Colloids and Surfaces B: Biointerfaces 125 (2015) 90–95
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A sensitive biosensor for the fluorescence detection of the acetylcholinesterase reaction system based on carbon dots Xiangling Ren a,c,1 , Jianfei Wei a,b,1 , Jun Ren a , Li Qiang a,b , Fangqiong Tang a,∗ , Xianwei Meng a,∗ a Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, NO. 29, Zhongguancun East Road, Haidian District, Beijing 100190, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c The State Key Laboratory of Bioelectronics, Southeast University, 210096, PR China
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Article history: Received 9 September 2014 Received in revised form 28 October 2014 Accepted 5 November 2014 Available online 22 November 2014 Keywords: Carbon dots Fluorescence Biosensor Hydrogen peroxide Acetylcholinesterase
a b s t r a c t The carbon dots (C-dots) with high fluorescence quantum yield were prepared using hydrothermal method. C-dots have been adopted as probes for the fluorescence turn-off detection of H2 O2 based on the special sensibility for the hydroxyl radical. And then the biosensors for the detection of substrate and enzymes activities were established in the acetylcholinesterase reaction system, which were related to the production of H2 O2 . Specifically, the proposed fluorescent biosensor was successfully applied to detect the concentration of choline (in the range from 0.025 to 50 M) and acetylcholine (in the range from 0.050 to 50 M), and the activity of choline oxidase (in the range from 1 to 75 U/L) and acetylcholinesterase (1 to 80 U/L). These results showed a sensitive, universal, nontoxic and eco-friendly detecting technique has been developed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fluorescence detection method has simple, convenient, rapid and real-time detection and other advantages. A lot of researches focus on quantum dots (QDs) biosensors in recent years due to their novel properties [1–3]. However, the biological toxicity of QDs limits their practical application in clinical analysis. There has been growing interest in the development of nontoxic, biocompatible, and highly fluorescent nanoparticles for biological applications. Carbon dots (C-dots) are one of the most promising class of fluorescent nanoparticles, whose excellent low-toxicity and eco-friendly characteristics are similar to other popular carbon nanomaterials, such as the fullerene, the carbon nanotube and graphene [4,5]. Moreover, the preparation of C-dots does not need for tedious, stringent, and costly preparation steps and it is easy to prepare in large-scale [4]. Because of these inherent advantages, significant progress has been achieved in biological labeling, bioimaging and related biomedical applications using C-dots as optical labels [6], since its first discovery in 2006 [7]. Furthermore, C-dots can
∗ Corresponding author. Tel.: +86 10 82543521; fax: +86 10 62554670. E-mail addresses: [email protected] (F. Tang), [email protected] (X. Meng). 1 These authors have contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.11.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
be designed for sensors, such as sensors for nitrite [8], phosphate [9], glutathione [10], ␣-fetoprotein [11], glucose and metal ions [12–17], and so on. However, the detection of biomolecules using C-dots is still rather scarce. The design of novel biomolecules biosensor using C-dots as fluorescent probes are highly required. Acetylcholinesterase (AChE) is an enzyme regulating acetylcholine (ACh) neurotransmitter that can catalytically break down ACh at cholinergic synapses, resulting in the termination of synaptic transmission. The decrease of AChE makes individuals prone to various nerve disorders including Alzheimer’s and Parkinson’s disease. Organophosphorus pesticides (OPs), and nerve gases such as sarin may cause health threats to humans and animals, which stems from the inhibition of AChE [18,19]. The substances in AChE reaction system are also significant in clinical analysis. ACh is an essential messenger involved in neurotransmission in both the peripheral and central nervous systems. The decrease of this compound makes individuals prone to various nerve disorders including Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. Choline is frequently added in food as a nutrient for humans. Hence, the quantitative determination of the ACh, choline and activity of AChE is significant in clinical analysis, such as human serum, brain extracts, amniotic fluid and pharmaceutical products [20–23]. Herein, we investigate a fluorescent biosensor to sensitive quantitative analysis of the AChE reaction system based on the C-dots. The C-dots used in the fluorescent detection is restricted because
X. Ren et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 90–95
the fluorescence intensity of C-dots is less susceptible to many active substance, such as H2 O2 . We first prove that the fluorescence intensity of C-dots is quenched by hydroxyl radical. Based on the powerful hydroxyl radical production in Fenton reaction, a biosensor adopting C-dots as probes for the fluorescence turnoff detection of H2 O2 is fabricated, which is of great importance in environmental, clinical and pharmaceutical fields [24,25]. Following that, the biosensor is also constructed for the detection of the choline, ACh, and activity of choline oxidase (ChOx) and AChE, due to these enzyme reactions are related with H2 O2. 2. Materials and methods 2.1. Materials Gelatin (average molecular weight 100,000), choline, ACh and AChE (EC 3.1.1.7, from Electrophorus electricus, electric eel., 425.94 units/mg) were purchased from Sigma–Aldrich. ChOx (EC 1.1.3.17, from Alcaligenes Sp. Lyophilized powder, 13 units/mg) was obtained from J&K Scientific Ltd. Ammonium iron (II) sulfate hexahydrate, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Xilong Chemical Co. Ltd. All reagents used were of analytical grade and used without further purification. All solutions were prepared using ultrapure water and the ultrapure water (0.22 m) was produced using a Millipore-Q water system.
