of 9 × 109 M−1. The high K sv value indicates the efficient quenching in the present study [37]. Additionally, positive deviation (concave toward Y-axis) in the Stern–Volmer plot was observed at higher NP concentration. This superlinear behavior indicates the presence of mixed quenching [28] in the present system.

Due to strong surface complexation with citrate, surface of the Ag NPs prepared in the present study is negatively charged [36, 38]. Chitosan-stabilized ZnS QDs, on the other hand, are positively charged as the pKa value of the chitosan amino group is estimated to be at ~6.3 [39]. The quenching of ZnS QDs by Ag NPs may arise from the formation of static quenching complex via attractive electrostatic interaction. In order to investigate this electrostatic interaction, UV-visible spectra of the Ag NPs in presence and absence of ZnS QDs were examined (Fig. 4a). Figure 4a clearly shows the simultaneous damping and broadening of the SPR band of Ag NPs in presence of ZnS QDs. This indicates the aggregation of Ag NPs in presence of ZnS QDs. The electrostatic interaction between chitosan-stabilized ZnS QDs and Ag NPs may lead to the formation of a QD–NP ensemble where a single Ag NP can quench a large number of ZnS QDs. This effect in turn could result in the extraordinary quenching efficiency observed here. The formation of electrostatic complex during the quenching process was further supported by the observation that the increase in ionic strength significantly reduces the quenching efficiency [37, 40]. Figure 4b shows the regaining of fluorescence from Ag NP-quenched ZnS QDs upon addition of salt (NaCl). This observation was consistent with the previous reports of reduced quenching efficiency due to ionic screening [37].

Fig. 4
figure 4

a Absorption spectra of Ag NPs (11.5 pM) in the presence and absence of ZnS QDs (1.4 μM). b The fluorescence recovery of ZnS QDs on addition of varying concentration of NaCl. Io and I are the integrated fluorescence intensity of QDs in absence and presence of Ag NPs, respectively

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However, the positive deviation of the Stern–Volmer plot indicates that additional mechanisms, in conjunction with electrostatic complex formation, are involved [28, 37] in the quenching of ZnS QD fluorescence by Ag NPs. The involvement of energy transfer and electron transfer mechanism in the interacting QD–NP pair has already been reported. Electron transfer mechanism which is only predominant in smaller (<5 nm) metal nanoparticles [41] does not seem to play significant role as the Ag NPs in the present study are much larger (~37 nm). On the other hand, energy transfer may significantly influence the present quenching process due to the excellent overlap between ZnS QDs emission and the SPR band of Ag NPs (Fig. 5).

Fig. 5
figure 5

Spectral overlap between the emission of ZnS QDs and absorbance of Ag NPs

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FRET can be defined as a process in which the excited-energy from the initially excited “donor” molecule is transferred to an “acceptor” molecule. FRET occurs without the appearance of a photon and is a result of long-range dipole–dipole interactions between the “donor” and “acceptor” [28]. In the present study, ZnS QDs and Ag NPs act as “donor” and “acceptor”, respectively. The rate of energy transfer depends upon (1) the extent of spectral overlap between the donor emission and the acceptor absorption, (2) the relative orientation of the transition dipole of the donor and the acceptor, (3) the distance between the donor and the acceptor, and (4) the quantum yield of the donor [28]. The Förster distance R o, defined as the distance at which resonance energy transfer is 50% efficient, is given by:

$$ {R_o} = {[8.8 \times {10^{{ - 25}}}{\kappa^2}{n^{{ - 4}}}\varphi J]^{{ \frac{1}{6} }}}\;({\hbox{in}}\;{\hbox{centimeters}}) $$
(3)

where κ 2 is the orientation factor between the emission dipole of the donor and the absorption dipole of the acceptor, n is the refractive index of the medium, ϕ is the quantum yield of the donor in absence of acceptor, and J (λ) is the overlap integral of the fluorescence emission spectra of the donor and the absorption spectra of the acceptor in units of M−1 cm3. J (λ) is given by [28]

