Author’s Accepted Manuscript

A novel and sensitive fluorescence sensor for

glutathione detection by controlling the surface
passivation degree of carbon quantum dots

Jiahong Pan, Zengyao Zheng, Jianying Yang,

Yaoyu Wu, Fushen Lu, Yaowen Chen, Wenhua
Gao
www.elsevier.com/locate/talanta

PII: S0039-9140(17)30133-9
DOI: http://dx.doi.org/10.1016/j.talanta.2017.01.033
Reference: TAL17215
To appear in: Talanta
Received date: 4 September 2016
Revised date: 8 January 2017
Accepted date: 10 January 2017
Cite this article as: Jiahong Pan, Zengyao Zheng, Jianying Yang, Yaoyu Wu,
Fushen Lu, Yaowen Chen and Wenhua Gao, A novel and sensitive fluorescence
sensor for glutathione detection by controlling the surface passivation degree of
carbon quantum dots, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.01.033
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A novel and sensitive fluorescence sensor for glutathione detection by

controlling the surface passivation degree of carbon quantum dots

Jiahong Pana, Zengyao Zhengb, Jianying Yangb, Yaoyu Wua, Fushen Lua, Yaowen Chena, Wenhua

Gaoa*

a
Department of Chemistry and Laboratory for Preparation and Application of Ordered Structural

Materials of Guangdong Province, Shantou University, Shantou, Guangdong 515063, P. R. China.

b
National Detergent and Cosmetics Products Quality Supervision and Inspection Center

(Guangdong), Shantou, Guangdong 515041, P. R. China.

*Corresponding author. Tel: +86-22-86502774; Fax: +86-22-82903941. E-mail:

whgao@stu.edu.cn

Abstract

A novel fluorescence sensor based on controlling the surface passivation degree of carbon

quantum dots (CQDs) was developed for glutathione (GSH) detection. First, we found that the

fluorescence intensity of the CQDs which was obtained by directly pyrolyzing citric acid would

increased largely after the surface passivation treatment by

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). In the light of this phenomenon, we

designed a simple, rapid and selective fluorescence sensor based on the surface passivated CQDs.

A certain and excess amount of EDC were mixed with GSH, part of EDC would form a stable

complex with GSH owing to the exposed sulfhydryl group of GSH. As the synthesized CQDs

were added into the above mixture solution, the fluorescence intensity of the (EDC/GSH)/CQDs

mixture solution could be directly related to the amount of GSH. Compared to other fluorescence

analytical methods, the fluorescence sensor we design is neither the traditional fluorescent “turn

on” probes nor “turn off” probes. It is a new fluorescence analytical method that target object
1
indirectly control the surface passivation degree of CQDs so that it can realize the detection of the

target object. Moreover, the proposed method manifested great advantages including short

analysis time, low cost and ease of operation.

Graphical Abstract

Schematic principle of GSH detection by using the surface passivated CQDs.

Keywords: surfaced passivation carbon quantum dots, GSH, fluorescence

1. Introduction

Reduced glutathione (GSH), a tripeptide, is a produced endogenously from glycine, L-cysteine,

and L-glutamic acid [1]. GSH plays great roles in biological systems and serves many cellular

functions such as the maintenance of intracellular redox states, detoxification, and metabolism

[2,3,4]. Meanwhile, the level of GSH in tissues is related to a variety of diseases, such as HIV,

AIDS, aging, cancer, Parkinson and autism in children [5,6,7,8,9]. Moreover, owing to its

important physiological properties e.g., as an antioxidant and detoxifier [10,11,12], GSH has been

widely used in health foods, cosmetics industries and pharmaceuticals [13]. Therefore, selective

and sensitive techniques for GSH detection are critically important. Currently, a variety of

detection techniques have been developed for GSH sensing, including high-performance liquid

chromatography (HPLC) [14], electrochemiluminescence (ECL) [15], surface-enhanced Raman

scattering (SERS) [16], electrochemistry (EC) [17], capillary electrophoresis (CE) [18], enzyme

linked immunosorbent assay (ELISA) [19], UV-Vis assays [20] and so on. Although these

strategies exhibit promising results for GSH detection, there are still some hindrances including

time consumption, low sensitivity, expensive equipment, and complex operations for skilled

technicians. Fluorescence spectroscopy is a powerful optical technology that can provide a good

flexible, sensitive, simple detection method. Over the past several years, great efforts have been

devoted to explore ideal fluorescence methods for GSH detection. For instance, a BODIPY-based

fluorescent sensor for GSH detection with high sensitive was developed [21]. Shao et al. [22]

proposed a fluorescence sensor for GSH detection using bis-spiropyran ligands as dipolar

