Size-dependent CdSe quantum dot–lysozyme interaction and effect on enzymatic activity

Abstract

Herein, protocols for modifying hydrophobic CdSe quantum dots with 3-mercaptopropionic acid (MPA) to generate hydrophilic moieties and their size-dependent (2.5 and 6.3 nm) interaction with lysozyme are reported comprehensively. The interaction of MPA-capped water-soluble quantum dots with lysozyme (Ly) was investigated, and a range of techniques such as static fluorescence spectroscopy and synchronous fluorescence spectroscopy were used to quantify QD–lysozyme binding isotherms, exchange rates, critical flocculation concentrations, and the compositions of mixed QD–lysozyme complexes. The results demonstrated that the binding of QDs with lysozyme induced conformational changes in lysozyme. QDs were able to enhance the enzymatic activity of lysozyme in a highly efficient dose-dependent manner. It was concluded that smaller-sized QDs were found to bind poorly to lysozyme, but produced a greater enhancement in enzymatic activity compared with larger QDs. In summary, a comprehensive characterization of the stability of lysozyme-bound QDs is a necessary step for their potential use as intracellular delivery vectors and imaging agents.

---

1. Introduction

Protein–QD interactions have become a topic of considerable interest owing to their similarity to protein–ligand (antibody opsonization) interactions in the living body.^1,2 It is crucial to understand how proteins (bioreceptors) interact with these inorganic nanomaterials, which are routinely used for applications such as drug delivery, targeting and bioimaging, etc. These interactions depend on many factors such as protein conformation and orientation. Researchers have investigated applications for nanocrystalline QDs as agents for cell imaging, and as potential qubits in quantum computing. Recently, Medintz et al. provided a generalized strategy for determining the orientation of a protein on a QD or other spherical nanoparticle.³ The binding of QDs to proteins and amino acids alters their secondary structure, which affects their functional properties.^4,5 The unusual optical properties of QDs make them ideal for in vivo and in vitro applications as fluorophores in a range of biological investigations, in which the use of traditional organic molecules as fluorescent labels provides inadequate long-term stability, with the simultaneous detection of multiple signals. The availability of water-dispersible QDs has enabled important applications in cell and deep-tissue imaging. Furthermore, these are efficient fluorescence resonance energy transfer (FRET) donors.⁶ Needless to say, photoluminescent low-dimensional semiconductor nanocrystals hold considerable promise for in vivo imaging.⁷

Wu et al. have reported a class of designer nanogels prepared by the in situ immobilization of CdSe quantum dots inside the core of temperature- and pH-responsive interpenetrating network matrices of hydroxypropyl cellulose (HPC)–poly(acrylic acid). The hydroxyl groups of the HPC chains were designed to immobilize the precursor Cd²⁺ ions in the nanogel networks. Furthermore, these gels stabilized the CdSe QDs that were formed in situ. These smart nanogels generated a strong emission at 741 nm for sensing pH-dependent environments and gave rise to a visible exciton emission at 592 nm for imaging mouse melanoma (B16F10) cells. Also, these hybrid nanogels could provide excellent stability as drug carriers with high drug-loading capacity for the model anticancer drug temozolomide.⁸ Savla et al. have reported the targeted and controlled delivery of a pH-responsive QD–mucin 1 aptamer–doxorubicin conjugate for the chemotherapy of ovarian tumors. DOX was conjugated to QDs through a pH-sensitive hydrazone bond to provide stability of the complex and drug release in the acidic environment prevailing inside cancer cells. The results showed that this bond was stable both at neutral and slightly basic pH. Furthermore, rapid hydrolysis in a mildly acidic pH environment was noted. The results demonstrated the high potential of the QD conjugate in the treatment of multi-drug-resistant ovarian cancer.⁹ Functionalization of the surface of a quantum dot (QD) with aptamers can recognize cocaine, which was studied in depth by Zhang and Johnson. The single-molecule detection and FRET between QD, Cy5 and Iowa Black RQ were exploited to develop a QD-based aptameric sensor. This sensor was capable of sensing the presence of cocaine in both signal-off and signal-on modes. In comparison with other aptameric sensors, this single-QD-based aptameric sensor had many advantages such as easy sample preparation, high sensitivity, and extremely low requirements for the sample.¹⁰

Jaiswal and Simon have reviewed the potential of QDs in biological applications. Improved synthesis protocols of water-stable QDs, the development of approaches for labeling cells efficiently with QDs, and concurrent improvements in the possibility of conjugation of QDs to selected biomolecules have generated considerable interest in their use in biology. There have been many successful applications of QDs in biology. Notwithstanding, several limitations remain that need to be overcome before these can be used routinely.¹¹ Nanoparticle-based imaging and targeting methods using nano core–shell structures and quantum dots have been studied in depth by Alivisatos et al. Emerging nanoelectronics-based sensing and controlled synthesis protocols for the applications of every nanoparticle system address their advantages and shortcomings in medical research.¹² Many investigations have illustrated the hidden potential of using quantum dots as new probes in vitro and in vivo. In a review, Alivisatos et al. have summarized the recent advances in the use of quantum dots at the cellular level, in immune labeling, cell tracking, in situ hybridization, FRET, in vivo imaging, etc.¹³

Therefore, it is important to understand protein–QD interactions at the molecular level. In particular, the enzymatic activity of proteins is of concern. In the present study, steady-state and synchronous fluorescence, UV-visible spectroscopy and CD spectroscopy were used to investigate the binding constant, binding sites, free energy of binding (entropy and enthalpy) and conformational changes induced by QDs in lysozyme. Thus, a complete profile of the enzymatic activity of lysozyme in its bound state with CdSe quantum dots was obtained, much of which yielded new information.

2. Materials and methods

Lysozyme (90%) was purchased from Sigma-Aldrich (batch no. L6876) and QDs were synthesized in our laboratory following the protocol described in ref. 4 and 5. A stock solution of lysozyme was prepared in phosphate buffer of a pH of 6.4 and a concentration of 10 mM. The protein concentration was calculated by dividing the absorbance at 280 nm by the molar extinction coefficient of lysozyme ε₂₈₀ = 2.64 mL mg⁻¹ cm⁻¹.¹⁴ All other reagents used were of analytical reagent grade and doubly distilled deionized water was used throughout the experiments.