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were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ). 2.6. Detection of the activity of ChOx The different amount of enzyme was added into PB solution of choline (0.2 mM) and the mixture obtained was kept for 5 min before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ). 2.7. Detection of the concentration of ACh AChE was added into PB solution with different concentration of ACh and ChOx, and the mixture obtained was kept for a period of time before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ).
2.2. Synthesis of water-soluble C-dots using hydrothermal method
2.8. Detection of the activity of AChE
Briefly, 1.1 g gelatin were dissolved in 20 mL water and transferred into a 50 mL Teflon-lined stainless steel autoclave. After that, the autoclave was kept in 240 ◦ C for 24 h. The product can be used after filtered with cylinder filtration membrane filter (0.22 m). The concentration of the C-dots was 8 mM. Transmission electron microscopy (TEM) (Model JEM-2100 and JEM-2100F, JEOL) was used to characterize the surface morphology of the as-prepared C-dots.
The different amount of AChE was added into PB solution of acetylcholine (0.2 mM) and ChOx and the mixture obtained was kept for 5 min before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ).
2.3. Electron spin resonance (ESR)
3. Results and discussion
120 L samples were prepared by mixing 100 L of Phosphate Buffer (PB) solution containing different concentration of H2 O2 with 20 L of 0.2 M dimethyl pyridine N-oxide (DMPO) in a 1 mL plastic tube. The prepared sample was transferred to a quartz capillary tube and placed in the ESR cavity. DMPO was used to trap the hydroxyl radical (• OH) radicals to form the DMPO/• OH spin adduct. Each sample was UV-irradiated at 254 nm for 2 min, and spectra were recorded afterwards. The ESR spectra were obtained on a Bruker ESP 300E.
3.1. Characterization of C-dots
2.4. Detection of the concentration of hydrogen peroxide 2.5 L C-dots was diluted into 1 mL PB solution with different concentration of hydrogen peroxide. After that, the solution was adjusted to pH 3.0 with 1 M HCl and measured the fluorescence intensity, which was defined as the fluorescence intensity of the starting point (F0 ). After the ammonium iron (II) sulfate hexahydrate solution was added to the mixture obtained, the fluorescence intensity was measured at different time (Ft ). 2.5. Detection of the concentration of choline ChOx was added into PB solution with different concentration of choline and the mixture obtained was kept for a period of time before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots
Fig. 1A shows a TEM image of the as-prepared C-dots, showing that the products consist of small particles which are well separated from each other. The diameter of the C-dots is about 4–10 nm. The inset in Fig. 1A shows the obvious lattice structure confirming the formation of C-dots. To evaluate the optical properties of C-dots, the emission and excitation spectra (Fig. 1B) were investigated. The fluorescence excitation spectrum shows a peak centered at 330 nm upon emission at 410 nm. The inset in Fig. 1B shows that the as-prepared C-dots solution is transparent brown in color under visible light, while it emits a blue fluorescence under UV light (254 nm). The fluorescence quantum yield for the as-prepared C-dots is determined by calibrating against quinine sulphate in 0.1 M H2 SO4 solution (Fig. S1). The fluorescence quantum yield of quinine sulfate in 0.1 M H2 SO4 solution is 54% and the fluorescence quantum yield of C-dots is 23%. 3.2. The special sensibility of C-dots for the hydroxyl radical Further research shows the prepared C-dots are insensitive to H2 O2 . There is no apparent change of the fluorescence intensity of C-dots storing in H2 O2 solution. However, the fluorescence intensity of C-dots decreases apparently, when the solution containing H2 O2 is exposed to UV light. Fig. 2A shows the change of
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X. Ren et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 90–95
Fig. 1. (A) TEM image and (B) the photoluminescence spectrum of the C-dots. The insets in (B) are the photographs of the solution of C-dots under natural light (1) and UV light (254 nm) (2).