2
molecule receptors. Among these fluorescence methods, organic dyes have been widely used as

fluorescent probes. Unfortunately, most of organic dyes have poor photostability, narrow

excitation spectra, and broad emission bands with red tailing, which have intrinsically confined

their further applications. Recently, fluorescent nanomaterials have attracted numerous attentions

for GSH detection such as quantum dots (QDs) [23], gold nanoparticles [24], upconversion

nanoparticles (UCNPs) [25], graphene oxide [26] and graphitic carbon nitride quantum dot

(g-CNQD) [27]. A fluorescent biosensor based on CdTe quantum dots for GSH detection was

proposed by Tan’ group [23]. However, the toxic heavy metal Cd2+ ions posed a big threat to

environment. Although upconversion nanoparticles (UCNPs) have several outstanding features

including high photostability, thermal stability and improved signal-to-noise ratios, UCNPs also

suffer some limitations such as complicated synthesis, high cost and overheating effect from a

relatively high power density NIR laser. Recently, Xu [27] established a sensing system for GSH

analysis by using graphitic carbon nitride quantum dot (g-CNQD), which is a novel fluorescence

carbon nanomaterial. Nevertheless, synthesis of graphitic carbon nitride quantum dot (g-CNQD)

needs harsh terms including high temperature of 550℃ and strong acid. Consequently, a novel

fluorescent nanomaterial with ideal environmental safety, high fluorescent intensity, simple

operability, and low-cost property is highly desirable for GSH analysis with low concentrations.

Carbon quantum dots (CQDs), a newly emerging form of carbon nanomaterials have inspired

intense research efforts in various fields which can be produced inexpensively by many

approaches [28]. Compared to traditional quantum dots and organic dyes, photoluminescent CQDs

are superior in terms of high aqueous solubility, robust chemical inertness, easy functionalization,

high resistance to photobleaching, low toxicity and good biocompatibility [29].Meanwhile,

compared with other fluorescent carbon nanomaterials such as graphene and g-CNQDs, CQDs

have much more sources of raw materials, and very good dispersibility in aqueous. These unique

characteristics have spurned intense interests in the use of CQDs for bioassays and biosensors,

which indicate that CQDs might be the most promising for quantifying trace amount of GSH.

Recently, a CQDs-MnO2 nanocomposites “turn-on” fluorescence sensor for GSH detection was

developed [30]. However, the synthesis procedure of MnO2 nanosheets and CQDs-MnO2

nanocomposites seems to be complicated, which limited its further applications in real time
3
analysis. To enhance the quantum yield of CQDs, many methods has been reported. Zhu et al [31]

proposed a hydrothermal route by adding different nitrogen source (including ethylenediamine,

ethylamine, n-heptylamine, urea and p-phenylenediamine) into citric acid solution in Teflon-lined

autoclave and maintaining 200℃ for 5 h. Woosung Kwon el at [32] reported the preparation of

highly photoluminescent CQDs via carbonization of glucose in reverse micelles followed by in

situ surface passivation. The maximum quantum yields of the CQDs achieved 35 percent

compared to quinine sulfate. Most fluorescent sensors were based on fluorescence resonance

energy transfer (FRET), which can transmit photo excitation energy from a donor fluorophore to

an acceptor fluorophore. It is time-consuming and complex to produce donor fluorophores and

acceptor fluorophore.