2.1. Synthesis of QDs

The chemicals needed for synthesis, namely, cadmium oxide (CdO), oleic acid, octadecene, mercaptopropionic acid (MPA), selenium powder, and trioctylphosphine (TOP), were purchased from Sigma-Aldrich (USA) and used as received. Deionized water bought from Organo Biotech Laboratories (India) was used as a solvent. CdSe quantum dots (QDs) were prepared from the CdO precursor and elemental Se using a kinetic growth protocol pioneered by Peng et al.^15–24 This particular method has several advantages compared with conventional synthesis protocols. The exact protocol used for the preparation of QDs of different sizes and their physical characterization is available in ref. 4 and 5. We successfully prepared QDs with different sizes, which was confirmed by UV-vis, dynamic light scattering and TEM data (see ref. 4 and 5 for more details).

An aqueous solution of lysozyme was prepared with a concentration of 0.01% (w/v) by dissolving a known amount of the protein powder in deionized water at 25 °C using a magnetic stirrer for almost 1 hour, and the pH of the protein solution was 6.0 ± 0.5. This produced optically clear and transparent solutions. These were stored in sterilized airtight glass bottles for future use. The pH of a dispersion of CdSe quantum dots was 9.0 ± 0.5. Keeping the protein concentration fixed, different protein–QDs complexes were obtained by varying the QDs concentration from 2.6 to 52.5 nM. The pH of these solutions changed to 8.0 ± 0.5 with the gradual addition of QDs to the stock solution of protein.

UV-vis absorption spectra were obtained using a spectrophotometer (Model CE-7300, Cecil Instruments, UK) operating in the wavelength range from 190 to 900 nm. FTIR spectra for all samples were recorded on an FT-IR/Raman spectrometer (1064 nm) attached to a microscope (Varian 7000 FT-Raman and Varian 600 UMA). Average particle sizing was performed by using a JEOL 2100F TEM (a digital TEM with an image analysis system at a maximum magnification of 150 000× operating at a voltage of 200 kV). This was also carried out by dynamic light scattering (DLS) techniques. Further details of the DLS setup that was used can be obtained from ref. 25. Zeta potential measurements were performed on an electrophoresis instrument (model ZC-2000, Microtec, Japan). The average values reported were based on triplicate measurements.

Steady-state fluorescence measurements were performed using a Varian Cary Eclipse fluorescence spectrophotometer with a spectral range of 190 to 1000 nm.

Circular dichroism (CD) experiments were carried out with an Applied Photophysics Chirascan instrument (USA) to estimate the secondary structure of proteins using the standard operating procedure. Each spectrum was the average of three successive scans. Appropriate baseline corrections were made to the CD spectra. The path length of the cuvette used in the CD experiments was 0.1 cm and the wavelength range used was from 200 to 280 nm. It has been reported that below 200 nm CD data are not very accurate for the analysis of the secondary structures of proteins.^26,27

Synchronous fluorescence spectra were recorded using the same spectrofluorometer at two different wavelength intervals (Δλ) of 15 and 60 nm, which give characteristic information for tyrosine (Tyr) and tryptophan (Trp) residues, respectively. The temperature was controlled during experiments using a constant-temperature cell holder connected to a constant-temperature water circulator (Varian, USA).

2.2. Lytic activity of lysozyme

The rate of lysis of Micrococcus lysodeikticus by lysozyme was measured as reported.^14,26,27 The activity of lysozyme was measured using a spectrophotometric turbidity assay. Lysozyme was dissolved in 66 mM potassium phosphate buffer (pH 6.23 or pH 5.0) in vials. The concentration of lysozyme was 50 mg mL⁻¹. A stock substrate solution of Micrococcus lysodeikticus (Sigma) was prepared in 66 mM potassium phosphate buffer with a concentration of 0.3 mg mL⁻¹. A portion (0.2 mL) of the lysozyme solution was mixed with 4.0 mL of the Micrococcus lysodeikticus solution in a cuvette. The change in absorbance at 450 nm, which corresponds to the hydrolysis of the cell wall substrate, was measured on a Shimadzu UV-vis 2500 spectrophotometer at 30 °C.

3. Results and discussion

3.1. Characterization of quantum dots

Fig. 1(a) illustrates the UV-vis absorption spectra of the CdSe QDs. The absorption edges clearly exhibit a blue shift with respect to the bulk band gap, which implies the possibility of quantum confinement.^28–30 For all the samples, a well-defined absorption (peak) maximum for the first electronic transition was recorded, which suggests that these nanocrystals had a narrow size distribution. The apparent particle diameters D of these nanoparticles were estimated from the first absorption maximum of the absorption spectrum using the following formula:³⁰

D = 59.60816 − 0.54736λ + 1.8873 × 10⁻³λ² − 2.85743 × 10⁻⁶λ³ + 1.62974 × 10⁻⁹λ⁴ (1)

where λ is the wavelength of the first excitonic absorption peak of the sample.

Fig. 1 (a) UV-vis and fluorescence spectra and (b and c) TEM images of CdSe QDs with sizes of 2.5 and 6.3 nm. The insets show the corresponding particle size histograms.

The results showed that the particle diameters of the as-prepared CdSe QDs were around 3.43 ± 0.08 and 5.09 ± 0.07 nm, which correspond to the first absorption maxima at 495 and 570 nm. The concentrations of the QD dispersions were calculated by the Beer–Lambert law. The zeta potentials of these QDs were measured using an electrophoresis instrument (Zeecom-2000, Microtek, Japan), which assigned values of ζ = −56 and −62 mV to these particles. The profiles are shown in Fig. S1(a) and (c) (ESI†). The particle size distribution and morphology of the QDs were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. Fig. 1(b) and (c) show TEM micrographs and particle size histograms of the two samples. Histograms of the DLS of the particles are shown in Fig. S1(b) and (d) (ESI†).