Fig. 2. (A) The effects of H2 O2 concentration on the fluorescence intensity of C-dots exposed to 254 nm UV light. (B) The effects of H2 O2 concentration on the formation of hydroxyl radical in the H2 O2 /UV system. Samples were mixture of 30 mM DMPO and various concentration of H2 O2 exposed to 254 nm UV light.
the fluorescence intensity of the C-dots solution containing different concentration of H2 O2 exposed to 254 nm UV light. It can be seen that the fluorescence intensity decreases linearly apparent with the increase of the concentration of H2 O2 . It is reasonable to assume that the production of chemically active species, i.e. • OH in the H2 O2 solution by UV light irradiation involves in the quenching process. The H2 O2 /UV/DMPO spin trap system is chosen to evaluate the • OH signal intensity by the electron spin-resonance spectroscopy (ESR) technique. According to Fig. 2B, it is found that the DMPO/• OH adduct signal intensity increases with the increase of the concentration of H2 O2 . Our findings show that • OH is present in the H2 O2 solution, and their number is inversely proportional to the fluorescence intensity of the C-dots. It is believed that the fluorescence intensity of C-dots is sensitive to the concentration of • OH.
3.3. Detection of H2 O2 based on C-dots The generation of • OH from H2 O2 could be more easily achieved in the presence of Fe2+ by Fenton reaction than UV light irritation. The Fe2+ reacts with H2 O2 could result in the formation of • OH (shown in Eq. (1)). Fe2+ + H2 O2 → Fe3+ + • OH + OH−
(1)
Based on the Fenton reaction and the special sensibility of Cdots for the • OH, the detection of H2 O2 is achieved. Fig. 3A shows the time-dependent fluorescence quenching of the C-dots in the presence of H2 O2 (30 M) and Fe2+ (0.34 mM). The results indicated the C-dots were quenched by • OH produced from Fenton reaction and the fluorescence intensity changed inconspicuously after 10 min, so we chose 10 min as the detection time. The control
Fig. 3. (A) Time-dependent fluorescence changes of C-dots in the presence of H2 O2 (30 M) and Fe2+ (0.34 mM) (0 min, 3 min, 5 min, 10 min, and 15 min), (B) The change of the fluorescence intensity of C-dots versus H2 O2 concentration in the presence of 0.34 mM Fe2+ .
X. Ren et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 90–95
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experiment, which was introduced to investigated the influence of Fe2+ and H2 O2 on the fluorescence response of C-dots, showed that the fluorescence intensity of C-dots changed inconspicuously in the presence of Fe2+ (0.5 mM) and H2 O2 (0.2 mM), separately (Fig. S2). We use F as a signal for the detection of H2 O2 [9], where F = F0 − Ft · F0 is the fluorescence intensity of the starting point, before the Fe2+ was introduced into the solution containing C-dots. After the Fe2+ was added to the mixture, the fluorescence intensity was measured at different time (Ft ). Fig. 3B shows the change of the fluorescence of C-dots with different concentration of H2 O2 for a fixed time interval of 10 min in the 0.05 M PB solution (which was adjusted to pH 3.0) containing 0.34 mM Fe2+ . It can be seen that the fluorescence intensity decreases linearly apparent with the increase of the concentration of H2 O2 . Hence, C-dots could be adopted as probes for the fluorescence turn-off detection of H2 O2 . 3.4. The detection of AChE reaction system Based on the detection of H2 O2 using C-dots as probes, we established biosensors for the detection of AChE reaction system using the fluorescent properties of C-dots (Fig. 4). The AChE reaction system includes two steps. The ACh can be catalyzed by AChE to choline (shown in Eq. (2)) and then choline was catalyzed by ChOx to H2 O2 (shown in Eq. (3)). AChE
Acetylcholine −→Choline + acetic acid ChOx
Choline + O2 + H2 O −→ betaine + 2H2 O2
(2) (3)
So hydrogen peroxide is generated from biocatalyzed reaction in the solution whose pH is suitable for enzyme firstly and then the pH for the solution is adjusted to 3.0, which is the optimum pH value for Fenton reaction [26–28]. Then, C-dots and Fe2+ is introduced into the solution and the H2 O2 is converted into • OH, which leads to turn-off of the fluorescence intensity of C-dots. The
Fig. 4. Scheme of the fluorescent biosensing platform for the detection of H2 O2 related substrates and enzymes based on C-dots.
change of fluorescence intensity before and after the addition of Fe2+ is determined. The proposed biosensor used for the detection of choline is investigated as an example. The sensitivity of the proposed choline biosensor is related to many factors, such as the concentration of ChOx and the time of enzyme reaction, so the experimental conditions are optimized for determination of the concentration of choline. We first investigated the influence of the enzyme concentration on F. The ChOx concentrations adopted in this study were 250, 500, 750 and 1000 U/L and the incubating time were constant (15 min) (Fig. S3A). The results clearly indicated that the optimum concentration of ChOx required for the detection of choline was 750 U/L. It was observed that there was significant increase in F when the ChOx concentration was increased below 750 U/L. When the ChOx concentration was greater than 750 U/L, there was no
Fig. 5. (A) Emission spectra of the C-dots solution with different concentration of choline (0, 0.025, 2.5, 10, 20, 30, 40, and 50 M); (B) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of choline; (C) Emission spectra of the C-dots solution with different concentration of ChOx (0, 1, 5, 25, 37.5, 62.5, 75 U/L); (D) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of ChOx.