In this study, we found that EDC would enhanced the fluorescence intensity of the CQDs

which was synthesized by directly pyrolyzing citric acid. The enhanced photoluminescence from

CQDs may attributed to the presence of surface energy traps that become emissive upon

stabilization as a result of the surface passivation [33]. Then we proposed a rapid fluorescence

sensor for GSH detection by controlling the surface passivation degree of the CQDs. The principle

of this strategy was demonstrated in Scheme 1. A certain and excess amount of EDC solution was

firstly mixed with GSH and the part of EDC would combined with GSH and form thiolester

linkages because of the exposed sulfhydryl group of GSH. Then the synthesized CQDs were

casted into the above mixture solution and the rest of the EDC would reacted with the synthesized

CQDs. Then fluorescence intensity of the surface passivated CQDs would be correlated with the

concentration of the GSH. Compared with previews fluorescent carbon quantum dots based

methods, the major advantages of the proposed method are the remarkable enhanced quantum

yield of the synthesized CQDs and the environmentally friendly rapid process. This rapid, facile

and effective method will show a potential application in the GSH monitoring of human serum or

detecting biological thiols in human blood serum, medical treatment, health care, functional foods

and cosmetics.

2. Materials and methods

2.1 Materials
4
GSH (reduced form), glycine (Gly), lysine (Lys), serine (Ser) and L-threonine (L-Threonine) were

purchased from Sangon Bioengineering Co., Ltd. (Shanghai, China). Citric acid (CA) was

purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China),

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) was purchased from

CCIS Reagent Co., Ltd. Phosphate buffer saline (PBS, 10 mM, pH 7.0) was used for all

experiments. The human serum samples were obtained from infirmary of Shantou University. All

other reagents not mentioned here were of analytical-grade purity and used as received. All

solutions were prepared using ultrapure water generated by a Millipore Mill-Q (Bedford, USA)

with an electric resistance≥18.2 MΩ·cm.

2.2 Characterization

The fluorescence spectra were collected with a F-7000 fluorescence spectrophotometer (Hitachi,

Japan) equipped with a 1 cm× 1 cm quartz cuvette under excitation of 360 nm. All pH

measurements were made with a PHS-3CA precision acidity meter (Dapu, China). UV-vis

measurements were carried out on a Lambda 950 spectrophotometer (PerkinElmer, USA). Fourier

Transform Infrared spectra (FTIR) were recorded on a Magna 750 FTIR spectrophotometer

(Nicolet, USA) as KBr pellets. Atomic force microscopy image (AFM) was obtained by

tapping-mode on a Nanoscope IIIa Digital Instruments with NSC15 tips (Veeco, USA). The

morphology and size of CQDs were characterized using a Tecnai F20 G2 S-TWIN transmission

electron microcopy (FEI, USA) operating at 200 kV. Samples for HRTEM (High Resolution

Transmission Electron Microcopy) were prepared on holy carbon coated 200 mesh copper grids.

2.3 Synthesis of CQDs

CQDs were prepared by directly pyrolyzing citric acid by following a reported method with a

minor modification [34]. In a typical synthesis, 2 g CA was put into a micro beaker (10 mL) with a

unsealed cover and heated to 200℃ using a muffle furnace. About 30 min later, the CA was

liquated and its color changed to orange. After the liquid was cool to room temperature in air

condition and the liquid concreted, 2 mL NaOH (100 mg·mL-1) was casted into the micro beaker

5
with magnetic stirring until the concretion dissolved completely. Then the solution was transferred

into a breaker containing 8 mL of 100 mg·mL-1 NaOH solution with continuous stirring, resulting

the 10 mL aqueous solution of CQDs. The pH of the final CQDs solution was nearly 7 and the

CQDs stock solution was stored at 4℃ before use.

2.4 Quantum yield (QY) measurements

The quantum yield (QY) of CQDs was obtained by the following equation [35]:

𝑌 𝐼𝑠 𝜂 2
𝑄 = 𝑄𝑆
𝑌𝑠 𝐼 𝜂𝑠2

Where Q is the QY, Y is the optical density, I is the measured integrated emission intensity, and η

is the refractive index of the solvent. The subscript “s” refers to the standard with known QY.