Analysis of the TEM data revealed the presence of nearly monodisperse QDs with average diameters of 2.5 ± 0.2 and 6.3 ± 0.4 nm, and the corresponding DLS study indicated that the hydrodynamic diameters were about 3.8 ± 0.2 and 6.8 ± 0.4 nm, respectively (Table S1†). The difference between the two sizes was marginal and may be due to the fact that the TEM data corresponded to a dehydrated sample and the loss of solvent affected the character of the dispersion. We can say that the average size observed by TEM was more likely to represent the actual particle size of QDs, whereas the DLS result referred to the size of hydrated QDs. The corresponding sizes determined from eqn (1) were 2.43 ± 0.08 and 5.09 ± 0.07 nm. We shall henceforth use the TEM data to specify the sizes of small and large QDs.

3.2. UV-visible absorbance

UV-visible absorption spectroscopy has been extensively used in the past to study changes in physical structure, and to identify complexation between ligands and protein molecules.³¹ The absorption spectra of lysozyme (5 μM) with increasing concentrations of QDs (from 2.6 to 52.5 nM) in phosphate buffer at a pH of 7.0 at five different temperatures are shown in Fig. S1 and S2 (ESI†). From these data, a double reciprocal plot for the interaction of lysozyme with QDs of different sizes at different temperatures was obtained, as shown in Fig. 2. The results clearly show that the absorbance decreased with the addition of quantum dots. Normally, the spectral range between 260 and 300 nm indicates a change in the microenvironment of the chromophore.³² Thus, we conclude that the lysozyme–QD interaction leads to the formation of a ground-state complex. The second inference indicated that the quenching mechanism was of a static nature.³³

Fig. 2 Double reciprocal plot for the interaction of lysozyme (5 μM) with QDs of sizes of (a) 2.5 nm and (b) 6.3 nm at different temperatures (298, 303, 308, 313 and 318 K).

3.3. Thermodynamic parameters and nature of the binding forces

Secondary force interactions such as hydrophobic forces, hydrogen bonding, van der Waals forces and Coulombic interactions govern binding between ligands and biomolecules.³⁴ Thermodynamic parameters such as enthalpy and entropy are indicators of the existence of non-covalent forces. For instance, changes in enthalpy (ΔH) and entropy (ΔS) may provide information about the nature of the binding forces. Ojha and Das³⁵ have categorized interactions based on these parameters. According to their proposition, when ΔH < 0 and ΔS < 0 the forces are van der Waals and hydrogen bonding interactions. Hydrophobic interactions are of significance in binding if ΔH > 0 and ΔS > 0. The values of these parameters were determined from the slope and intercept of the linear van't Hoff plot (ln K versus 1/T) given by eqn (2):³⁶where K is the binding constant, which is analogous to the Stern–Volmer quenching constant K_SV at the corresponding temperature, R is the universal gas constant and T is the experimental temperature on the absolute scale. The changes in free energy (ΔG) were estimated from the following relationship and are plotted in Fig. 3:

ΔG = ΔH − TΔS (3)

and

Fig. 3 Plot of Gibbs free energy for the interaction of lysozyme (5 μM) with QDs of sizes of 2.5 and 6.3 nm at different temperatures (298, 303, 308, 313 and 318 K). The change in enthalpy ΔH and change in entropy ΔS were determined from the intercept and slope of a straight line fitted by least squares to the data points, as described by eqn (3).

The values of ΔG (ΔH − TΔS) for the interaction between QDs and lysozyme are summarized in Table 1. Normally, the presence of hydrogen bonding in the binding mechanism results in a negative value of ΔH.³⁷ The data shown in Table 1 imply that both hydrophobic forces and hydrogen bonding interactions were important in the binding between QDs and lysozyme, a result which is supported by a molecular docking study.³⁸ The observed negative value of the free energy of binding implied a spontaneous interaction.

Table 1 Binding constants (K_b) and thermodynamic parameters for the lysozyme–QDs system at five temperatures

S. No.	Temperature (K)	Kb2.5 nm (×10^5 L mol^−1)	ΔG2.5 nm (kJ mol^−1)	Kb6.3 nm (×10^5 L mol^−1)	ΔG6.3 nm (kJ mol^−1)
1	298	8.27	−33.74	8.39	−33.77
2	303	12.3	−35.56	22.3	−36.81
3	308	15.3	−36.44	65.4	−40.17
4	313	22.8	−38.08	214	−43.88
5	318	39.9	−40.17	340	−45.82

---

3.4. Steady-state fluorescence spectroscopy

Fluorescence spectroscopy measurements yield considerable information about the binding mechanism in general. Parameters such as the binding constant, number of binding sites, and intermolecular distances are easily obtainable from these spectroscopy data.³⁹ The three distinct intrinsic fluorophore residues, namely, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), when present in proteins may provide information about the conformation, dynamics and intermolecular interactions of the proteins concerned. Of these residues, Trp and Tyr are mostly used as intrinsic fluorophores. The fluorescence signal arising from Phe is ignored owing to its low molar extinction coefficient and poor quantum yield.^40,41 Fluorescence quenching spectra recorded for lysozyme (5 μM) solutions in the presence of QDs of different sizes (concentrations from 2.6 to 52.5 nM) at 298 K are shown in Fig. S3 (ESI†).

The fluorescence spectral profile revealed that the fluorescence of lysozyme was gradually quenched, accompanied by marginal blue shifts from 343 to 340 nm, with increases in the QD concentration for a given lysozyme content. This could simply be due to errors of the fluorometer. This clearly indicated the complexation of lysozyme with QDs, and a concomitant shift in the fluorophore towards a more hydrophobic environment.⁴² Experiments have revealed two distinct types of quenching mechanism, static and dynamic, which are identifiable from their temperature-dependent binding constant data. For instance, in static quenching an increase in temperature decreases the binding constant because of the reduced stability of the ground-state complex. In the dynamic quenching process, an increase in the binding constant with temperature leads to an increase in the rate of collisions.^42–44 Dynamic quenching mostly results from collisions between the fluorophore and the quencher, whereas static quenching arises from the generation of a ground-state complex between the fluorophore and the quencher.⁴² The fluorescence intensities that were determined from the lysozyme samples were adequately corrected by determining the absorbance of the QD at the emission and excitation wavelengths. This was carried out using the following equation:^45,46

F_cor = F_obsd10(A₁ + A₂)/2 (5)

where F_cor and F_obsd are the corrected and observed fluorescence intensities, respectively, and A₁ and A₂ are the values of absorbance at the excitation and emission wavelengths, respectively.