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apparent change with the increase of ChOx concentration. We also investigated the influence of the time for enzyme reaction on F. In order to optimize the time, different times selected for the study were 5, 15, 30 and 45 min (Fig. S3B). It could be seen that the F increased gradually up to a maximum value with the increasing of time to 30 min. When the time for enzyme reaction was prolonged to 45 min, the F changed slightly compared with that of 30 min. As a result, the time of enzyme reaction was determined as 30 min. Furthermore, the time for Fenton reaction is 10 min. After the experimental conditions were optimized, the F at different concentration of choline was recorded, and was used to plot working curve. Fig. 5A shows the degree of quenching effect of the fluorescence of C-dots with different concentration of choline for a fixed time interval of 10 min. The resulting calibration curve for choline displayed good linearity for concentrations ranging from 0.025 to 50 M with a correlation coefficient of 0.9939 (Fig. 5B). The detection limit was as low as 0.025 M, which is lower than that for the choline biosensor adopting other fluorescent materials as probes [20,21]. The proposed biosensor also can be used to detect the activity of ChOx. In order to detect the activity of the ChOx, the concentration of choline has to be constant. Fig. 5C shows the degree of quenching
effect of the fluorescence of C-dots with different concentration of ChOx for a fixed time interval of 10 min, while the concentration of choline was 0.2 mM. The resulting calibration curve for ChOx displayed good linearity for concentrations ranging from 1 to 75 U/L with a correlation coefficient of 0.9975 (Fig. 5D). We also constructed a biosensor for the detection of ACh or AChE activity in the similar way. Fig. 6A shows the degree of fluorescence quenching effect of C-dots interaction with enzyme reaction solution with different concentrations of ACh, 750 U/L ChOx, and 1500 U/L AChE. Fig. 6B shows good linearity for concentrations ranging from 0.05 to 50 M with a correlation coefficient of 0.9987. The detection of AChE also achieved based on the fluorescence quenching effect of C-dots interaction with enzyme reaction solution with different concentrations of AChE, 0.2 mM ACh, and 750 U/L ChOx (Fig. 6C). Fig. 6D shows good linearity for concentrations ranging from 1 to 80 U/L with a correlation coefficient of 0.9937. 3.5. The reproducibility and anti-interference ability The reproducibility study is one of the most important evaluations for the biosensor. The reproducibility was evaluated by
Fig. 6. (A) Emission spectra of the C-dots solution with different concentration of ACh (0, 0.05, 2.5, 5, 10, 20, 30, 40, and 50 M); (B) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of ACh; (C) Emission spectra of the C-dots solution with different concentration of AChE (0, 1, 5, 10, 20, 30, 40, 50, 60, 80 U/L); (D) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of AChE.
Fig. 7. The reproducibility (A) and anti-interference ability (B) of the biosensor.
X. Ren et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 90–95
comparing the different response of the determination of 20 M ACh (see Fig. 7A). The RSD (relative standard deviation) was 4.23% for 6 assays. Fig. 7B shows the F of 20 M ACh (see “C” in Fig. 7B). Then the additions of 20 M lactic acid (“C + LA” in Fig. 7B), 20 M ascorbic acid (“C + AA” in Fig. 7B), 20 M KCl (see “C + KCl” in Fig. 7B), and 20 M sodium citrate (see “C + SC” in Fig. 7B) did not cause observable changes of theF of the biosensor. It was proved that the biosensor can provide credible anti-interference ability. 4. Conclusions In summary, we have successfully introduced a facile optical method for the detection H2 O2 and H2 O2 -related substrate concentration and enzyme activity through the luminescence quenching of C-dots by • OH. The high fluorescence quantum yield (ca. 23%) C-dots are prepared using hydrothermal method involving gelatin as precursor. It is proved that the as-prepared C-dots are sensitive to • OH. Based on this finding, we established an efficient fluorescent biosensing platform using the Fenton reaction. Specifically, the proposed fluorescent biosensing platform is successfully applied to detect the concentration of H2 O2 , choline, ACh, and the activity of ChOx and AChE. In comparison with conventional QDs-based sensing method [20,29–36], our platform has two advantages. First, the C-dots have low-toxicity and eco-friendly characteristics, which are particular interests and significances for the design, preparation and application of nanomaterials. Secondly, the excellent photostability of the C-dots will enhance the anti-interference performance of the sensors. Therefore, the simplicity, low-toxicity, anti-interference, and eco-friendly characteristics of the analytic method, support potential future applications of the proposed fluorescent biosensing platform. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 61178035, 61171049, and 81171454), National Hi-Tech. Research and Development Program of China (No. 2011AA02A114), and the State Key Laboratory of Bioelectronics of Southeast University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.11.007.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A sensitive biosensor for the fluorescence detection of the acetylcholinesterase reaction system based on carbon dots Xiangling Ren a,c,1 , Jianfei Wei a,b,1 , Jun Ren a , Li Qiang a,b , Fangqiong Tang a,∗ , Xianwei Meng a,∗ a Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, NO. 29, Zhongguancun East Road, Haidian District, Beijing 100190, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c The State Key Laboratory of Bioelectronics, Southeast University, 210096, PR China