For these aqueous solutions, η/ηs=1. In order to minimize reabsorption effects, make sure that the

absorbance of carbon quantum dots aqueous solution in 10 mm quartz cuvette was below 0.1 at

360 nm. Quinine sulfate (0.1 M H2SO4 as solvent; QY= 0.54) were chosen as standards.

2.5 Procedures for GSH detection

EDC and GSH were prepared in pH 7.0 PBS buffer (10 mM). The experiment testing process is

below: First, 100 μL EDC (200 μM) was added to 1 mL different concentrations (1.0~100 μM) of

GSH for 10 min reaction. Then 10 μL CQDs stock solution was introduced to the mixture and

incubated for another 10 min. Last, the fluorescence intensity of the mixture solutions was

recorded with an excitation wavelength of 360 nm, a slit of 10 nm and PMT voltage 450 V. The

procedures for all the optimization experiments are the same as described above. Here, we must

emphasized that adding order is important. If GSH were added into the EDC treated CQDs, the

fluorescence intensity of the EDC treated CQDs would not change. Because the surface

passivation process had already completed.

2.6 Specificity investigation

For the selectivity study, an aliquot of the stock solutions of nontarget samples (KCl，MgCl2，Gly,

6
Lys, Ser, L-Thr, L-Arg) (10 mM) were prepared in pH 7.0 PBS buffer (10 mM). 1 mL interfering

substances were mixed with 100 μL EDC (200 μM) for 10 min reaction. Fluorescence spectra

were recorded with excitation at 360 nm after 10 μL CQDs stock solution was introduced to the

mixture for another 10 min incubation.

3. Results and discussion

3.1 Characterization of the synthesized CQDs

HRTEM was used to characterize the morphology and size of CQDs. As shown in Fig 1(A-B), the

CQDs are nearly spherical and well separated from each other with the size smaller than 5 nm.

The AFM result indicates that the CQDs (Fig 2) are mostly nanoparticles of ~4 nm in size. The

photoluminescence emission spectra of the synthesis CQDs with various excitation wavelengths

were shown in Fig S1. When the excitation wavelength increased from 320 to 400 nm, the

wavelength of the maximum emission shifted from 444 to 475 nm. As the excitation wavelength

increase, the emission peak position shifts to longer wavelength increases. The maximum

fluorescence emission intensity can be obtained when it is excited at 360 nm. Then the intensity

gradually decreases. The excitation wavelength dependence of the emission wavelength and

intensity is a common phenomenon observed in other reported carbon quantum dots [31]. FTIR

was used to characterize the obtained CQDs (Fig 3A). These CQDs are highly water soluble due

to the existence of carboxyl groups on their surface, which can be proved from the fourier

transform infrared spectrum. The synthesis CQDs show stretching vibrations of C-OH at 3442.50

cm-1 and C-H at 2931.90 cm-1 and absorption of stretching vibration C=O at 1710.65 cm-1 and

1582.06 cm-1. Moreover, the as-synthesized CQDs are very stable for several months at room

temperature without any precipitations, which facilitates their further application.

3.2 Sensing mechanism

As shown in Fig 3B, the optical properties of the synthesized CQDs and the surface passivated

CQDs were investigated by UV-vis absorption spectra. From Fig 3B, the GSH (a) and EDC (b)

7
show no obvious absorption peak. The UV-vis absorption of the synthesized CQDs (c) in the

figure shows a slight peak at about 330 nm. After surface passivation by EDC, these CQDs

solution (d) have a strong UV absorption peak at 361 nm. The presence of GSH in EDC solution

would decreased the efficiency of the surface passivation treatment for the synthesized CQDs. As

shown in the Fig 3B curve d and e, the intensity of absorption peak at 361 nm of the

(EDC/GSH)/CQDs mixture solution which was made by adding the synthesized CQDs into the

EDC/GSH mixture solution is weaker than the EDC/CQDs mixture solution. Meanwhile, the

photoluminescence (PL) intensity of the synthesized CQDs increased much after surface

passivation, which can be confirmed from the fluorescence spectra (Fig S2). From the Fig S2, the

quantum yields (QY) of the synthesis CQDs (curve a) is determined to be 0.8% with quinine

sulfate (curve f) as a reference. With the increase concentration 2.5 to 25 mM of the EDC, the QY

of the surface passivation CQDs increase from 18.83% to 41.10%. The relative high QY implies

that the CQDs should be well surface-passivated.

