Highly sensitive, stable, and precise detection of dopamine with carbon dots/tyrosinase hybrid as fluorescent probe

Abstract

A carbon dots/tyrosinase (CDs/TYR) hybrid as a low-cost fluorescent probe for the detection of dopamine exhibits high sensitivity, stability, and precision. The detection limit of dopamine is as low as 6.0 × 10⁻⁸ mol L⁻¹, and the wide detection range is from 1.318 × 10⁻⁴ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹. This kind of fluorescent probe did not need enzyme immobilization and modification, and the experiment results could be revealed as soon as the probe–sample incubation was completed. More importantly, these test results are comparable to that of the present clinical fluorescence detection and high-performance liquid chromatography (HPLC), and the results show that the three methods agreed well with each other.

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1. Introduction

Dopamine is an important neurotransmitter in the brain,¹ which is a chemical released by nerve cells for send signals to other nerve cells. So the dopamine is implicated in locomotion and motivation.² In spite of it being a simple organic chemical in the catecholamine and phenethylamine families, dopamine plays a number of vital roles in the brains and bodies of animals. A deficiency of dopamine in the brain can cause pathogenesis of neurological disorders such as schizophrenia, Huntington's disease, and Parkinson's disease.^3–5 Traditionally, high-performance liquid chromatography (HPLC)^6,7 and capillary electrophoresis techniques⁸ have been widely used for the determination of dopamine. However, these methods need complicated pre-concentration, time-consuming steps, high-cost instruments, and sophisticated equipment, which considerably limit their wide applicability. Compared with these conventional analytical techniques, the fluorescent biosensor,⁹ as a new class of detection method, has attracted a great deal of attention for the detection of dopamine with high sensitivity. The semiconductor-based quantum dots (QDs) have been proven as powerful inorganic fluorescent probes.¹⁰ For example, Yuan et al. used CdTe quantum dots as a fluorescent indicator to test for dopamine,¹¹ but the potential high toxicity and poor biocompatibility were unavoidable. Therefore, it is highly desirable to identify a kind of low-toxicity and environmentally friendly material to use instead of semiconductor QDs. However, to further improve the sensitivity, specificity and simplicity of testing procedures, a simple, highly sensitive, stable, and precise detection method for dopamine is still a huge challenge.¹²

Fluorescent amorphous carbon dots (CDs) are superior in terms of high aqueous solubility, robust chemical inertness, easy functionalization, high resistance to photobleaching, low toxicity, good biocompatibility, and using inexpensive and abundant raw materials.^13–18 Based on these advantages, CDs play a large role in a variety of bio-applications. To date, there have been many reports, suggesting that CDs should be an excellent candidate for biological imaging,¹⁹ but there have been few studies on the sensitive detection of dopamine with CDs as a high-sensitivity fluorescent biosensor.

Here, we report a CDs/tyrosinase (CDs/TYR) hybrid (synthesis process shown in Scheme 1), as a fluorescent probe which exhibits high efficiency and is sensitive, stable, precise and inexpensive, for the detection of dopamine (DOPA). In the present system, dopamine can be oxidized by TYR to form dopamine quinone, which can subsequently be quickly oxidized to obtain dopaminechrome in phosphate buffer (PB). During the process of dopamine oxidation, the fluorescence of CDs could be efficiently quenched because of their excellent electron-accepting properties. Using the CDs/TYR hybrid as a fluorescent probe, the detection limit of dopamine is about 6.0 × 10⁻⁸ mol L⁻¹ and the linear detection range is very wide, i.e. from 1.318 × 10⁻⁴ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹. Our further experiments confirmed that this kind of CDs/TYR hybrid fluorescent probe without enzyme immobilization and modification could give the test results as soon as the probe–sample incubation was completed. Notably, these quantitative detection results of clinical samples are comparable to those of the present clinical fluorescence detection and HPLC methods.

Scheme 1 Synthesis process of CDs/TYR hybrids and as fluorescent probes in the detection of DOPA.

2. Experimental section

2.1 Instruments and chemicals

Transmission electron microscopy (TEM) images were carried out using a FEI/Philips Tecnai 12 BioTWIN TEM. The UV-visible spectra were measured with an Agilent 8453 UV-vis diode array spectrophotometer, while the electron-accepting and -donating capacity of CDs study was conducted with a Horiba Jobin Yvon FluoroMax-4 luminescence spectrometer. The Fourier transform infrared (FTIR) spectrum of CDs was obtained with a Varian Spectrum GX spectrometer. PL study was carried out on a Fluorolog-TCSPC luminescence spectrometer. The human urine samples used the HP Agilent 1100 series HPLC.

