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Article

Portable and Visual Detection of Cytochrome c with Graphene Quantum Dots–Filter Paper Composite

by
Liangtong Li
1,
Yongjian Jiang
1,
Ni Wang
1,
Yusheng Feng
1,
Binbin Chen
2 and
Jian Wang
1,*
1
Key Laboratory of Biomedical Analytics (Southwest University), Chongqing Science and Technology Bureau, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
2
School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen 518172, China
*
Author to whom correspondence should be addressed.
Chemosensors 2024, 12(8), 167; https://doi.org/10.3390/chemosensors12080167
Submission received: 9 June 2024 / Revised: 15 July 2024 / Accepted: 14 August 2024 / Published: 19 August 2024
(This article belongs to the Special Issue Advanced Sensing Technology for Biomarker Detection and Medical Diagnosis)
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Abstract

:
As a significant biomarker during the apoptosis process, cytochrome c (Cyt c) is considered as a critical component in the inherent apoptotic pathway, but the simple and portable detection still remains challengeable. In this work, a portable and visual sensing platform for Cyt c was developed based upon the fluorescence quenching of graphene quantum dots (GQDs), which could be finished within a few seconds. Herein, the absorption spectrum of Cyt c matched the emission spectrum of GQDs well, which could cause the fluorescence quenching of GQDs via the inner filter effect (IFE) in the range of 1–50 μg/mL with the limit of detection as low as 0.1 μg/mL. Furthermore, the intracellular Cyt c was imaged to observe the apoptosis process of cancer cells induced by staurosporine. To achieve the portable and visual detection of Cyt c, GQDs were deposited on the filter paper to form the solid platform, which displayed a gradual fluorescence quenching when different concentrations of Cyt c were present. Compared to the conventional methods, the proposed assay is low-cost, fast, portable, and visual, which will be useful for the investigation of mitochondrial dysfunction and apoptotic cell death.

Graphical Abstract

1. Introduction

As is well known, as a heme protein, cytochrome c (Cyt c) is closely related to the inner mitochondrial membrane [1,2], which functions as an electron carrier essential for adenosine triphosphate (ATP) production [3]. Under normal circumstances, the outer membrane of mitochondria is permeable to metabolites but impermeable to proteins, causing the translocation of Cyt c from the mitochondrial intermembrane gap to the cytoplasm, which is a unique event in early apoptosis [4]. This is a programmed cell death [2] that occurs in all living cells and is regulated by genes, which can be characterized by a series of cellular morphological changes, such as chromosome condensation, nuclear division, cell atrophy, and the formation of apoptotic bodies [5]. Therefore, the development of assays of Cyt c is highly significant in biology.
By now, researchers have devoted increasing effort to developing a wide range of Cyt c assays, such as Western blotting [4], high-performance liquid chromatography [5], electrochemistry [6], chemiluminescence [7], fluorometry [8], and colorimetry [9], but some of them are mostly limited by the defects of complex modification, long reactions, and low sensitivity. Furthermore, the above-mentioned assays detect Cyt c in the solution, which are inconvenient for point-of-care testing. Thus, it is highly desirable to develop fast, portable, and visual solid-phase-based platforms for Cyt c detection.
At present, a considerable number of devices are developed as portable sensors [10,11,12], including a sodium carboxymethyl cellulose matrix [13], paper-based device [14], pregnancy test kit [15], glucose meter [16], and so on. Among them, the paper-based device has received numerous concerns due to portability and convenience. In the portable sensors, the fluorescent carbon dots (CDs) are usually acted as the signal reporter because of their advantages [17], such as high photostability, good biocompatibility, low cost, and facile modification [18]. In particular, as a kind of CDs with unique structural, physicochemical, and photochemical properties, graphene quantum dots (GQDs) have been developed as a popular material for biology, devices, catalysis, and sensing applications [19].
To achieve the effective sensing based on GQDs, a broad range of mechanisms have been investigated, including fluorescence resonance energy transfer (FRET) [20], electron transfer (ET) [21], inner filter effect (IFE) [22], and so forth. Over the past few years, IFE has usually been applied in biochemical detection based on the principle that the excitation or emission spectra of chromophores match the absorption spectra of quenchers, which leads to a fluorescence quenching [23]. Particularly, IFE just requires the complementary overlapping between the absorption spectrum of quenchers and the excitation or emission spectra of fluorophores, which does not require any chemically covalent linkage between the quencher and fluorophore; that is, neither the donor nor the receptor need to be maintained in a specific distance range. Therefore, IFE is much simpler than FRET or ET systems; it is more convenient and has been widely applied in biochemical sensing [22].
Since the spectral overlapping plays a critical role in the IFE process, the nanoprobe with the appropriate optical feature is vital. Herein, GQDs were precisely prepared by a hydrothermal method with p-phenylenediamine and ethylenediamine as carbon and nitrogen sources, which presented the emission spectrum perfectly matching the absorption spectrum of Cyt c, supplying a great possibility to establish an assay of Cyt c based on IFE. Thus, a very fast and convenient fluorimetric method was developed for the visual and portable detection of Cyt c based on the quenching of GQDs on solid filter paper (Scheme 1). The results found that the GQDs displayed a high photobleaching resistance, low toxicity, and good biocompatibility, which were further fabricated with filter paper to form the portable sensing platform for the fast and visual detection of Cyt c. The economic and portable platform is considered to be promising in point-of-care testing.