The quenching mechanism was analyzed using the following Stern–Volmer equation:⁴⁷

where F₀ and F are the fluorescence intensities in the absence and presence of QD, K_SV is the Stern–Volmer quenching constant, [QD] is the concentration of QD, K_q is the quenching rate constant of the bimolecular reaction and τ₀ is the average lifetime of molecules in the absence of QD, of which the value is about 10⁻⁸ s.⁴⁸ The fluorescence quenching spectra are depicted in Fig. S4 (ESI†). Fig. 4 shows Stern–Volmer plots of the QD–lysozyme system at different temperatures. The values of K_q obtained from the Stern–Volmer plot (Table 2) decreased with an increase in temperature, which confirms the involvement of static quenching rather than dynamic quenching in the QD–lysozyme system.

Fig. 4 Logarithmic plots (Stern–Volmer plots) derived from fluorescence data for lysozyme (5 μM) as a function of the concentration of QDs of sizes of 2.5 and 6.3 nm (∼0 to 60 nM) at 298 K. The binding constant K and number of binding sites n were determined from the intercept and slope of a straight line fitted by least squares to the data points, as described by eqn (7).

Table 2 Stern–Volmer quenching constants (K_SV) and quenching rate constants (K_q) of the lysozyme–QD system

S. No.	Size of QDs (nm)	Number of binding sites	KSV (×10^10 L mol^−1)	Kq (×10^18 L mol^−1 s^−1)	R^2
1	2.5	1.34	1.74	1.74	0.99
2	6.3	1.49	40.74	40.74	0.98

---

In addition, the maximum collision rate constant for various quenchers with the biopolymer is higher than 2 × 10¹⁰ L mol⁻¹ s⁻¹.⁴⁴ The values of K_q indicate a static quenching mechanism. As listed in Table 2, the values of K_q (≈K_SV/τ₀) were found to be higher than 2 × 10¹⁰ L mol⁻¹ s⁻¹, which indicates that the fluorescence quenching of lysozyme by QD was initiated via a static mechanism.

3.5. Binding constant and number of binding sites

For fluorescence quenching, when QDs bind independently to a set of equivalent sites on lysozyme, the binding parameters can be obtained from the formula:^49,50where K_b and n are the binding constant and number of binding sites, respectively. A plot of versus log[QD] at five different temperatures gives a straight line (Fig. 2). The intercept of the curve is represented by K_b, whereas the slope of the curve is equal to n. The values of the quenching rate constant k_q and the numbers of binding sites n are listed in Table 2. The number of binding sites n approximately equals 1, which indicates that there is on average one binding site on lysozyme for QD. From Table 1, it was also found that the value of K_b increased with an increase in temperature, which is in accordance with the static quenching mechanism.⁵¹

All the binding experiments were performed in a buffer solution at a pH of 6.4, in which lysozyme possessed a zeta potential of approximately +7.5 mV. Also, the zeta potentials of the small and large QDs were strongly negative at ζ = −56 and −62 mV. Hence, a priori, it is difficult to disprove the presence of electrostatic interactions. To confirm this, we performed the binding experiments at different ionic strengths (I = 0, 0.001, 0.01, and 0.1 N NaCl). The binding constant was found to be marginally reduced owing to the presence of salt (See Fig. S5–S7, ESI†).

There was universal quenching of fluorescence, which was caused by QD–protein binding. From Fig. S5–S7 (ESI†), the percentage decrease in the values of the binding constant was much less than 1% for both QDs, which implies that there was negligible screening of the interaction due to mobile ions. Therefore, the role of electrostatic interactions was very marginal.

We need to differentiate between hydrophobic and electrostatic binding. Based upon energetic considerations, Ross et al. (1981) reviewed the thermodynamics of protein association processes for the examples that were best characterized in terms of their chemistry and structure. They accounted for the signs and magnitudes of the thermodynamic parameters (such as ΔH, ΔS and ΔG) for protein association (protein–protein or protein–ligand) reactions in terms of known molecular forces and the thermochemistry of small-molecule interactions.⁵² In an environment with a low dielectric constant, ΔH₀ should be substantially negative and ΔS₀ should be substantially negative. However, in the case of ionic bonding ΔH₀ should be significantly positive and ΔS₀ should also be positive. Therefore, electrostatic binding is primarily determined by a positive change in entropy accompanied by a negative change in enthalpy (predominantly of negative sign).⁵²

Our thermodynamics calculations showed the dominance of hydrophobic interactions in the binding of QD to lysozyme [see Table IV in Ross et al. (1981) and also Kumari et al. (2014), Mir et al. (2014) and Patel et al. (2014)].^52–55 Furthermore, the zeta potential of the complex was recorded as a function of the QD concentration, which again implied the absence of electrostatic interactions (no inversion of charge, data not shown).

At this stage, it is imperative to compare our results with those of Li et al. (2014), who have studied the strong electrostatic interaction between graphene oxide (negatively charged) and lysozyme (from a mixture of binary and ternary proteins) using a range of techniques, namely, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV-vis absorption and fluorescence spectroscopy, etc.⁵⁶ They have demonstrated how strongly graphene oxide (GO) interacts with lysozyme. This strong electrostatic interaction also enables the selective adsorption of lysozyme on GO from a mixture of binary and ternary proteins. This selectivity was confirmed by SDS-PAGE, UV-vis absorption spectroscopy and fluorescence spectroscopy. The adsorbed lysozyme could be released from the surface of GO by adding NaOH solution and then precipitating GO with CaCl₂. The rapid reduction in fluorescence intensity reveals the existence of a strong electrostatic interaction between GO and lysozyme, in which the pH plays a central role in the quenching effect. According to Wetter et al. (1951), the pH must be below the isoelectric point (≈10.5), to generate more net positive charge on the protein concerned.⁵⁷

The ionic strength and pH value are extremely important for determining the charge of lysozyme. At a pH of ≈6.5, lysozyme possesses a weakly positive charge, which reduces the contribution of electrostatic interactions in spite of the high charge density on both QDs (ζ = −56 mV (large) and −62 mV (small)). The marginal reduction in quenching at a higher ionic strength indicates the weakly electrostatic nature of the QD–lysozyme interaction. Eliminating the possibility of π–π stacking interactions, it should be realized that some weak interactions may also exist such as van der Waals and hydrogen bonding. Hydrophobic interactions play the major role (from calculations of the thermodynamic parameters); on investigating further, the ionic strength of NaCl further neutralized the surface charge. The strong quenching of QD on lysozyme is predominantly due to the hydrophobic interaction (not due to electrostatic attraction) between them. This elucidates the difference between our results and the conclusions of Li et al. (2014).