3.3 Optimization of experimental conditions

The parameters including the time of surface passivation treatment and media pH are optimized

for the CQDs based detection system. The time dependent fluorescence of the reaction between

the synthesized CQDs and EDC is investigated. As shown in Fig 4A, the fluorescence intensity of

the surface passivated CQDs change largely from 0 to 10 min and gradually become stable from

10 to 40 min. To ensure the rapid detection of GSH, 10 min is chosen as the optimum incubation

time.

Besides, the effect of the concentration of EDC on the fluorescence enhancement is also

systemically investigated. The higher concentration of EDC were adding, the higher fluorescence

intensity of the CQDs were achieved. However, high concentration of EDC meant that the

sensitivity of detection for GSH would decrease. After a series of optimization trials, 200 mM

EDC was chosen as the suitable concentration.

In addition, the fluorescence intensity of the surface passivated CQDs is closely related to the

pH value of media. Fig. 4B shows that the fluorescence intensity of the synthesized CQDs reached

the maximum value in the buffer solution of pH 7.0. This pH is close to physiological pH, which

8
retains the activity of GSH. pH 7.0 substrate solution was chosen for the detection. Meanwhile, it

is worthwhile mentioning that these pH-sensitive surface passivated CQDs have great potential for

monitoring pH changes.

3.4 Measurement of GSH

To demonstrate the applicability of this developed fluorescence sensor for GSH detection, we

tested the response of this assay to different concentrations of GSH under optimal conditions.

According to Fig 5, the fluorescence signal of CQDs was gradually decreased as the concentration

of GSH varied from 0 to 200 μM. The plot of the fluorescence decreased efficiency (F0-F)/F0

against the concentration of GSH from 1.0 to 200 μM is shown in Fig 5 inset image. Meanwhile, a

good linear relationship over the range from 1.0 to 50 μM with a correlation coefficient square of

0.9951 was obtained (Fig 6A, F and F0 are fluorescence intensities in the presence and absence of

GSH, respectively), the calibration curve can be express as (F0-F)/F0=0.0322+5002.443C (C: mol

L-1). The limit detection (3σ/s) for GSH is 0.943 μM, where σ represents the standard deviation of

twenty blank measurements, and s is the slope of the calibration curve. This proposed sensing

procedure is comparable to or even better than the previous reports for GSH sensing (see Table 1).

Analysis of diluted human serum containing spike amounts of GSH showed that the recovery rate

is from 96.7%~101.8% with a RSD of around 3.17% (Table 2). This definitely demonstrate the

accuracy and reliability of the present fluorescence method for detecting GSH in human serum.

3.5 Effect of potential interfering substances

To demonstrate the selectivity of the proposed method, we investigate the fluorescence response

of surface passivated CQDs system to GSH at a concentration of 10 μM in presence of different

interfering substances including some electrolytes and amino acid. As shown in Fig 6B, the

fluorescence decrease ratio in the presence of 10 μM GSH were strikingly larger than that of other

amino electrotypes, acids. This result revealed that a wide range of electrolytes and amino acids

(without -SH) did not combined with EDC, which indicates that this proposed assay is highly

selective toward GSH over other nontarget samples. Although cysteine (Cys) and homocysteine

(HCys) also can cause fluorescence response to this system, whose content (μM levels) is much

9
lower than that of GSH (mM levels) in biological systems [36, 37].