2. Materials and Methods

2.1. Detection of Cytochrome c in Solution

To detect Cyt c in a solution, 50 μL of PBS (pH 7.24), 50 μL of GQDs (0.18 mg/mL), and 50 μL of Cyt c at different concentrations were mixed in a 1.5 mL tube, which was diluted to 0.5 mL with ultrapure water and kept at 37 °C for 2 min, which were detected to obtain the emission spectra excited at 410 nm with an F-2500 fluorescence spectrophotometer.

2.2. Portable and Visual Detection of Cytochrome c on Graphene Quantum Dots-Modified Filter Paper

To obtain the portable and visual sensing paper for Cyt c detection, the filter paper was immersed into GQDs’ solution (1 mg/mL) for 30 min and then dried at 60 °C for 5 min, which was cut into 1 cm × 1 cm square pieces. Subsequently, 50 μL of Cyt c at various concentrations was dropped on the above sensing material, which was further imaged at 365 nm with a WFH-204B ultraviolet analyzer (Hangzhou, China). In order to verify the specificity, 50 μL of different substance solutions was dropped onto 1 cm × 1 cm sensing paper, which was further imaged at 365 nm with a WFH-204B ultraviolet analyzer.

2.3. Determination of Cytochrome c in Cell Lysate

To compare the apoptosis, cell lysate was obtained and detected by the following steps: MCF-7 cells were suspended in 20 mL of deionized water, which could be broken due to the great osmotic pressure difference between the osmotic pressure in the cells and water, leading to the release of the substances in MCF-7 cells. Then, the precipitation was removed by centrifugation and the resultant solution was detected by the standard addition method according to the steps in the detection of cytochrome c in a solution.

2.4. Fluorescence Activation Imaging of Cytochrome c during Cell Apoptosis

To obtain Cyt c during cell apoptosis, MCF-7 cells were used by the following process: Firstly, MCF-7 cells were incubated with GQDs (40 μg/mL) for 2 h in Dulbecco’s modification of Eagle’s medium (DMEM). Subsequently, DMEM containing staurosporine (1 μM) was incubated with MCF-7 cells containing GQDs for 2 h to induce apoptosis. The normal state, pre-apoptotic state, and apoptotic state of MCF-7 cells were imaged by disk scanning unit living cell confocal scanning fluorescence microscopy in the FAM channel.

