Study of the bioconjugation of ternary alloyed ZnCdTe nanocrystals to Concanavalin A

Highlights

• Conjugation of ZnCdTe nanocrystals to Concanavalin A created cluster-like motif.

• Increasing amounts of protein decreased semiconductor photoluminescence.

• Quenching was only partial and protein remains active.

• Excitation profile of Concanavalin A changes with conjugation.

Abstract

This work describes the first spectroscopic study of the covalent binding of Concanavalin A with ZnCdTe semiconductor nanocrystals, together with structural characterization of the bioconjugates. Glutathione-capped ZnCdTe nanocrystals were prepared in an aqueous medium and conjugated to Concanavalin A using glutaraldehyde as the coupling agent. Morphological characterization of the bioconjugates revealed the formation of nanoparticle clusters resulting from the binding of protein molecules and the formation of bridges between two or more ZnCdTe nanocrystals. This caused a progressive decrease in the photoluminescence intensity of the nanocrystals when the amount of protein was increased. Nonetheless, the emission intensity remained at a satisfactory level after bioconjugation. The presence of the protein also greatly reduced the thermal quenching of quantum dot photoluminescence at higher temperatures, indicating that changes in the protein conformation might favor passivation of the nanocrystal surface.

Introduction

The use of semiconductor nanocrystals as fluorescent markers for proteins [1], [2] is motivated by the potential advantages offered by these nanomaterials, compared to fluorescent dye molecules. These include large Stokes shifts, broad absorption bands, sharp and intense emission bands, and the ability to tune the spectroscopic properties by changing the particle size [3], [4]. Spectroscopic studies can be used to evaluate the interaction of proteins with nanomaterials, simulating the presence of the nanoparticles in a biological medium, and to determine the effects of adsorption or bioconjugation on the protein structure. In the case of protein interactions, bovine serum albumin (BSA) is useful as a model protein due to its similarity to human serum albumin (HSA), which is the major soluble protein present in the circulatory system [5]. Several previous studies have described the use of spectroscopic techniques to investigate the interaction [6], [7], [8], [9] and bioconjugation of BSA [10], [11], [12] with different semiconductor nanocrystals. Spectroscopic data can also help to elucidate the behavior of nanoparticles in biological media, where proteins can be adsorbed on their surfaces and cause changes in structure and stability. For instance, Chakrabarti et al. used spectroscopic data to find that BSA retained its native structure after binding to ZnO-polyethyleneimine, with only minor alterations [13].

Lectins, a class of carbohydrate-recognizing proteins, are potential candidates for use in diagnostic methods and as highly specific sensors for glucose and other sugars. As a result, the bioconjugation of lectins with semiconductor nanocrystals is attracting increasing attention. Wang et al. [14] reported the conjugation of glutathione-stabilized CdTe nanocrystals to Concanavalin A for the detection of glucose. Santos et al. [15] conjugated Concanavalin A to polyphosphate-stabilized CdS-Cd(OH)₂ quantum dots for the recognition of mammary tumors. Xu et al. [16] prepared a conjugate of MPA-capped CdTe nanocrystals and Concanavalin A for in situ evaluation of cell surface carbohydrate. Tang et al. [17], [18] designed a nanobiosensor in which Concanavalin A-conjugated CdTe quantum dots were coupled to gold nanoparticles capped with thiolated β-cyclodextrin. The coupled system exhibited Förster resonance energy transfer (FRET), which could be reversed in the presence of glucose, restoring the photoluminescence of the quantum dots.

Despite the efforts to prepare and utilize bioconjugates based on lectins and semiconductor nanocrystals, to our knowledge there has been no evaluation of the effect of bioconjugation on the spectroscopic properties of both species. This is needed in order to understand and maintain the functionality expected for each component of the conjugate.

Composition-tunable alloyed semiconductor nanocrystals have emerged as potential candidates for use in biological studies, due to the possibility of partially replacing toxic cadmium ions by zinc, as well as improving both the photoluminescence quantum yield and the control of emissions over the visible range [19]. Ideal systems for use in biological applications must fulfill requirements that include compatibility with aqueous media, which can be achieved by direct aqueous synthesis, and also present biocompatibility and high quantum yield. Among the semiconductor nanocrystals that can be successfully synthesized in an aqueous medium, CdTe is one of the most widely studied compositions because it offers satisfactory control of size distributions, a relatively high quantum yield, and emissions covering almost the entire visible range [20]. However, its use in biological applications is restricted due to the release of cadmium ions, which can be cytotoxic to living cells, although this limitation could potentially be alleviated by the use of alloyed semiconductor nanocrystals [21]. Another important advantage of alloyed ternary and quaternary nanocrystals is the dependence of the band gap energy on both particle size and composition. This means that it is possible to design the band gap energy by changing the composition while keeping the nanocrystal size unchanged [22]. Higher quantum yields may also be achieved by a suitable choice of elements, because differences in the nature of the chemical bonds can inhibit defect formation. For example, the inclusion of ZnSe in the CdSe lattice increases covalency and inhibits deformation and generation of defects [23]. Teng and co-workers [24] reported the first aqueous synthesis of alloyed Zn_xCd_1−xTe and observed that the fluorescence peak showed a blue shift when the proportion of Zn²⁺ was increased, which was attributed to the tuning effect of the wider band gap of the ZnTe semiconductor. In addition, higher quantum yields have been achieved by the aqueous synthesis of glutathione-capped Zn_1−xCd_xTe [21].

The present work concerns the preparation of glutathione-capped ZnCdTe alloyed nanocrystals (with equal nominal proportions of Zn and Cd), covalently bioconjugated to Concanavalin A. For both species, the effects of bioconjugation were evaluated using absorption and photoluminescence spectroscopy analyses. The bioconjugates were characterized by FTIR spectroscopy and TEM microscopy, both of which revealed that the structure of the bioconjugate contained short chains of nanoparticles connected by protein bridges.