3.6. Synchronous fluorescence spectra

Synchronous fluorescence is a standard technique for the simultaneous characterization of multi-component samples without any pretreatment.^58–61 One of the major advantages of this technique is that the analysis can be carried out directly under ambient conditions.

The selection of the wavelength interval is a major experimental factor when the synchronous fluorescence technique is used. This selection was made empirically by taking into account the excitation and emission maxima for the three chosen analytes (phenol, resorcinol and hydroquinone), and scans were recorded from Δλ = 5 to 40 nm.⁵¹ For Δλ ≤ 5 nm, the peaks could not be reliably separated, whereas for Δλ ≥ 40 nm the fluorescence intensity decreased sharply. For the scan at Δλ = 15 nm, the peaks displayed a good peak shape with synchronous scanning between λ_exc = 230–360 nm and λ_em = 245–375 nm. This selected spectral region had 130 distinct values of wavelength for each sample.

Synchronous fluorescence spectroscopy happens to be a sensitive technique for studying alterations in the molecular environment of fluorophore residues.⁶² Reductions in spectral bandwidth combined with spectral simplification and perturbation effects observed in synchronous spectra provide characteristic information on Tyr and Trp residues when Δλ is fixed at 15 and 60 nm, respectively.⁶³ Positional synchronous maxima for these residues are usually observed on binding, which yields information on changes in polarity around these fluorophores and thus on their proximity to the ligand concerned.⁶⁴ The synchronous fluorescence spectra of lysozyme (as a function of concentration) in the presence of QDs are illustrated in Fig. S8 and S9 (ESI†). The results indicate that on an increase in the concentration of QDs, the fluorescence intensity of Trp (Fig. S9, ESI†) decreased more significantly than that of Tyr (Fig. S8, ESI†) which implies that QDs quenched the fluorescence emission of lysozyme mostly by quenching the Trp residue. This is clearly indicated by Fig. 5. In addition, a minor red shift in the wavelength of the maximum emission was noticed, which revealed that quantum dots changed the hydrophobicity of the microenvironment around the Trp residue, which also changed the physical conformation of lysozyme.⁶⁵

Fig. 5 Dependence on size of the quenching of the intensity of synchronous fluorescence at Δλ = 15 nm (tyrosine) and Δλ = 60 nm (tryptophan) of lysozyme (5 μM) in the absence and presence of QDs (2.6 to 52.5 nM). Note the remarkable quenching due to the complexation of the protein with QD particles of sizes of 6.3 nm and also the invariance in quenching between Δλ = 15 nm (Tyr) and Δλ = 60 nm (Trp).

3.7. Circular dichroism

Circular dichroism measurements performed in the far UV region were used to study changes in the secondary structure of proteins^66,67 and changes in the ellipticity measured at 222 nm can be used to quantify the α-helix content.⁶⁸ The CD spectra recorded for lysozyme samples in the absence and presence of QD are shown in Fig. S10 (ESI†) and exhibit two negative bands at 208 and 218 nm, which are characteristic of the alpha-helix structure of proteins.⁶⁹ The helix content was observed to decrease from 36.43% (when untreated) to 32.89%, 28.53% and 24.09% when the QD contents were 20, 33 and 50 mM, respectively. At higher QD concentrations, the α-helix content was less (Fig. 6), which implied changes in the secondary structure of the protein during its interaction with QD. This clearly implies binding of QD to the protein surface, which caused the protein to reveal its hydrophobic residues. At higher QD concentrations, disruption of the α-helix structure of the protein was noticed. This resulted in the appearance of more solvent-compatible structures such as β-sheets and random coils.

Fig. 6 Dependence of secondary structure (helicity) of lysozyme (5 μM) on the concentration of QDs of sizes of 2.5 and 6.3 nm (2.6 nM to 52.5 nM) at 298 K. Note the remarkable alterations in the secondary structure due to complexation of the protein with QD particles of sizes of 2.5 and 6.3 nm, respectively.

3.8. Effect of QD on the enzymatic activity of lysozyme

Fig. S11 (ESI†) shows the effect of the concentration of QD on the enzymatic activity of lysozyme. A normalized reading of the optical density (OD) of Micrococcus lysodeikticus was used to indicate the activity of lysozyme. As can be seen, the higher the concentration, the higher was the activity of lysozyme. The enzyme became highly activated (an almost two fold increase) when the concentration of QD increased from 2.6 to 52.5 nM. This is summarized in Fig. 7.

Fig. 7 Effect of concentration of QDs of sizes of 2.5 and 6.3 nm (2.6 to 52.5 nM) on the enzymatic activity of lysozyme (5 μM).