3. Results and Discussion

3.1. The Features of Graphene Quantum Dots

In this work, GQDs presented with unique structural and optical characteristics (Figure 1). The morphological and structural features of GQDs were obtained by microscopic images and the X-ray diffraction (XRD) spectrum, respectively. The average size of about 2.5 nm and an in-plane lattice spacing of 0.22 nm (Figure 1A) were observed in high-resolution transmission electron microscopic (HRTEM) imaging, corresponding to the (100) plane of graphite carbon, suggesting the graphite-like structure of as-prepared carbon nanomaterials [24]. Furthermore, a diffraction peak at 24° was found in the XRD spectrum. According to the Bragg equation, this diffraction peak corresponded to the lattice spacing at 0.22 nm (Figure 1B). Additionally, as a powerful tool to observe the nanomaterials [25], atomic force microscope (AFM) imaging showed that the height of GQDs was about 1 nm (Figure 1C,D), further inferring that GQDs consisted of about three layers of graphene [24].
In spectroscopy, GQDs presented the characteristic absorption at around 400 nm (Figure 1E), which was attributed to the n-π∗ transition [26]. Moreover, a yellow fluorescence emission of GQDs was found at 550 nm when excited at 410 nm, and a slight excitation-dependent emission phenomenon was displayed when the excitation wavelength ranged from 260 nm to 410 nm (Figure S1), which was possibly related to the homogeneous size and surface state [26]. In addition, the absolute quantum yield of GQDs was measured up to 56.9%, which could be qualified as an effective optical probe for the biosensing and bioimaging [27].
It was found in X-ray photoelectron spectroscopy (XPS) (Figure 1F) that carbon (C1s, 286.2 eV), nitrogen (N1s, 398.7 eV), and oxygen (O1s, 531.4 eV) were the main ingredients, confirming that the nitrogen element was successfully doped into the GQDs. In detail, C-C/C=C, C=O/C=N, and C=O groups corresponded to the peaks of C1s at 284.8 eV, 286.3 eV, and 287.1 eV, respectively (Figure S2A). Moreover, the peaks at 399.3 eV and 401.1 eV in the N1s spectrum corresponded to the N-H bond and graphite nitrogen (Figure S2B), and the peaks at 531.3 eV, 532.4 eV, and 533.2 eV in the O1s spectrum were attributed to C=O, C-OH, and C-O (Figure S2C), respectively [28]. The above results indicated that GQDs with surface functional groups were successfully prepared.

3.2. Stability of Graphene Quantum Dots

The high stability is one excellent feature of GQDs, which is superior to the traditional organic fluorescent dyes [29]. GQDs kept the strong fluorescence well in the anti-photobleaching capability investigation, which were irradiated at 365 nm for 2 h (Figure S3A), indicating the high photostability of GQDs. Moreover, the fluorescence intensity of GQDs showed no remarkable change even when the concentration of the NaCl solution was as high as 2.5 M (Figure S3B), suggesting that GQDs were highly stable in complex samples with high ionic strength. Additionally, the fluorescence intensity of GQDs remained well in the presence of anions or cations (Figure S3C,D), indicating that GQDs were provided with the strong interference ability. Therefore, the stable characteristics of the GQDs were conducive to the biochemical detections in complex samples.

3.3. Detection of Cytochrome c in Solution

3.3.1. The Working Conditions’ Optimization for Cytochrome c Detection

To obtain a better analytical performance for Cyt c detection, the important factors including acidity, reaction temperature, and time were taken into consideration to optimize. The fluorescence of GQDs was kept very stable within 30 min in the interaction kinetic investigation. Meanwhile, once GQDs were mixed with Cyt c, the fluorescence was decreased immediately and reached a stable state after a few seconds (Figure S4A). The above results suggested that the decrease in fluorescence intensity of GQDs was induced by the fast reaction with Cyt c, instead of the unstable optical property of GQDs. Furthermore, the fluorescence property of GQDs was slightly dependent on the acidity [30], revealing a little lower fluorescence intensity under acidic conditions than under neutral or alkaline conditions (Figure S4B), which was possibly related to the protonation and deprotonation of some functional groups. Moreover, considering cell activity in the following work, the neutral condition (pH 7.24) was the optimal acidity for the following work. Additionally, the temperature exerted a little effect on the fluorescence quenching (Figure S4C), which showed the stable fluorescence of GQDs when temperature was in the range of 25–65 °C. However, fluorescence quenching was slightly prevented when temperature was higher than 37 °C, which was due to the denaturation of Cyt c at higher temperature. Therefore, the optimal temperature for the quenching reaction should be controlled within 37 °C.