Dopamine and TYR (845 U mg⁻¹) were purchased from Worthington Biochemical Co. Ltd (USA). Carbon rods (diameter 5 mm) were purchased from Shanghai Moyang electronic and carbon Co. Ltd. (Shanghai, China). Anhydrous ethanol (analytical grade), polyethylene glycol (PEG-200), 2,4-dinitrotoluene and N,N-diethylaniline were purchased from Sigma-Aldrich. All other chemicals used in this work were of analytical grade. Except when specifically stated, the detection buffer was PB buffer (pH = 6.8, 0.05 M sodium phosphate). Milli-Q ultrapure water (Millipore, ≥18 MΩ cm) was used throughout.

2.2 Preparation of the CDs

All the chemicals were purchased from Sigma-Aldrich. In a typical experiment, 3.0 g NaOH was added to a mixed solution of 50 mL PEG-200 and 10 mL distilled water to form a clear solution. Then, the mixed solution would become brown after electrolysis (voltage is 15 V) for 6 h. To remove the impurities, the crude solution was given dialysis treatment using a semi-permeable membrane (MWCO 1000). Finally, the obtained solution changed to a golden yellow color, indicating that we produced the CDs.

2.3 Adsorption equilibrium measurements

In order to study the proton (H⁺) adsorption capacity of CDs, the concentration of 0.005 M HCl solution was selected to investigate the adsorption behavior of CDs. Due to the excellent water solubility of CDs, the adsorption experiments were conducted with a dialysis method. The CDs solution was dialyzed using a semi-permeable membrane (MWCO 1000) in a 600 mL beaker, and the dialysate was 0.005 M HCl (500 mL). Notably, the CDs were treated previously by the dialysis method before use; therefore, the CDs would not dialyze out of the semi-permeable membrane. If CDs display good adsorption behaviour for H⁺, H⁺ would gradually cross the semi-permeable membrane and dialyze into the CDs solution. After stirring on a shaker for predetermined time intervals, the residual concentration of HCl solution was determined by titrating with 0.005 M NaOH solution.

2.4 Analytes sensing by PL detection

For all experiments, the tests were repeated at least three times in order to ensure the accuracy of the measurement. All the PL spectra were recorded on the same fluorescence spectrophotometer. The excitation wavelength was fixed at 370 nm with a scan rate of 5 nm s⁻¹. For the detection of DOPA, 500 μL CDs solution was mixed with 20 mL (0.2 mg mL⁻¹) TYR solution, then the DOPA solutions with different concentrations were added into the 2 mL CDs/TYR mixture. After that, the mixed solution was diluted with PB to 4 mL and incubated at 40 °C for 40 min. Finally, the mixture was measured by a fluorescence spectrophotometer.

2.5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Mixtures of CDs/TYR hybrids and the CDs were loaded onto SDS polyacrylamide gels (20%) and electrophoresed at 50 mA under an air atmosphere. Imaging of gels was carried out by UV illumination and visible light.

3. Results and discussion

3.1 The structure and properties of CDs

Fig. 1a shows the transmission electron microscopy (TEM) image of the obtained CDs, revealing that these CDs are uniform and monodisperse with a diameter of 3.0 ± 0.5 nm. These CDs can be well dispersed in water without further ultrasonic treatment. The inset image in Fig. 1a is the digital photograph of the CDs aqueous solution under visible and UV (at 365 nm excitation) light, respectively, and the green fluorescent color is easily observed by the naked eye. Fourier transform infrared (FTIR) spectroscopy was employed to identify the functional groups of the as-prepared CDs (Fig. 1b, red line). The peaks around 3400, 1640, and 1080 cm⁻¹ correspond to the vibrations of O–H, C=O, and C–O bonds, which indicates that the surfaces of the obtained CDs were occupied by hydrophilic groups (–OH and –COOH) from PEG. These hydrophilic groups led to the good water dispersibility of CDs, which greatly widens their applications in aqueous systems. The UV-vis absorption spectrum of CDs (Fig. 1b, black line) shows that the CDs have an absorption peak around 251–270 nm. The absorption band represents the typical absorption of an aromatic pi system, which is similar to that of polycyclic aromatic hydrocarbons.²⁰

Fig. 1 (a) Typical TEM image of CDs, insets are the digital photos of CDs under visible and UV (365 nm) light. (b) UV-vis absorption and FTIR spectra of CDs. The PL spectra of CDs with different excitation wavelengths in water (c) and PB (pH = 6.8) (d).