3.9. Phenomenology of differential binding

It was observed that smaller QDs were found to bind poorly to lysozyme, but produced greatly enhanced enzymatic activity compared with larger QDs, which in turn exhibited stronger binding. The thermodynamics of binding of the smaller QDs to lysozyme revealed values of ΔH = 58 kJ mol⁻¹ and ΔS = 307 J K⁻¹ mol⁻¹, whereas for the larger QDs these values were 152 kJ mol⁻¹ and 623 J K⁻¹ mol⁻¹. The corresponding free energy of binding was ΔG ≈ 34 kJ mol⁻¹ for small as well as large QDs at 25 °C. A pertinent question is: did coulombic interactions play any role in QD–lysozyme binding? The zeta potentials of QDs of sizes of 2.5 and 6.3 nm were −56 and −62 mV, respectively. This implies that the smaller QDs had a typical surface charge density that was ∼6 times higher than that of the larger QDs. On the other hand, lysozyme has a hydrodynamic radius of 20.5 nm (ref. 70) and a pI = 11.35. The binding studies were carried out at pH = 8, where lysozyme carries a net positive charge.⁷¹ Regardless of the magnitude of this charge, an electrostatic interaction between negatively charged QDs and positively charged lysozyme becomes a finite possibility. Because the surface charge density of smaller QDs is about 6 times higher than that of their larger counterparts, the smaller QDs will preferentially bind to the surface of lysozyme. This was observed in our experiments. The interaction between CdSe quantum dots and lysozyme is shown schematically in Fig. 8. Electrostatic interactions between QD and lysozyme cause a local neutralization of charge on the surface of lysozyme, and this will release counterions into the bulk solvent, thereby increasing its entropy. We observed this effect. Therefore, the possibility of electrostatic interactions between QDs and lysozyme cannot be ruled out completely. The smaller QDs bind more efficiently to lysozyme, but exhibit enhanced enzymatic activity when compared with larger QDs. We appreciate the observation made by the referee. This is due to the differential enzymatic behavior of the lysozyme–QD complex formed in this way. In an earlier work, we (Tyagi et al. (2015)) have discussed the size-dependent antimicrobial activity of these QDs. The results showed the individual contribution of QDs towards the enhanced enzymatic activity.⁷²

Fig. 8 Schematic of adsorption of lysozyme on CdSe QD.

4. Conclusions

The interactions of MPA-coated CdSe quantum dots (QDs) with lysozyme were studied by fluorescence, UV-visible, and circular dichroism spectroscopy. The results showed that the QDs quenched the fluorescence of lysozyme. The interaction process (binding) was mostly dominated by hydrophobic forces, which induced conformational changes in the protein involved.

The present study provides important quantitative data on the binding affinity of lysozyme with QDs via UV-visible spectroscopy, circular dichroism, and steady-state and synchronous fluorescence (quenching) measurements. The calculated thermodynamic parameters reveal that apart from electrostatic forces, hydrophobic interactions play a major role in stabilizing the lysozyme–QD complex. As a result, QD is able to enhance the enzymatic activity of lysozyme and activate the enzyme. Both synchronous fluorescence and circular dichroism spectra confirmed that the formation of a complex between the QDs and lysozyme caused conformational changes in the structure of the protein. It was concluded that smaller QDs were found to bind poorly to lysozyme, but produced greatly enhanced enzymatic activity compared with larger QDs. The comprehensive physical characterization and stability of lysozyme–QD complexes provide an important step in their potential use as imaging agents and intracellular delivery vectors.

Acknowledgements

KD acknowledges receipt of a junior research fellowship from the Council of Scientific and Industrial Research, Government of India. KR is thankful for the Department of Science and Technology, Government of India Inspire Faculty Award. We are thankful to the Advanced Research Instrumentation Facility of the University for allowing us access to the TRFS and TEM facility. This work was supported by a research grant received from the Department of Science and Technology, Government of India.