a r t i c l e
i n f o
Article history: Received 9 September 2014 Received in revised form 28 October 2014 Accepted 5 November 2014 Available online 22 November 2014 Keywords: Carbon dots Fluorescence Biosensor Hydrogen peroxide Acetylcholinesterase
a b s t r a c t The carbon dots (C-dots) with high fluorescence quantum yield were prepared using hydrothermal method. C-dots have been adopted as probes for the fluorescence turn-off detection of H2 O2 based on the special sensibility for the hydroxyl radical. And then the biosensors for the detection of substrate and enzymes activities were established in the acetylcholinesterase reaction system, which were related to the production of H2 O2 . Specifically, the proposed fluorescent biosensor was successfully applied to detect the concentration of choline (in the range from 0.025 to 50 M) and acetylcholine (in the range from 0.050 to 50 M), and the activity of choline oxidase (in the range from 1 to 75 U/L) and acetylcholinesterase (1 to 80 U/L). These results showed a sensitive, universal, nontoxic and eco-friendly detecting technique has been developed. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fluorescence detection method has simple, convenient, rapid and real-time detection and other advantages. A lot of researches focus on quantum dots (QDs) biosensors in recent years due to their novel properties [1–3]. However, the biological toxicity of QDs limits their practical application in clinical analysis. There has been growing interest in the development of nontoxic, biocompatible, and highly fluorescent nanoparticles for biological applications. Carbon dots (C-dots) are one of the most promising class of fluorescent nanoparticles, whose excellent low-toxicity and eco-friendly characteristics are similar to other popular carbon nanomaterials, such as the fullerene, the carbon nanotube and graphene [4,5]. Moreover, the preparation of C-dots does not need for tedious, stringent, and costly preparation steps and it is easy to prepare in large-scale [4]. Because of these inherent advantages, significant progress has been achieved in biological labeling, bioimaging and related biomedical applications using C-dots as optical labels [6], since its first discovery in 2006 [7]. Furthermore, C-dots can
∗ Corresponding author. Tel.: +86 10 82543521; fax: +86 10 62554670. E-mail addresses: [email protected] (F. Tang), [email protected] (X. Meng). 1 These authors have contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.11.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
be designed for sensors, such as sensors for nitrite [8], phosphate [9], glutathione [10], ␣-fetoprotein [11], glucose and metal ions [12–17], and so on. However, the detection of biomolecules using C-dots is still rather scarce. The design of novel biomolecules biosensor using C-dots as fluorescent probes are highly required. Acetylcholinesterase (AChE) is an enzyme regulating acetylcholine (ACh) neurotransmitter that can catalytically break down ACh at cholinergic synapses, resulting in the termination of synaptic transmission. The decrease of AChE makes individuals prone to various nerve disorders including Alzheimer’s and Parkinson’s disease. Organophosphorus pesticides (OPs), and nerve gases such as sarin may cause health threats to humans and animals, which stems from the inhibition of AChE [18,19]. The substances in AChE reaction system are also significant in clinical analysis. ACh is an essential messenger involved in neurotransmission in both the peripheral and central nervous systems. The decrease of this compound makes individuals prone to various nerve disorders including Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. Choline is frequently added in food as a nutrient for humans. Hence, the quantitative determination of the ACh, choline and activity of AChE is significant in clinical analysis, such as human serum, brain extracts, amniotic fluid and pharmaceutical products [20–23]. Herein, we investigate a fluorescent biosensor to sensitive quantitative analysis of the AChE reaction system based on the C-dots. The C-dots used in the fluorescent detection is restricted because
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the fluorescence intensity of C-dots is less susceptible to many active substance, such as H2 O2 . We first prove that the fluorescence intensity of C-dots is quenched by hydroxyl radical. Based on the powerful hydroxyl radical production in Fenton reaction, a biosensor adopting C-dots as probes for the fluorescence turnoff detection of H2 O2 is fabricated, which is of great importance in environmental, clinical and pharmaceutical fields [24,25]. Following that, the biosensor is also constructed for the detection of the choline, ACh, and activity of choline oxidase (ChOx) and AChE, due to these enzyme reactions are related with H2 O2. 2. Materials and methods 2.1. Materials Gelatin (average molecular weight 100,000), choline, ACh and AChE (EC 3.1.1.7, from Electrophorus electricus, electric eel., 425.94 units/mg) were purchased from Sigma–Aldrich. ChOx (EC 1.1.3.17, from Alcaligenes Sp. Lyophilized powder, 13 units/mg) was obtained from J&K Scientific Ltd. Ammonium iron (II) sulfate hexahydrate, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Xilong Chemical Co. Ltd. All reagents used were of analytical grade and used without further purification. All solutions were prepared using ultrapure water and the ultrapure water (0.22 m) was produced using a Millipore-Q water system.