3.3.2. The Analytical Performance of the Proposed Method for Cytochrome c Sensing

Under the optimal working conditions, Cyt c could effectively quench the fluorescence of GQDs, and a higher Cyt c concentration led to a greater reduction degree of GQDs’ fluorescence (Figure 2A). Furthermore, the decrease in fluorescence intensity at 550 nm fit a good linear relationship, I = 2953–41.94 c (c, μg/mL), with the correlation coefficient R2 = 0.9924 when Cyt c concentration was in the range of 1~50 μg/mL (Figure 2B) with the limit of detection (LOD) as low as 0.1 μg/mL (7.69 nM, 3σ/k). Generally, Cyt c in apoptotic cells is in the range of 1–10 μM [8]; thus, LOD of this proposed method was lower than that in apoptotic cells, which was sensitive enough to detect Cyt c in apoptotic cells. Furthermore, compared with some other methods (Table S1), this proposed sensing method supplied the advantages of high sensitivity, easy operation, and rapid response, suggesting the potential in real-time imaging of the Cyt c translocation event in apoptotic cells.
The selectivity is an important issue for the assays because of the potential interferences. In this work, Cyt c in cells was detected, and thus some potential interferences, such as proteins as well as amino acids (Figure 2C), were demonstrated. The result confirmed that other species did not lead to the remarkable changes in the fluorescence intensity or fluorescence images (Figure S5), suggesting that the proposed method exhibited high specificity toward Cyt c sensing, which will be discussed in the subsequent work.

3.4. The Portable Detection of Cytochrome c on Solid Filter Paper

In order to develop the portable sensing platform for Cyt c detection, GQDs were loaded onto the filter paper, which offered the advantages of convenience, low cost, small sample volume, and easy operation. To avoid the interference from filter paper, filter paper was immersed into water for 30 min and then dried at 60 °C for 5 min, which were then not able to emit yellow-green fluorescence (1 in Figure 3A). While illuminated with a UV lamp at 365 nm, yellow-green fluorescence could be observed from the filter paper–GQD composite (2 in Figure 3A), which could be effectively quenched by Cyt c, presenting a gradual and colorful quenching trend with increasing the concentration of Cyt c (Figure 3A). These colorful images could be developed for the portable and visual detection of Cyt c. However, the other biomolecules, such as L-Cysteine and alkaline phosphatase (ALP), were not able to induce the remarkable fluorescence quenching of the filter paper–GQD composite, suggesting the excellent selectivity of the filter paper–GQD composite towards Cyt c detection (Figure 3B).

3.5. Determination of Cytochrome c in Cell Lysate

Cyt c could be produced in the apoptotic process, but could not be found in the non-apoptotic cell lysate [31]. To confirm this point, the spiked recovery experiment was carried out on the cell lysate. The result suggested that no Cyt c was found in non-apoptotic cell lysate, and the recovery rates were in the range of 92.0–98.5% with the relative standard deviation (RSD) less than 5% (Table 1), indicating that this method was effective for Cyt c detection in actual samples.

3.6. Cellular Imaging during Apoptosis

Before observing fluorescence imaging during apoptosis, the cytotoxicity of GQDs is an important issue [26]. The result showed that the cell viability was greater than 90% when the concentrations of GQDs were in the range from 1 μg/mL to 200 μg/mL, which demonstrated the high biocompatibility of GQDs (Figure S6).
Cell imaging was carried out with a DSU living cell confocal scanning fluorescence microscopy, where the green-yellow fluorescence signal was extremely strong and visible to the naked eyes without staurosporine (STS) treatment because of the low concentration of Cyt c in living cells [8]. Moreover, the pre-apoptotic cells displayed a significant fluorescence after uptaking GQDs. However, when it entered the apoptosis process induced by STS, the fluorescence disappeared (Figure 4), indicating that the overexpression of Cyt c during the apoptosis process was induced by apoptosis inducer STS and triggered the fluorescence quenching [8]. Therefore, the apoptosis cells could be effectively distinguished with GQDs.