References

1. M. Wellington, J. M. Bliss and C. G. Haidaris, Enhanced phagocytosis of Candida species mediated by opsonization with a recombinant human antibody single-chain variable fragment, Infect. Immun., 2003, 71, 7228–7231.
2. J. Hostetter, R. Kagan and E. Steadham, Opsonization effects on Mycobacterium avium subsp. paratuberculosis–macrophage interactions, Clin. Diagn. Lab. Immunol., 2005, 12, 793–796.
3. I. L. Medintz, J. H. Konnert, A. R. Clapp, I. Stanish, M. E. Twigg, H. Mattoussi, J. M. Mauro and J. R. Deschamps, A fluorescence resonance energy transfer-derived structure of a quantum dot–protein bioconjugate nanoassembly, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 9612–9617.
4. A. Tyagi, K. Rawat, A. K. Verma and H. B. Bohidar, Mechanistic evaluation of the size dependent antimicrobial activity of water soluble QDs, Anal. Methods, 2016, 8, 1060–1068.
5. I. A. Mir, K. Das, K. Rawat and H. B. Bohidar, Hot Injection versus Room Temperature Synthesis of CdSe Quantum Dots: A Differential Spectroscopic and Bioanalyte Sensing Efficacy Evaluation, Colloids Surf., A, 2016, 494, 162–169.
6. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Quantum dot bioconjugates for imaging, labelling and sensing, Nat. Mater., 2005, 4, 435–446.
7. M.-K. So, C. Xu, A. M. Loening, S. S. Gambhir and J. Rao, Self-illuminating quantum dot conjugates for in vivo imaging, Nat. Biotechnol., 2006, 24, 339–343.
8. W. Wu, M. Aiello, T. Zhou, A. Berliner, P. Banerjee and S. Zhou, In situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery, Biomaterials, 2010, 31, 3023–3031.
9. R. Savla, O. Taratula, O. Garbuzenko and T. Mink, Tumor targeted quantum dot–mucin 1 aptamer–doxorubicin conjugate for imaging and treatment of cancer, J. Controlled Release, 2011, 153(1), 16–22.
10. C.-y. Zhang and L. W. Johnson, Single Quantum-Dot-Based Aptameric Nanosensor for Cocaine, Anal. Chem., 2009, 81(8), 3051–3055.
11. J. K. Jaiswal and S. M. Simon, Potentials and pitfalls of fluorescent quantum dots for biological imaging, Trends Cell Biol., 2004, 14(9), 497–504.
12. N. G. Portney and M. Ozkan, Nano-oncology: drug delivery, imaging, and sensing, Anal. Bioanal. Chem., 2006, 384, 620–630.
13. A. P. Alivisatos, W. Gu and C. Larabell, Quantum Dots as Cellular Probes, Annu. Rev. Biomed. Eng., 2005, 7, 55–76.
14. C. Tanford and K. C. Aune, Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. III. Dependence on temperature, Biochemistry, 1970, 9, 206–211.
15. A. C. S. Samia, C. Xiaobo and C. Burda, Semiconductor quantum dots for photodynamic therapy, J. Am. Chem. Soc., 2003, 125, 15736–15737.
16. A. Samia, S. Dayal and C. Burda, Quantum Dot-based Energy Transfer: Perspectives and Potential for Applications in Photodynamic Therapy, Photochem. Photobiol., 2006, 82, 617–625.
17. P. Z. Adam and X. Peng, Nearly monodisperse and shape-controlled CdSe nanocrystals via alternative routes: nucleation and growth, J. Am. Chem. Soc., 2002, 124, 3343–3353.
18. P. Z. Adam and X. Peng, Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor, J. Am. Chem. Soc., 2001, 123, 183–184.
19. X. Peng, Green Chemical Approaches toward High-Quality Semiconductor Nanocrystals, Chem.–Eur. J., 2002, 8, 334–339.
20. L. Qu, P. Z. Adam and X. Peng, Alternative routes toward high quality CdSe nanocrystals, Nano Lett., 2001, 1, 333–337.
21. L. Qu and X. Peng, Control of photoluminescence properties of CdSe nanocrystals in growth, J. Am. Chem. Soc., 2002, 124, 2049–2055.
22. W. Y. L. Ko, H. G. Bagaria, S. Asokan, K. J. Lina and M. S. Wong, CdSe tetrapod synthesis using cetyltrimethylammonium bromide and heat transfer fluids, J. Mater. Chem., 2010, 20, 2474–2478.
23. W. W. Yu and X. Peng, Formation of high-quality CdS and other II–VI semiconductor nanocrystals in non-coordinating solvents: tunable reactivity of monomers, Angew. Chem., Int. Ed., 2002, 41, 2368–2371.
24. T. Kippeny, L. A. Swafford and S. J. Rosenthal, Semiconductor nanocrystals: a powerful visual aid for introducing the particle in a box, J. Chem. Educ., 2002, 79, 1094–1100.
25. K. Rawat and H. B. Bohidar, Universal charge quenching and stability of proteins in 1-methyl-3-alkyl(hexyl/octyl)imidazolium chloride ionic liquid solutions, J. Phys. Chem. B, 2012, 116, 11065–11074.
26. D. Shugar, The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme, Biochim. Biophys. Acta, 1952, 8, 302–309.
27. A. Toumadje, S. W. Alcorn and W. C. Johnson, Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures, Anal. Biochem., 1992, 200, 321–331.
28. C. B. Murray, D. J. Norris and M. G. Bawendi, Synthesis and characterization of nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites, J. Am. Chem. Soc., 1993, 115, 8706–8715.
29. J. Jasieniak, L. Smith, J. V. Embden and P. Mulvaney, Re-examination of the size-dependent absorption properties of CdSe quantum dots, J. Phys. Chem. C, 2009, 113, 19468–19474.
30. P. Qin, R. Liu and Y. Teng, Perfluorodecanoic acid binding to hemoproteins: new insights from spectroscopic studies, J. Agric. Food Chem., 2011, 59, 3246–3252.
31. X. Pan, R. Liu, P. Qin, L. Wang and X. Zhao, Spectroscopic studies on the interaction of acid yellow with bovine serum albumin, J. Lumin., 2010, 130, 611–617.
32. X. Guo, X. Han, J. Tong, C. Guo, W. Yang, J. Zhu and B. Fu, The investigation of the interaction between piracetam and bovine serum albumin by spectroscopic methods, J. Mol. Struct., 2010, 966, 129–135.
33. J. Tian, J. Liu, Z. Hu and X. Chen, Interaction of wogonin with bovine serum albumin, Bioorg. Med. Chem., 2005, 13, 4124–4129.
34. Z. Chi, R. Liu and H. Zhang, Noncovalent interaction of oxytetracycline with the enzyme trypsin, Biomacromolecules, 2010, 11, 2454–2459.
35. B. Ojha and G. Das, The interaction of 5-(alkoxy)naphthalen-1-amine with bovine serum albumin and its effect on the conformation of protein, J. Phys. Chem. B, 2010, 114, 3979–3986.
36. M. H. Rahman, T. Maruyama, T. Okada, K. Yamasaki and M. Otagiri, Study of interaction of carprofen and its enantiomers with human serum albumin—I: mechanism of binding studied by dialysis and spectroscopic methods, Biochem. Pharmacol., 1993, 46, 1721–1731.
37. Q. Yang, X. M. Zhou and X. G. Chen, Combined molecular docking and multi-spectroscopic investigation on the interaction between eosin B and human serum albumin, J. Lumin., 2011, 131, 581–586.
38. Y.-P. Wang, Y.-l. Wei and C. Dong, Study on the interaction of 3,3-bis(4-hydroxy-1-naphthyl)-phthalide with bovine serum albumin by fluorescence spectroscopy, J. Photochem. Photobiol., A, 2006, 177, 6–11.
39. Y. Shu, M. Liu, S. Chen, X. Chen and J. Wang, New insight into molecular interactions of imidazolium ionic liquids with bovine serum albumin, J. Phys. Chem. B, 2011, 115, 12306–12314.
