Dual-enzyme carbon dot nanoenzymes for the detection of glutathione and hydrogen peroxide

Summary of entire text

The maintenance of redox homeostasis depends on the delicate balance between reactive oxygen species and antioxidants.

H₂O₂, as a signaling molecule, participates in immune regulation, cell proliferation, and signal transduction.

Its abnormal accumulation is closely related to aging, cancer, inflammation, and neurodegenerative diseases;

Glutathione (GSH) is the most abundant thiol antioxidant in cells, protecting cells from oxidative damage through the thiol-disulfide cycle.

The two respectively represent the oxidation end and the reduction end of the cellular redox network, and their reliable quantification is of great significance for evaluating oxidative stress status and disease progression.

Recently, Professor Dong Chuan and Professor Gong Xiaojuan from the Institute of Environmental Science of Shanxi University reported a Fe-doped carbon dot (Fe-CD) nanozyme with Fe-pyrazine N dual active sites.

By adjusting the amount of ethylenediamine (EDA) in the synthesis process, the team achieved precise control of the abundance of pyrazine N species and the ratio of Fe²⁺/Fe³⁺, making Fe-CD₂.₀ simultaneously possess photoactivated oxidase-like (OXD-like) activity and H₂O₂-activated peroxidase-like (POD-like) activity.

The two catalytic pathways can be orthogonally triggered by light and H₂O₂, respectively, for independent detection of glutathione and H₂O₂;

The TMB oxidation product simultaneously generates absorption, fluorescence quenching, and photothermal signals, thereby constructing a multi-modal sensing platform integrating colorimetry, fluorescence, smartphone imaging, and photothermal readout.

Research results

The Fe-CD nanozyme was synthesized in one step using citric acid as the carbon source, ethylenediamine as the nitrogen source and coordinating regulator, and FeCl₃ as the iron source, through a 220°C hydrothermal reaction for 12 hours.

During the synthesis process, EDA first coordinated with Fe³⁺ to regulate the local coordination environment, and then reacted with citric acid to form Fe-doped carbon dots.

Keeping the amounts of citric acid and Fe³⁺ unchanged, and gradually increasing the EDA amount from 0.25 mmol to 4.0 mmol, three representative samples, Fe-CD₁.₀, Fe-CD₂.₀, and Fe-CD₃.₀, were obtained.

The most remarkable feature of the Fe-CD nanozyme is that its dual enzymatic activity can be orthogonally activated by two independent stimuli.

Without external stimulation, Fe-CD₂.₀ has almost no catalytic activity for TMB;

After adding H₂O₂ or providing light, a clear absorption peak appears at 652 nm, corresponding to the initiation of the POD-like and OXD-like pathways respectively.

The assignment of the active sites was confirmed through selective poisoning experiments.

After KSCN sealed the Fe sites, both catalytic pathways were significantly inhibited;

While the protonation of pyridine N by H₃PO₄ had negligible effect on activity.

This indicates that the Fe center and pyrrole N species - rather than pyridine N - are the dominant sites in the catalytic process.

Based on the above results, the team proposed the orthogonal activation mechanism:

Light selectively initiates the OXD-like pathway of O₂→·O₂⁻, while H₂O₂ initiates the POD-like pathway of H₂O₂→·OH.

Both pathways share TMB as the substrate, but are independently triggered by different external stimuli, providing a mechanism basis for orthogonal sensing of glutathione and H₂O₂.

The TMB_ox generated by the catalytic oxidation of TMB by Fe-CD₂.₀ exhibits absorption characteristics spanning the visible, near-infrared, and ultraviolet regions.

This results in three independent yet homologous signal outputs.

The absorption in the visible region at 652 nm is used for colorimetric analysis;

The broad absorption near 808 nm endows TMB_ox with photothermal conversion capabilities.

Under 808 nm laser irradiation, the system temperature reaches a steady state within approximately 2 minutes, and the temperature increase is proportional to the degree of TMB oxidation;

The absorption band of TMB_ox overlaps significantly with the excitation band of Fe-CD₂.₀ (approximately 360 nm).

Through the internal filtering effect, the fluorescence of Fe-CD₂.₀ is quenched, and the fluorescence lifetime remains essentially unchanged (approximately 6.89 ns) in the presence of TMB_ox, ruling out dynamic or static quenching mechanisms.

The three signals - absorption, fluorescence quenching, and photothermal - originate from the same catalytic event but are read out through different physical transduction mechanisms, forming a mutually corroborating sensing framework.

In glutathione detection, glutathione reduces TMB_ox back to TMB, resulting in a decrease in 652 nm absorption, restoration of Fe-CD₂.₀ fluorescence, and a decrease in the photothermal temperature rise;

For H₂O₂ detection, the trend is the opposite: an increase in H₂O₂ concentration promotes the formation of TMB_ox, leading to enhanced absorption, fluorescence quenching, and enhanced photothermal signals.

The five readout modes - microplate OD, microplate FI, smartphone visible light image, smartphone ultraviolet image, and infrared thermal image - all exhibit good linearity and sensitivity.

The detection limits for glutathione and H₂O₂ are both lower than 3.16 μM.

In HeLa cells, the team verified the detection capability of this platform for intracellular glutathione and H₂O₂ through RIPA lysis and PMA stimulation respectively.

For glutathione, the increase in cell density reduced the TMB_ox produced by the photoactivated OXD-like pathway and decreased the absorption;

For H₂O₂, PMA stimulation induced the release of endogenous H₂O₂ and activated the POD-like pathway. The increase in cell density enhanced the absorption.

The measurement results of both analytes were highly consistent with the commercial kit method.

In MCF-7 and HepG2 cells, this platform also provided consistent glutathione and H₂O₂ levels with the reference method, with spiked recovery rates ranging from 96.84% to 108.91%.

The quantitative results of the five independent readout modes for glutathione and H₂O₂ in HeLa cells were close to each other, with relative standard deviations ranging from 1.53% to 9.51%, and recovery rates ranging from 93.54% to 109.11%.

Since all the readouts were from the same orthogonal activation catalytic process and only the physical transduction method was different, this multi-mode design provided built-in cross-validation capabilities, reducing the risk of misjudgment caused by a single signal output.