Nitrogen and sulfur co-doped carbon dots with bright fluorescence for intracellular detection of iron ion and thiol

Abstract

Carbon dots (CDs) have been widely used in recent years because of their excellent water solubility and abundant surface functional groups. However, compared with quantum dots or biological probes, the quantum yield of CDs is lower, and the fluorescence mainly concentrated in the blue-green range, which significantly limits the biological applications of CDs. Heteroatoms doping is the most common method to improve the luminescence of CDs. In this work, nitrogen and sulfur co-doped luminescent CDs were successfully synthesized by microwave assisted method using glutathione (GSH) and p-phenylenediamine (PPD) as raw materials. It can emit bright green fluorescence in ethanol solution, and the maximum emission wavelength is 535 nm when excited at 374 nm, and the absolute quantum yield is as high as 63%. Iron ion (Fe³⁺) can interact with the functional groups on the surface of the CDs to form CDs/Fe³⁺, which is a non-fluorescence complex, and Fe³⁺ can be reduced to ferrous ion (Fe²⁺). In other words, the reaction mechanism of CDs and Fe³⁺ is a combination of dynamic quenching and static quenching. The fluorescence of CDs quenched by Fe³⁺ can be restored by thiol, because there is a stronger binding force between sulfhydryl (-SH) on the surface of thiol and Fe³⁺, which enables CDs to be released. In addition, the CDs has good biocompatibility and stability, indicating that it has excellent potential in bioimaging. This discovery will expand the application of CDs in the fields of biosensing and imaging.

Introduction

As the most abundant transition metal element in the earthcrust,[1] iron is exist widely in the human body. It is an essential nutrient for organisms. It can participate in many biological metabolic processes, such as photosynthesis,[2] respiration,[3] nitrogen fixation [4] DNA biosynthesis,[5] oxygen transport,[6] electron transfer,[7] etc. Excessive or lack of iron content in the human body can lead to the occurrence of some diseases, such as Parkinson’s syndrome,[8] Alzheimer’s disease,[9] anemia[10] and cancer.[11] Iron is also an essential element for the formation of heme. Iron ion (Fe³⁺) combines with transferrin to form serum iron, then transported to the bone marrow or other iron-demanding tissues for use. As a kind of critical biochemical indicator, the serum iron content can objectively evaluate and diagnose many diseases.[12] In addition, in the “Standards for drinking water quality” promulgated by our country in 2007, it is clearly stipulated that the iron content in the water shall not exceed 0.3 mg/mL. Since ferrous ion (Fe²⁺) is unstable and can be easily oxidized to Fe³⁺, iron mainly exists in the form of Fe³⁺ in nature. Therefore, it is necessary to develop a sensitive, convenient, and low-cost Fe³⁺ detection method for actual water samples and organisms.

So far, there have been many reports in the literature about Fe³⁺ detection methods, including colorimetry,[13] atomic absorption spectrometry,[14] and inductively coupled plasma mass spectrometry.[15] However, these methods have certain limitations, such as long test time, the need for complex sample preparation, and expensive experimental equipment. These shortcomings limit the broad application of the above methods. In recent years, the rapid development of fluorescence spectroscopy has received widespread attention at home and abroad because of its advantages of high sensitivity, convenience and good reproducibility.[16], [17], [18] At present, there have been reports in the literature that chemical sensors based on organic small molecule probes,[19] semiconductor quantum dots,[20] metal nanoclusters,[21] and metal–organic frameworks[22] have been used for the detection of Fe³⁺. However, these materials also have some shortcomings that cannot be ignored. For example, the preparation routes are commonly complicated, and heavy metal raw materials used in the synthesis process may cause environmental pollution, poor water solubility and poor photostability would also limit their biological applications.

Carbon dots (CDs) is a quasi-zero-dimensional carbon-based material with a size of less than 20 nm. Since it was first discovered in 2004,[23] it has been rapidly developed because of its good biocompatibility, good stability, wide range of raw materials, easy preparation, and low cost.[24], [25] CDs are widely used in many aspects, such as catalysis,[26], [27] bioimaging,[28] biomedicine,[29] information encryption[30] and antibacterial.[31] Due to its good optical properties and abundant surface functional groups, CDs are also widely used in biosensing.[32] However, compared with quantum dots and probes, CDs have some shortcomings, such as low quantum yield and the luminescence mainly concentrated in the range of blue-green light. To improve the luminescence properties of CDs, scientists have made a lot of efforts, such as doping with heteroatoms and increasing the conjugated structure.[33], [34], [35] Among them, the doping of nitrogen and sulfur atoms is widespread. Literatures show that the doping of nitrogen and sulfur atoms can effectively increase the quantum yield, reduce the energy gap, and make the luminescence redshift.[36], [37]

In this work, we used glutathione (GSH) and p-phenylenediamine (PPD) as the carbon source and water as the solvent to successfully synthesize a nitrogen and sulfur co-doped CDs by microwave assisted method, and achieved the specific detection of Fe³⁺ and thiol. Fe³⁺ not only forms a complex with sulfhydryl (-SH) and amino (–NH₂) group on the surface of the CDs, leading to the quenching of the fluorescence of the CDs, but also can be reduced by the CDs to form Fe²⁺. In other words, the reaction mechanism of CDs and Fe³⁺ is a combination of dynamic quenching and static quenching. In addition, the addition of thiol can restore the fluorescence of CDs quenched by Fe³⁺, because the -SH rich in thiol has a stronger binding force with Fe³⁺, so that the CDs are released and the fluorescence of the CDs is restored (Scheme 1).