3.7. The Fluorescence Quenching Mechanism Investigation

Generally, fluorescence quenching can be classified as static and dynamic quenching effects, or both, which can form a ground-state complex model and follows the Stern–Volmer equation theory [31].
F 0 / F = 1 + K S V [ Q ]
wherein [Q] is the quencher’s concentration. F0 and F are fluorescence intensities in the absence and presence of the quencher. In this work, the corrected fluorescence intensity ratio (F0/F) of GQDs was linear with the concentration of Cyt c, which could be expressed as F0/F = 0.927 + 0.0336c (c, μg/mL) with R2 = 0.9964, indicating that the quenching case in this work followed the Stern–Volmer equation, as well as the quenching constant of Stern–Volmer being KSV = 0.0336. Usually, KSV always keeps in the range of 102–103 in a dynamic quenching mechanism [32]. However, the KSV was 0.0336 in this work, which was far lower than KSV in a dynamic quenching. Therefore, it could be inferred that the quenching mechanism of GQDs by Cyt c was related to a static quenching effect.
In order to clearly explore the quenching mechanism of GQDs, the following investigations were carried out. In the absorption spectrum, there were two absorption peaks of Cyt c at 408 nm and 530 nm (Figure 5A), which were greatly overlapped by GQDs’ excitation at 410 nm and emission at 550 nm, respectively. Therefore, the reason for fluorescence quenching was likely to be related to IFE [22]. Furthermore, in the Fourier transform infrared (FTIR) spectra, no characteristic peaks of GQDs were changed in the presence of Cyt c, indicating that no new chemical bonds between Cyt c and GQDs were formed (Figure 5B). Since the fluorescence lifetime of GQDs remained unchanged in the presence of Cyt c (Figure 5C), the FRET quenching process was unreasonable. In conclusion, the fluorescence quenching of GQDs is most likely related to the IFE process.
Furthermore, the proportion of IFE in the quenching of GQDs’ fluorescence by Cyt c was calculated (Table S2), suggesting that the quenching mechanism was greatly attributed to IFE (>90%) due to the great spectral overlapping, which supplied a remarkable specificity for Cyt c sensing (Figure 5D).

4. Conclusions

In summary, the visual and portable detection of Cyt c was realized by loading graphene quantum dots onto filter paper. Furthermore, Cyt c was detected in the cell lysate and imaged in apoptotic cells, which was fast and straightforward. Finally, the careful quenching mechanism investigation found that the fluorescence quenching of Cyt c was related to the inner filter effect induced by Cyt c due to the tremendous spectral overlapping, which endowed this assay with high specificity. This proposed method is fast, portable, and visual, which lays a good foundation for the development of a more convenient and rapid solid-phase detection system for point-of-care testing.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/chemosensors12080167/s1, Figure S1. The emission spectra of GQDs at different excitation wavelengths in the range of 360 nm to 430 nm; Figure S2. The XPS of GQDs; Figure S3. Stability of graphene quantum dots; Figure S4. The working conditions’ optimization for Cyt c detection; Figure S5. The colorful images for selectivity investigation; Figure S6. The cell viability under different concentrations of GQDs; Table S1. An overview on recently reported nanomaterial-based assays for Cyt c; Table S2 [2,33,34,35,36,37,38,39,40]. Inner filter effect of Cyt c on fluorescence of GQDs.

Author Contributions

L.L.: Investigation, Formal analysis, Writing—original draft. Y.J.: Investigation, Conceptualization. N.W.: Investigation, Conceptualization. Y.F.: Investigation, Funding acquisition. B.C.: Conceptualization, Formal analysis, Writing—review and editing. J.W.: Methodology, Conceptualization, Writing—review and editing, Funding acquisition, Formal analysis, Project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This work is financially supported by the National Natural Science Foundation of China (No. 22174115), the Graduate Education and Teaching Reform Research Project of Chongqing (No. yjg223038), and Fundamental Research Funds for the Central Universities (SWU-XDJH202321), as well as the Innovation and Entrepreneurship Project of Southwest University (X202210635081).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article.

Acknowledgments

The authors would like to thank the Ministry of Education Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University) for supporting this research.

Conflicts of Interest

The authors declare no conflicts of interest.