3.6 Determination of GSH in human serum

To explore the feasibility of this proposed method in complicated biological environment, the

procedure was applied to detection GSH spiked in diluted human serum. Due to high

concentration of GSH in human serum (mM levels), the human serum was diluted so that original

GSH concentration could fall in the standard calibration curve of this method, which also proved

that our assay in feasible in real samples. After that, 5 μM, 10 μM, 15 μM GSH were spiked into

the diluted serum samples, respectively, and then was mixture the EDC solutions. Then the CQDs

was casted into the mixture and incubated for 10 min. All the data are based on three duplicated

measurements and shown in Table 2, the original GSH concentration in the human serum sample

was determined as 1.543 μM. Furthermore, recoveries of the known spiked amounts of GSH in

human serum were obtained in the range of 96.7%~101.8%. Apparently, successfully detection

GSH in human serum displays the promise of the fluorescence sensor for GSH measuring in

biological environments.

4. Conclusion

In this work, we developed a new fluorescence assay for GSH detection based on controlling the

surface passivation degree of the CQDs. This novel proposed method offered a linear GSH

detection range of 1.0-50.0 μM, with a detection limit of 0.943 μM. Taking full advantages of the

surface passivated CQDs, this assay shows remarkable properties including very fast and simple,

cost-effective as well as environmental-friendly, which suggest that this strategy has a great

potential to be utilized as an efficient tool for analyzing GSH level in biological environment,

health foods, pharmaceuticals, and cosmetics. Moreover, our work also provides a new way to

design the fluorescence sensors.

Acknowledgments

We are grateful for the financial support from the Natural Science Foundation of Guangdong

Province (No. 2014A030313480), the Science & Technology Project of Guangdong Province (No.

10
2013B030600001 & No. 2016A020223014) and the Guangdong High Education Fund of Science

and Technology Innovation (No. 2013KJCX0078).

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Figures:

Scheme 1. Schematic principle of GSH detection by using the surface passivated CQDs.

Figure 1. HRTEM (A and B) images of CQDs.

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Figure 2. AFM images of CQDs

Figure 3. (A) FTIR spectrum of CA (a), CQDs (b); (B) UV-vis absorption spectra: (a) GSH, (b) EDC, (c) the
synthesized CQDs, (d) the synthesized CQDs mixed with EDC solution, (e) the synthesized CQDs mixed with
EDC and GSH mixture solution.

Figure 4. (A) Relationship between fluorescence intensity and the time surface passivation treatment for CQDs; (B)
The effect of pH on the fluorescence CQDs in the presence of EDC.

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 Figure 5. (A) Fluorescence spectra of the CQDs/EDC system in the prescence of different concentration od GSH
(λexc=360 nm). Inset: Relation ship between relative fluorescence intensity and GSH.

Figure 6. (A) Calibration curve of relative fluorescence intensity and GSH; (B) Selectivity of the EDC/CQDs
system toward GSH. The concentration of GSH and interferences were both 10 μM.

Table 1 Comparison of different optical nanosensors for GSH determination

Methods Linear range LOD Note Ref
g-C3N4-MnO2 fluorescence 200-500 μM Not given Special equipment, sophisticated technique 38
g-CNQDs-Hg2+ fluorescence 0.16-16 μM 37 nM Time-consuming, complicated operation 26
AuNCs-Hg(II) fluorescence 0-250 nM 9.4 nM Low selectivity, costly material 39
CdSe-ZnS QDs-MV2+ fluorescence 5-250 μM 0.6 μM Complicated operation, 40
Fe3O4-ABTS-H2O2 colorimetry 3.0-30 μM Not given Time-consuming 41
EDC/CQDs fluorescence 1.0-50 μM 0.943 μM Fast, cost-effective, easy operation, This
environmental friendly work

Table 2 Determination of GSH in human serum

Sample Found in sample Added Total found (μM) Recovery (%) RSD (%)
(μM) (μM) n=3 n=3
Human serum 1 1.54 5.00 6.37 96.7 3.1
10.00 11.72 101.8 2.7
15.00 16.46 99.48 3.7
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Human serum samples were diluted 50 times by 10 mM pH 7.4 PBS before detection. Each data was given as the
average value obtained from three independent experiments

Highlights
 The carbon quantum dots with surface passivation treatment by

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC).

 The simple and environmental friendly synthesis process of carbon quantum dots.

 A novel, rapid and sensitive measurement for GSH without complicated procedures.

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