40. L. He, X. Wang, B. Liu, J. Wang, Y. Sun, E. Gao and S. Xu, Study on the interaction between promethazine hydrochloride and bovine serum albumin by fluorescence spectroscopy, J. Lumin., 2011, 131, 285–290.
41. Y. Z. Zhang, B. Zhou, X. P. Zhang, P. Huang, C. H. Li and Y. Liu, Interaction of malachite green with bovine serum albumin: determination of the binding mechanism and binding site by spectroscopic methods, J. Hazard. Mater., 2009, 163, 1345–1352.
42. D. Li, J. Zhu and J. Jin, Spectrophotometric studies on the interaction between nevadensin and lysozyme, J. Photochem. Photobiol., A, 2007, 189, 114–120.
43. F. Ding, W. Liu, Y. Li, L. Zhang and Y. Sun, Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence, J. Lumin., 2010, 130, 2013–2021.
44. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, 2007.
45. Z. Chi, R. Liu and H. Zhang, Noncovalent interaction of oxytetracycline with the enzyme trypsin, Biomacromolecules, 2010, 11, 2454–2459.
46. P. D. Ross and S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry, 1981, 20, 3096–3102.
47. Y.-P. Wang, Y.-l. Wei and C. Dong, Study on the interaction of 3,3-bis(4-hydroxy-1-naphthyl)-phthalide with bovine serum albumin by fluorescence spectroscopy, J. Photochem. Photobiol., A, 2006, 177, 6–11.
48. M. Bihari, T. P. Russell and D. A. Hoagland, Dissolution and dissolved state of cytochrome c in a neat, hydrophilic ionic liquid, Biomacromolecules, 2010, 11, 2944–2948.
49. A. Airinei, R. I. Tigoianu, E. Rusu and D. O. Dorohoi, Fluorescence quenching of anthracene by nitroaromatic compounds, Digest Journal of Nanomaterials and Biostructures, 2011, 6, 1265.
50. A. Ray, B. K. Seth, U. Pal and S. Basu, Nickel (II)-Schiff base complex recognizing domain II of bovine and human serum albumin: spectroscopic and docking studies, Spectrochim. Acta, Part A, 2012, 92, 164–174.
51. M. F. Pistonesi, M. S. Di Nezio, M. E. Centurión, M. E. Palomeque, A. G. Lista and B. S. F. Band, Determination of phenol, resorcinol and hydroquinone in air samples by synchronous fluorescence using partial least-squares (PLS), Talanta, 2006, 69, 1265–1268.
52. P. D. Ross and S. Subramanian, Thermodynamics of Protein Association Reactions: Forces Contributing to Stability?, Biochemistry, 1981, 20(11), 3096–3102.
53. M. Kumari, J. K. Maurya, U. K. Singh, A. B. Khan, M. Ali, P. Singh and R. Patel, Spectroscopic and docking studies on the interaction between pyrrolidinium based ionic liquid and bovine serum albumin, Spectrochim. Acta, Part A, 2014, 124, 349–356.
54. M. U. H. Mir, J. K. Maurya, S. Ali, S. Ubaid-Ullah, A. B. Khan and R. Patel, Molecular interaction of cationic gemini surfactant with bovine serum albumin: a spectroscopic and molecular docking study, Process Biochem., 2014, 49(4), 623–630.
55. R. Patel, J. K. Maurya, M. U. H. Mir, M. Kumari and N. Maurya, An insight into the binding between ester-functionalized cationic gemini surfactant and lysozyme, J. Lumin., 2014, 154, 298–304.
56. S. Li, J. J. Mulloor, L. Wang, Y. Ji, C. J. Mulloor, M. Micic, J. Orbulescu and R. M. Leblanc, Strong and Selective Adsorption of Lysozyme on Graphene Oxide, ACS Appl. Mater. Interfaces, 2014, 6(8), 5704–5712.
57. L. R. Wetter and H. F. Deutsch, Immunological Studies on Egg White Proteins: IV. Immunochemical and Physical Studies of Lysozyme, J. Biol. Chem., 1951, 192, 237–242.
58. M. Del Olmo, C. Diez, A. Molina, I. De Orbe and J. L. Vilchez, Resolution of phenol, o-cresol, m-cresol and p-cresol mixtures by excitation fluorescence using partial least-squares (PLS) multivariate calibration, Anal. Chim. Acta, 1996, 335, 23–33.
59. A. Espinosa-Mansilla, A. M. de la Peña, F. Salinas and D. G. Gómez, Partial least squares multicomponent fluorimetric determination of fluoroquinolones in human urine samples, Talanta, 2004, 62, 853–860.
60. D. Cozzolino, M. J. Kwiatkowski, M. Parker, W. U. Cynkar, R. G. Dambergs, M. Gishen and M. J. Herderich, Prediction of phenolic compounds in red wine fermentations by visible and near infrared spectroscopy, Anal. Chim. Acta, 2004, 513, 73–80.
61. U. S. Mote, S. L. Bhattar, S. R. Patil and G. B. Kolekar, Interaction between felodipine and bovine serum albumin: fluorescence quenching study, Luminescence, 2010, 25, 1–8.
62. J. Q. Lu, F. Jin, T. Q. Sun and X. W. Zhou, Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin–copper(II) complex, Int. J. Biol. Macromol., 2007, 40, 299–304.
63. J. C. Fan, X. Chen, Y. Wang, C. P. Fan and Z. C. Shang, Binding interactions of pefloxacinmesylate with bovine lactoferrin and human serum albumin, J. Zhejiang Univ., Sci., B, 2006, 7, 452–458.
64. G. Zhang, N. Zhao and L. Wang, Probing the binding of vitexin to human serum albumin by multispectroscopic techniques, J. Lumin., 2011, 131, 880–887.
65. C. Sun, J. Yang, X. Wu, X. Huang, F. Wang and S. Liu, Unfolding and refolding of bovine serum albumin induced by cetylpyridiniumbromide, Biophys. J., 2005, 88, 3518–3524.
66. M. Dockal, D. C. Carter and F. Rüker, Conformational transitions of the three recombinant domains of human serum albumin depending on pH, J. Biol. Chem., 2000, 275, 3042–3050.
67. Y.-H. Chen, J. T. Yang and H. M. Martinez, Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion, Biochemistry, 1972, 11, 4120–4131.
68. J. Corbin, N. Méthot, H. H. Wang, J. E. Baenziger and M. P. Blanton, Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy, J. Biol. Chem., 1998, 273, 771–777.
69. Z. C. Shang, P. G. Yi, Q. L. Yu and R. L. Lin, Reaction mechanism between ciprofloxacin hydrochloride and bovine serum albumin, Acta Phys.-Chim. Sin., 2001, 17, 48–52.
70. V. Calandrini, D. Fioretto, G. Onori and A. Santucci, Role of hydrophobic interactions on the stabilization of native state globular proteins, Chem. Phys. Lett., 2000, 124, 344–348.
71. M. Bostrom, D. R. M. Williams and B. W. Ninham, Specific ion effects: why the properties of lysozyme in salt solutions follow Hofmeister series, Biophys. J., 2003, 85, 686–694.
72. A. Tyagi, K. Rawat, A. K. Verma and H. B. Bohidar, Mechanistic evaluation of the size dependent antimicrobial activity of water soluble QDs, Anal. Methods, 2016, 8, 1060–1068.

---

Footnote

† Electronic supplementary information (ESI) available: The zeta potential, time autocorrelation function, size distribution, hydrodynamic radius (R_h) and physical characteristics of CdSe QDs and the temperature- and salt-dependent absorption, fluorescence (steady and synchronous), CD spectra and enzymatic activity of lysozyme in the absence and presence of CdSe QDs recorded in this study. See DOI: 10.1039/c6ra07368a

---