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Scheme 1. The portable detection of cytochrome c with graphene quantum dots on filter paper.
Scheme 1. The portable detection of cytochrome c with graphene quantum dots on filter paper.
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Figure 1. Structural and optical properties of GQDs. (A) HRTEM image of GQDs. Inset: lattice spacing of GQDs. (B) XRD spectrum of GQDs. (C) AFM image of GQDs. (D) Height distribution of GQDs. (E) Absorption (blue), excitation (black), and emission (red) spectra of GQDs. (F) Full XPS spectrum of GQDs.
Figure 1. Structural and optical properties of GQDs. (A) HRTEM image of GQDs. Inset: lattice spacing of GQDs. (B) XRD spectrum of GQDs. (C) AFM image of GQDs. (D) Height distribution of GQDs. (E) Absorption (blue), excitation (black), and emission (red) spectra of GQDs. (F) Full XPS spectrum of GQDs.
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Figure 2. The sensitivity and selectivity of the proposed fluorescence method for Cyt c detection. (A) The fluorescence spectra of GQDs in the presence of Cyt c at different concentration. (B) The linear relationship between the fluorescence intensity of GQDs at 550 nm and the concentration of Cyt c. Conditions: GQDs, 0.18 mg/mL; pH, 7.24; t, 2 min; T, 37 °C. (C) The selectivity of GQDs for the Cyt c analysis among various biomolecules. 1, QGDs; 2, GQDs + Cyt c (50 μg/mL); 3, GQDs + alkaline phosphatase (25 μg/mL); 4, QGDs+ L-Cysteine (120 μg/mL); 5, QGDs+ L-Asparagine (13 μg/mL); 6, QGDs + L-Serine (110 μg/mL); 7, QGDs + bovine serum albumin (190 μg/mL); 8, QGDs + glutathione (310 μg/mL); 9, QGDs+ human serum albumin (200 μg/mL); 10, QGDs + bemoglobin (23 μg/mL). Conditions: GQDs, 0.18 mg/mL; pH, 7.24; t, 2 min; T, 37 °C. Error bars indicate the standard deviation (n = 3).
Figure 2. The sensitivity and selectivity of the proposed fluorescence method for Cyt c detection. (A) The fluorescence spectra of GQDs in the presence of Cyt c at different concentration. (B) The linear relationship between the fluorescence intensity of GQDs at 550 nm and the concentration of Cyt c. Conditions: GQDs, 0.18 mg/mL; pH, 7.24; t, 2 min; T, 37 °C. (C) The selectivity of GQDs for the Cyt c analysis among various biomolecules. 1, QGDs; 2, GQDs + Cyt c (50 μg/mL); 3, GQDs + alkaline phosphatase (25 μg/mL); 4, QGDs+ L-Cysteine (120 μg/mL); 5, QGDs+ L-Asparagine (13 μg/mL); 6, QGDs + L-Serine (110 μg/mL); 7, QGDs + bovine serum albumin (190 μg/mL); 8, QGDs + glutathione (310 μg/mL); 9, QGDs+ human serum albumin (200 μg/mL); 10, QGDs + bemoglobin (23 μg/mL). Conditions: GQDs, 0.18 mg/mL; pH, 7.24; t, 2 min; T, 37 °C. Error bars indicate the standard deviation (n = 3).
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Figure 3. Sensitivity and selectivity for Cyt c analysis on filter paper. (A) Fluorescence images of GQDs in presence of Cyt c at different concentration. 1, Control; 2, QGDs; 3, QGDs+ Cyt c (0.05 μg/mL); 4, QGDs+ Cyt c (0.25 μg/mL); 5, QGDs+ Cyt c (0.5 μg/mL); 6, QGDs+ Cyt c (2.5 μg/mL); 7, QGDs+ Cyt c (5 μg/mL); 8, QGDs+ Cyt c (25 μg/mL); 9, QGDs+ Cyt c (50 μg/mL); 10, QGDs+ Cyt c (500 μg/mL). Conditions: pH, 7.24; t, 2 min; T, 37 °C. (B) Selectivity for Cyt c analysis among various biomolecules. 1, QGDs; 2, GQDs + Cyt c (50 μg/mL); 3, GQDs + alkaline phosphatase (25 μg/mL); 4, QGDs+ L-Cysteine (120 μg/mL); 5, QGDs+ L-Asparagine (13 μg/mL); 6, QGDs+ L-Serine (110 μg/mL); 7, QGDs+ bovine serum albumin (190 μg/mL); 8, QGDs + glutathione (310 μg/mL); 9, QGDs+ human serum albumin (200 μg/mL); 10, QGDs + bemoglobin (23 μg/mL). Conditions: pH, 7.24; t, 2 min; T, 37 °C.