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were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ). 2.6. Detection of the activity of ChOx The different amount of enzyme was added into PB solution of choline (0.2 mM) and the mixture obtained was kept for 5 min before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ). 2.7. Detection of the concentration of ACh AChE was added into PB solution with different concentration of ACh and ChOx, and the mixture obtained was kept for a period of time before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ).
2.2. Synthesis of water-soluble C-dots using hydrothermal method
2.8. Detection of the activity of AChE
Briefly, 1.1 g gelatin were dissolved in 20 mL water and transferred into a 50 mL Teflon-lined stainless steel autoclave. After that, the autoclave was kept in 240 ◦ C for 24 h. The product can be used after filtered with cylinder filtration membrane filter (0.22 m). The concentration of the C-dots was 8 mM. Transmission electron microscopy (TEM) (Model JEM-2100 and JEM-2100F, JEOL) was used to characterize the surface morphology of the as-prepared C-dots.
The different amount of AChE was added into PB solution of acetylcholine (0.2 mM) and ChOx and the mixture obtained was kept for 5 min before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots were added to the obtained mixture and measured the fluorescence intensity (F0 ). What calls for special attention was the volume of the mixture is 1 mL. After the ammonium iron (II) sulfate hexahydrate solution (0.34 mM) was added to the mixture obtained, the fluorescence intensity was measured at 10 min (Ft ).
2.3. Electron spin resonance (ESR)
3. Results and discussion
120 L samples were prepared by mixing 100 L of Phosphate Buffer (PB) solution containing different concentration of H2 O2 with 20 L of 0.2 M dimethyl pyridine N-oxide (DMPO) in a 1 mL plastic tube. The prepared sample was transferred to a quartz capillary tube and placed in the ESR cavity. DMPO was used to trap the hydroxyl radical (• OH) radicals to form the DMPO/• OH spin adduct. Each sample was UV-irradiated at 254 nm for 2 min, and spectra were recorded afterwards. The ESR spectra were obtained on a Bruker ESP 300E.
3.1. Characterization of C-dots
2.4. Detection of the concentration of hydrogen peroxide 2.5 L C-dots was diluted into 1 mL PB solution with different concentration of hydrogen peroxide. After that, the solution was adjusted to pH 3.0 with 1 M HCl and measured the fluorescence intensity, which was defined as the fluorescence intensity of the starting point (F0 ). After the ammonium iron (II) sulfate hexahydrate solution was added to the mixture obtained, the fluorescence intensity was measured at different time (Ft ). 2.5. Detection of the concentration of choline ChOx was added into PB solution with different concentration of choline and the mixture obtained was kept for a period of time before adjusting the solution pH to 3.0 with 1 M HCl. 2.5 L C-dots
Fig. 1A shows a TEM image of the as-prepared C-dots, showing that the products consist of small particles which are well separated from each other. The diameter of the C-dots is about 4–10 nm. The inset in Fig. 1A shows the obvious lattice structure confirming the formation of C-dots. To evaluate the optical properties of C-dots, the emission and excitation spectra (Fig. 1B) were investigated. The fluorescence excitation spectrum shows a peak centered at 330 nm upon emission at 410 nm. The inset in Fig. 1B shows that the as-prepared C-dots solution is transparent brown in color under visible light, while it emits a blue fluorescence under UV light (254 nm). The fluorescence quantum yield for the as-prepared C-dots is determined by calibrating against quinine sulphate in 0.1 M H2 SO4 solution (Fig. S1). The fluorescence quantum yield of quinine sulfate in 0.1 M H2 SO4 solution is 54% and the fluorescence quantum yield of C-dots is 23%. 3.2. The special sensibility of C-dots for the hydroxyl radical Further research shows the prepared C-dots are insensitive to H2 O2 . There is no apparent change of the fluorescence intensity of C-dots storing in H2 O2 solution. However, the fluorescence intensity of C-dots decreases apparently, when the solution containing H2 O2 is exposed to UV light. Fig. 2A shows the change of
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Fig. 1. (A) TEM image and (B) the photoluminescence spectrum of the C-dots. The insets in (B) are the photographs of the solution of C-dots under natural light (1) and UV light (254 nm) (2).