$$ J(\lambda ) = \int\limits_0^{ \propto } {{F_{\rm{D}}}(\lambda )} \,{\varepsilon_{\rm{A}}}(\lambda ) {\lambda^4}d\lambda $$
(4)

where, F D(λ) is the corrected fluorescence intensity of the donor with total intensity normalized to unity, ε A(λ) is the decadic extinction coefficient of the acceptor at λ, which is in unit of M−1 cm−1. In the present case, ϕ = 0.095, n = 1.33, κ 2 = 2/3. According to Eqs. (3) and (4), values of the above parameters were found to be \( J = 2.2 \times {10^{{ - {9}}}}{{\hbox{M}}^{{ - {1}}}}{\hbox{c}}{{\hbox{m}}^{{3}}} \) and R o = 18.4 nm. The value of R o is higher than the conventional Förster distance which could be due to large size and higher molar extinction coefficient of Ag NPs. This also indicates the involvement of NSET process [42].

Were the energy transfer between the excited ZnS QDs and Ag NPs responsible for emission quenching, characteristic signature can be found in the emission decay profile of the ZnS QDs in time-resolved fluorescence measurements [43, 44]. Time-correlated single-photon counting provides an accurate measurement of energy transfer, excluding both ground-state complex formation and inner filter effect. Figure 6 shows the emission decay of ZnS QDs at 427 nm both in presence and absence of Ag NPs. The resulting fluorescence can be fitted to exponential curve in order to derive the decay time constants, which can be used to calculate the lifetime of the fluorophore. It is evident from Fig. 6 that ZnS QDs follows a multi-exponential decay, characteristic of semiconductor QDs [45]. The decay was analyzed using biexponential kinetics

$$ I(t) = \sum\limits_i {{\alpha_i}\exp ({{{ - t}} \left/ {{{\tau_i}}} \right.})} $$
(5)

where, α i and τ i are the pre-exponential factors and excited-state fluorescence lifetimes associated with the ith component, respectively. The lifetimes (τ 1 and τ 2) and pre-exponential factors (α 1 and α 2) of ZnS QDs emission at different Ag NPs concentration, is summarized in Table 1. For the biexponential decay the average lifetime < τ > was determined from the equation [28]

$$<\tau>=\sum\limits_i{\frac{{{\alpha_i}\tau_{{_i}}^2}}{{{\alpha_i}{\tau_i}}}}$$
(6)
Fig. 6
figure 6

Fluorescence decay profile of ZnS QDs in absence and presence of Ag NPs

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Table 1 Life-time data obtained using the biexponential model for the ZnS QDs in absence and presence of Ag NPs
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ZnS nanocrystal show an average lifetime of 47.05 ns in the absence of Ag NPs, which decreases with increase in Ag NPs concentration due to the availability of more number of acceptors. The decrease in average lifetime of the ZnS QDs confirms the involvement of an additional mode of quenching, other than Coulombic interaction, possibly through energy-transfer process between the ZnS QD—Ag NP pair. Moreover, this explains the positive deviation observed in the Stern–Volmer plot.

Conclusion

In summary, water dispersible chitosan-stabilized ZnS QDs of ca. 3 nm were prepared in an environment friendly method. The interaction of ZnS QDs with citrate-capped Ag NPs resulted in the fluorescence quenching of the QDs in concentration-dependent manner. The corresponding Stern–Volmer plot for the present fluorophore–quencher pair indicated mixed nature of quenching with a high K sv value (9 × 109 M−1) which demonstrates the efficient quenching of ZnS QDs by Ag NPs. The Coulombic interaction between the positively charged ZnS QDs and negatively charged Ag NPs led to the formation of electrostatic complex that contribute to the static mechanism of the quenching process. Furthermore, the involvement of energy transfer process due to excellent spectral overlap between ZnS QDs emission and SPR band of Ag NPs was confirmed by the time-resolved fluorescence measurements. The R o value calculated for the present donor-acceptor pair was found to be 18.4 nm which was greater than the generally observed Förster distance. The implication of the present findings may lead to the development of long-range FRET- or NSET-based methods for studying biological multi-component system.