Figure 3. Sensitivity and selectivity for Cyt c analysis on filter paper. (A) Fluorescence images of GQDs in presence of Cyt c at different concentration. 1, Control; 2, QGDs; 3, QGDs+ Cyt c (0.05 μg/mL); 4, QGDs+ Cyt c (0.25 μg/mL); 5, QGDs+ Cyt c (0.5 μg/mL); 6, QGDs+ Cyt c (2.5 μg/mL); 7, QGDs+ Cyt c (5 μg/mL); 8, QGDs+ Cyt c (25 μg/mL); 9, QGDs+ Cyt c (50 μg/mL); 10, QGDs+ Cyt c (500 μg/mL). Conditions: pH, 7.24; t, 2 min; T, 37 °C. (B) Selectivity for Cyt c analysis among various biomolecules. 1, QGDs; 2, GQDs + Cyt c (50 μg/mL); 3, GQDs + alkaline phosphatase (25 μg/mL); 4, QGDs+ L-Cysteine (120 μg/mL); 5, QGDs+ L-Asparagine (13 μg/mL); 6, QGDs+ L-Serine (110 μg/mL); 7, QGDs+ bovine serum albumin (190 μg/mL); 8, QGDs + glutathione (310 μg/mL); 9, QGDs+ human serum albumin (200 μg/mL); 10, QGDs + bemoglobin (23 μg/mL). Conditions: pH, 7.24; t, 2 min; T, 37 °C.
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Figure 4. Fluorescence imaging of cells in different states and under different imaging conditions.
Figure 4. Fluorescence imaging of cells in different states and under different imaging conditions.
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Figure 5. The investigations of the fluorescence quenching mechanism. (A) The absorption spectrum of Cyt c. (B) The FTIR spectra of the GQDs, Cyt-c, and GQDs-Cyt c. (C) The fluorescence lifetime of GQDs (black) in the presence of Cyt c (red). (D) Observed suppressed efficiency (E%) measurements for GQDs in the presence of Cyt c, E = 1 − F/F0. F0 and F are the steady-state fluorescence intensities of GQDs in the absence and presence of Cyt c, respectively.
Figure 5. The investigations of the fluorescence quenching mechanism. (A) The absorption spectrum of Cyt c. (B) The FTIR spectra of the GQDs, Cyt-c, and GQDs-Cyt c. (C) The fluorescence lifetime of GQDs (black) in the presence of Cyt c (red). (D) Observed suppressed efficiency (E%) measurements for GQDs in the presence of Cyt c, E = 1 − F/F0. F0 and F are the steady-state fluorescence intensities of GQDs in the absence and presence of Cyt c, respectively.
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Table 1. Detection of cytochrome c in cell lysate.
Table 1. Detection of cytochrome c in cell lysate.
SamplesDetected (μg/mL)Spiked (μg/mL)Average Found (μg/mL)Average Recovery (%, n = 3)RSD
(%, n = 3)
1None10.009.2092.04.33
2None50.0047.4094.83.28
3None100.098.5098.54.67
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Li, L.; Jiang, Y.; Wang, N.; Feng, Y.; Chen, B.; Wang, J. Portable and Visual Detection of Cytochrome c with Graphene Quantum Dots–Filter Paper Composite. Chemosensors 2024, 12, 167. https://doi.org/10.3390/chemosensors12080167

AMA Style

Li L, Jiang Y, Wang N, Feng Y, Chen B, Wang J. Portable and Visual Detection of Cytochrome c with Graphene Quantum Dots–Filter Paper Composite. Chemosensors. 2024; 12(8):167. https://doi.org/10.3390/chemosensors12080167

Chicago/Turabian Style

Li, Liangtong, Yongjian Jiang, Ni Wang, Yusheng Feng, Binbin Chen, and Jian Wang. 2024. "Portable and Visual Detection of Cytochrome c with Graphene Quantum Dots–Filter Paper Composite" Chemosensors 12, no. 8: 167. https://doi.org/10.3390/chemosensors12080167

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