Fig. 2. (A) The effects of H2 O2 concentration on the fluorescence intensity of C-dots exposed to 254 nm UV light. (B) The effects of H2 O2 concentration on the formation of hydroxyl radical in the H2 O2 /UV system. Samples were mixture of 30 mM DMPO and various concentration of H2 O2 exposed to 254 nm UV light.
the fluorescence intensity of the C-dots solution containing different concentration of H2 O2 exposed to 254 nm UV light. It can be seen that the fluorescence intensity decreases linearly apparent with the increase of the concentration of H2 O2 . It is reasonable to assume that the production of chemically active species, i.e. • OH in the H2 O2 solution by UV light irradiation involves in the quenching process. The H2 O2 /UV/DMPO spin trap system is chosen to evaluate the • OH signal intensity by the electron spin-resonance spectroscopy (ESR) technique. According to Fig. 2B, it is found that the DMPO/• OH adduct signal intensity increases with the increase of the concentration of H2 O2 . Our findings show that • OH is present in the H2 O2 solution, and their number is inversely proportional to the fluorescence intensity of the C-dots. It is believed that the fluorescence intensity of C-dots is sensitive to the concentration of • OH.
3.3. Detection of H2 O2 based on C-dots The generation of • OH from H2 O2 could be more easily achieved in the presence of Fe2+ by Fenton reaction than UV light irritation. The Fe2+ reacts with H2 O2 could result in the formation of • OH (shown in Eq. (1)). Fe2+ + H2 O2 → Fe3+ + • OH + OH−
(1)
Based on the Fenton reaction and the special sensibility of Cdots for the • OH, the detection of H2 O2 is achieved. Fig. 3A shows the time-dependent fluorescence quenching of the C-dots in the presence of H2 O2 (30 M) and Fe2+ (0.34 mM). The results indicated the C-dots were quenched by • OH produced from Fenton reaction and the fluorescence intensity changed inconspicuously after 10 min, so we chose 10 min as the detection time. The control
Fig. 3. (A) Time-dependent fluorescence changes of C-dots in the presence of H2 O2 (30 M) and Fe2+ (0.34 mM) (0 min, 3 min, 5 min, 10 min, and 15 min), (B) The change of the fluorescence intensity of C-dots versus H2 O2 concentration in the presence of 0.34 mM Fe2+ .
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experiment, which was introduced to investigated the influence of Fe2+ and H2 O2 on the fluorescence response of C-dots, showed that the fluorescence intensity of C-dots changed inconspicuously in the presence of Fe2+ (0.5 mM) and H2 O2 (0.2 mM), separately (Fig. S2). We use F as a signal for the detection of H2 O2 [9], where F = F0 − Ft · F0 is the fluorescence intensity of the starting point, before the Fe2+ was introduced into the solution containing C-dots. After the Fe2+ was added to the mixture, the fluorescence intensity was measured at different time (Ft ). Fig. 3B shows the change of the fluorescence of C-dots with different concentration of H2 O2 for a fixed time interval of 10 min in the 0.05 M PB solution (which was adjusted to pH 3.0) containing 0.34 mM Fe2+ . It can be seen that the fluorescence intensity decreases linearly apparent with the increase of the concentration of H2 O2 . Hence, C-dots could be adopted as probes for the fluorescence turn-off detection of H2 O2 . 3.4. The detection of AChE reaction system Based on the detection of H2 O2 using C-dots as probes, we established biosensors for the detection of AChE reaction system using the fluorescent properties of C-dots (Fig. 4). The AChE reaction system includes two steps. The ACh can be catalyzed by AChE to choline (shown in Eq. (2)) and then choline was catalyzed by ChOx to H2 O2 (shown in Eq. (3)). AChE
Acetylcholine −→Choline + acetic acid ChOx
Choline + O2 + H2 O −→ betaine + 2H2 O2
(2) (3)
So hydrogen peroxide is generated from biocatalyzed reaction in the solution whose pH is suitable for enzyme firstly and then the pH for the solution is adjusted to 3.0, which is the optimum pH value for Fenton reaction [26–28]. Then, C-dots and Fe2+ is introduced into the solution and the H2 O2 is converted into • OH, which leads to turn-off of the fluorescence intensity of C-dots. The
Fig. 4. Scheme of the fluorescent biosensing platform for the detection of H2 O2 related substrates and enzymes based on C-dots.
change of fluorescence intensity before and after the addition of Fe2+ is determined. The proposed biosensor used for the detection of choline is investigated as an example. The sensitivity of the proposed choline biosensor is related to many factors, such as the concentration of ChOx and the time of enzyme reaction, so the experimental conditions are optimized for determination of the concentration of choline. We first investigated the influence of the enzyme concentration on F. The ChOx concentrations adopted in this study were 250, 500, 750 and 1000 U/L and the incubating time were constant (15 min) (Fig. S3A). The results clearly indicated that the optimum concentration of ChOx required for the detection of choline was 750 U/L. It was observed that there was significant increase in F when the ChOx concentration was increased below 750 U/L. When the ChOx concentration was greater than 750 U/L, there was no
Fig. 5. (A) Emission spectra of the C-dots solution with different concentration of choline (0, 0.025, 2.5, 10, 20, 30, 40, and 50 M); (B) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of choline; (C) Emission spectra of the C-dots solution with different concentration of ChOx (0, 1, 5, 25, 37.5, 62.5, 75 U/L); (D) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of ChOx.
