Sensitive and selective sensing system of metallothioneins based on carbon quantum dots and gold nanoparticles

Highlights

• An approach based on the CQDs and the AuNPs was developed for detecting matallothioneins.

• Sensing system was realized by the principle of FRET, and the CQDs acted as the donor while AuNPs acted as the acceptor.

• This approach was sensitive, convenient and non-cytotoxicity for matallothioneins detection.

Abstract

In this study, we developed a sensitive and selective sensing system for the detection of metallothioneins (MTs). The system is based on the fluorescence resonance energy transfer (FRET) between carbon quantum dots (CQDs) and gold nanoparticles (AuNPs). In this method, the fluorescence emission of CQDs was quenched by AuNPs due to FRET. When MTs were added to the CQD–AuNP system, the strong combination between thiol group and Au made the CQDs release from AuNPs, and the fluorescence of CQDs was recovered. The CQD–AuNP system can detect the MTs in aqueous solution (pH 3.0, citrate–HCl buffer) selectively and sensitively with a short response time (15 min). Results show that the fluorescence recovery efficiency has a good linear relationship with the MTs concentration in the range of 12–210 nmol L⁻¹, and the limit of detection was 5.25 × 10⁻⁹ mol L⁻¹. Furthermore, the sensing system was utilized to determine MTs in human urine samples with satisfactory results. The proposed system exhibits the advantages of high sensitivity, high selectivity, easy operation and most importantly, low cost and non-cytotoxicity to detect protein MTs.

Introduction

Metallothioneins (MTs) are a kind of low-molecular-weight (Mr < 10 kDa), cysteine-rich, metal-binding proteins that are widely found in various animals, higher plants, microorganisms, and various tissues and organs of humans [1,2]. As a class of heavy metal-binding proteins, MTs can be stably coordinated in the form of metal–thiolate clusters by thiol groups in cysteine residues and d¹⁰ metal ions, such as Zn²⁺, Cd²⁺, and Hg²⁺, to form a nontoxic or low-toxic complex [3]. MTs play an important role in the detoxification of heavy metals, scavenging of free radicals, regulation of intracellular balance, and maintenance of the homeostasis of metal ion concentrations. In addition, MTs are associated with several physiological processes, including the growth and development of the body, delaying aging, and certain diseases.

Various organisms have been reported to induce the expression of MTs in the body through exposure to heavy metals [4]. MTs are considered as potential biomarkers for heavy metal pollution in the environment because of their ability to respond to heavy metal contamination and the statistical correlation between MTs content in aquatic animals and heavy metal content in the environment [5]. Several studies have found a statistically significant relationship between the MTs content in human urine and the degree of environmental pollution caused by heavy metals [4,6,7]. As biomarkers for heavy metal pollution, MTs have attracted considerable attention in the fields of environmental science and bioscience toxicology. Hence, the establishment of a rapid, sensitive, convenient, and nontoxic method for the direct detection of MTs has important practical significance for the development of preventive management measures and timely monitoring of environmental pollution.

At present, there have been some reports on the detection of MTs, such as metal saturation determination [8], atomic absorption spectroscopy (AAS) [9], immunization [10], and high-performance liquid chromatography (HPLC) [11]. However, most of these methods have their own limitations, such as the lack of sufficient selectivity and sensitivity, the need for expensive complex equipment, or the involvement of cumbersome procedures. Although electrochemical techniques [12,13] have high sensitivity and can be quantified at very low concentration levels, the potential toxic vapor of mercury likely limits its wide application.

In recent years, fluorescence spectroscopy techniques have shown unique advantages in analytical and bioanalytical studies due to their operational simplicity, high sensitivity, and stability [14,15]. Although fluorescence spectroscopy can perform sensitive analysis on biological macromolecules, it cannot easily detect proteins that do not have native fluorescence, such as MTs. Fluorescence resonance energy transfer (FRET) technology is a nonradiative phenomenon, in which the energy is transferred from the excited donor fluorophore to the ground-state acceptor through dipole–dipole coupling process [16,17]. FRET has been used for the detection of many biological molecules [[18], [19], [20]] and ions [21,22]. In addition, it has high detection sensitivity and precision and low cost. An excellent donor–acceptor pair is a significant factor in improving the efficiency of FRET. Carbon QDs (CQDs) are a new class of carbon nanomaterials with a nearly spherical shape and sizes below 10 nm [23]. Compared with semiconductor QDs or organic fluorescent dyes, CQDs have apparent advantages in terms of aqueous solubility, chemical stability, photostability, low cytotoxicity, and favorable biocompatibility [24,25]. Therefore, CQDs are widely used in sensing compounds due to its wide range of excitation spectra and adjustable emission spectrum [26,27]. Gold nanoparticles (AuNPs) can be considered as an efficient acceptor of FRET due to their high extinction coefficient and intense electronic absorption bands in the visible region [28].

In the present study, we used nitrogen-doped CQDs as fluorescent probes and AuNPs as a fluorescent quencher to construct a FRET fluorescence sensor to detect MTs. The AuNPs modified with citrate were negatively charged and could adsorb positively charged CQDs onto the surface of AuNPs to form CQD–AuNP nanocomposites via electrostatic interaction. The fluorescence quenching of CQDs was caused by the FRET between CQDs and AuNPs. However, when MTs were added, the adsorbed CQDs were released from the surface of AuNPs due to the specific binding of MTs to AuNPs. As a result, the CQDs fluorescence was restored. On the basis of this principle, a novel and simple “turn-on” fluorescence sensor can be constructed for the detection of MTs.