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apparent change with the increase of ChOx concentration. We also investigated the influence of the time for enzyme reaction on F. In order to optimize the time, different times selected for the study were 5, 15, 30 and 45 min (Fig. S3B). It could be seen that the F increased gradually up to a maximum value with the increasing of time to 30 min. When the time for enzyme reaction was prolonged to 45 min, the F changed slightly compared with that of 30 min. As a result, the time of enzyme reaction was determined as 30 min. Furthermore, the time for Fenton reaction is 10 min. After the experimental conditions were optimized, the F at different concentration of choline was recorded, and was used to plot working curve. Fig. 5A shows the degree of quenching effect of the fluorescence of C-dots with different concentration of choline for a fixed time interval of 10 min. The resulting calibration curve for choline displayed good linearity for concentrations ranging from 0.025 to 50 M with a correlation coefficient of 0.9939 (Fig. 5B). The detection limit was as low as 0.025 M, which is lower than that for the choline biosensor adopting other fluorescent materials as probes [20,21]. The proposed biosensor also can be used to detect the activity of ChOx. In order to detect the activity of the ChOx, the concentration of choline has to be constant. Fig. 5C shows the degree of quenching
effect of the fluorescence of C-dots with different concentration of ChOx for a fixed time interval of 10 min, while the concentration of choline was 0.2 mM. The resulting calibration curve for ChOx displayed good linearity for concentrations ranging from 1 to 75 U/L with a correlation coefficient of 0.9975 (Fig. 5D). We also constructed a biosensor for the detection of ACh or AChE activity in the similar way. Fig. 6A shows the degree of fluorescence quenching effect of C-dots interaction with enzyme reaction solution with different concentrations of ACh, 750 U/L ChOx, and 1500 U/L AChE. Fig. 6B shows good linearity for concentrations ranging from 0.05 to 50 M with a correlation coefficient of 0.9987. The detection of AChE also achieved based on the fluorescence quenching effect of C-dots interaction with enzyme reaction solution with different concentrations of AChE, 0.2 mM ACh, and 750 U/L ChOx (Fig. 6C). Fig. 6D shows good linearity for concentrations ranging from 1 to 80 U/L with a correlation coefficient of 0.9937. 3.5. The reproducibility and anti-interference ability The reproducibility study is one of the most important evaluations for the biosensor. The reproducibility was evaluated by
Fig. 6. (A) Emission spectra of the C-dots solution with different concentration of ACh (0, 0.05, 2.5, 5, 10, 20, 30, 40, and 50 M); (B) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of ACh; (C) Emission spectra of the C-dots solution with different concentration of AChE (0, 1, 5, 10, 20, 30, 40, 50, 60, 80 U/L); (D) The corresponding change of fluorescence intensity of C-dots solution versus the concentration of AChE.
Fig. 7. The reproducibility (A) and anti-interference ability (B) of the biosensor.
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comparing the different response of the determination of 20 M ACh (see Fig. 7A). The RSD (relative standard deviation) was 4.23% for 6 assays. Fig. 7B shows the F of 20 M ACh (see “C” in Fig. 7B). Then the additions of 20 M lactic acid (“C + LA” in Fig. 7B), 20 M ascorbic acid (“C + AA” in Fig. 7B), 20 M KCl (see “C + KCl” in Fig. 7B), and 20 M sodium citrate (see “C + SC” in Fig. 7B) did not cause observable changes of theF of the biosensor. It was proved that the biosensor can provide credible anti-interference ability. 4. Conclusions In summary, we have successfully introduced a facile optical method for the detection H2 O2 and H2 O2 -related substrate concentration and enzyme activity through the luminescence quenching of C-dots by • OH. The high fluorescence quantum yield (ca. 23%) C-dots are prepared using hydrothermal method involving gelatin as precursor. It is proved that the as-prepared C-dots are sensitive to • OH. Based on this finding, we established an efficient fluorescent biosensing platform using the Fenton reaction. Specifically, the proposed fluorescent biosensing platform is successfully applied to detect the concentration of H2 O2 , choline, ACh, and the activity of ChOx and AChE. In comparison with conventional QDs-based sensing method [20,29–36], our platform has two advantages. First, the C-dots have low-toxicity and eco-friendly characteristics, which are particular interests and significances for the design, preparation and application of nanomaterials. Secondly, the excellent photostability of the C-dots will enhance the anti-interference performance of the sensors. Therefore, the simplicity, low-toxicity, anti-interference, and eco-friendly characteristics of the analytic method, support potential future applications of the proposed fluorescent biosensing platform. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 61178035, 61171049, and 81171454), National Hi-Tech. Research and Development Program of China (No. 2011AA02A114), and the State Key Laboratory of Bioelectronics of Southeast University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.11.007.
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