Fig. 1c shows the photoluminescence (PL) spectra of CDs with different excitation wavelengths in water. The strongest emission peak was located at 500 nm with the excitation wavelength at 400 nm. By increasing the excitation wavelength from 300 to 420 nm, the emission spectra from CDs were gradually red-shifted to higher wavelengths accompanied by decreased fluorescence intensity, which is similar to the previous reports.^20–23 In the PL test, the detailed PL spectra of CDs in PB (pH = 6.8) were also investigated with different excitation wavelengths. As shown in Fig. 1d, in PB, the optimal excitation wavelength was obtained at 370 nm with the strongest emission peak at 460 nm. These results indicated that the CDs possess stable and strong fluorescence in PB solution, and may serve as an excellent fluorescent probe for biodetection.

To further confirm the fluorescence stability of CDs, the effects of different temperature, pH, stabilization time and ionic strength in NaCl aqueous solution on the PL intensity of CDs were investigated in PB solution. As shown in Fig. 2a, when the temperature increased from 10 °C to 60 °C, it was found that the PL intensity of CDs was almost the same, which indicated that the temperature has no obvious effect on the PL intensity of CDs. Fig. 2b reveals the effect of different pH on the fluorescence intensity of CDs and the fluorescence intensity was pH-dependent. Under strongly acidic (pH < 6.0) or alkaline (pH > 9.0) conditions, the PL of CDs was quenched, whereas the CDs showed the strongest PL emission intensity when the pH value was 8.0, which is similar to the physiological pH environment (pH = 7–8). These results indicate that the CDs with excellent fluorescence properties can be regarded as good candidates for potential biological applications. Fig. 2c displays the effect of stabilization time on the PL intensity of CDs, showing that the CDs were very stable over 6 h in air. Fig. 2d shows the influence of different ionic strength in NaCl aqueous solution on the PL intensity of CDs; moreover, as shown, there was no obvious change in PL intensity even in an aqueous solution with high ionic strength (2 M NaCl).

Fig. 2 The effects of different temperature (a), pH (b), stabilization time (c) and ionic strength in NaCl aqueous solution (d) on the normalized fluorescence intensity of CDs in PB solution.

In the experiments, the PL quantum yield of CDs was about 38% and the result was obtained according to the Williams method.²⁴ Typically, the quantum yield of CDs was measured according to the established procedure using quinine sulfate in 0.10 M H₂SO₄ solution as the standard. The absorbance was measured on a Perkin Elmer LS 55 spectrophotometer. Absolute values are calculated according to the following equation:

where Q is the quantum yield, m is the slope of the plot of integrated fluorescence intensity vs. absorbance and n is the refractive index (taken here as 1.33, the refractive index of distilled water). The subscript refers to the reference fluorophore, quinine sulphate solution. In order to minimize re-absorption effects, absorbance in the 1 cm quartz cuvette was kept below 0.15 at the excitation wavelength of 375 nm. All the above results revealed that the fluorescence of CDs is extremely stable under high temperature (60 °C), acid or alkaline conditions, and high ionic strength, which makes them highly suitable for practical biological detection.

3.2 The electron-accepting and -donating capacity of CDs

The experiments indicated that the CDs could act as both electron donors and electron acceptors under visible light. Fig. 3 shows the photoluminescence decays (485 nm excitation, monitored with a 550 nm narrow bandpass filter) of CDs, which were quenched by the known electron donor N,N-diethylaniline (DEA, 0.88 V vs. NHE) and electron acceptor 2,4-dinitrotoluene (−0.9 V vs. NHE, the full fluorescence spectra are shown in Fig. S1†), with the observed Stern–Volmer quenching constants (K_SV = τF°k_q) from linear regression of 35 M⁻¹ and 20 M⁻¹, respectively. The above results clearly indicate that the PL of CDs can be quenched highly efficiently by either electron acceptors or electron donors, which confirms that CDs are excellent as both electron acceptors and electron donors.

Fig. 3 (a and b) Photoluminescence quenching spectra (485 nm excitation) of the CDs in toluene without and with the quenchers (2,4-dinitrotoluene and DEA, both 0.05 M). (c and d) Photoluminescence decays (485 nm excitation, monitored with 550 nm narrow bandpass filter) of the CDs with 2,4-dinitrotoluene and DEA, respectively. Insets: Stern–Volmer plots for the quenching of photoluminescence quantum yields (485 nm excitation) of the CDs by (c) 2,4-dinitrotoluene and (d) DEA.

3.3 The proton (H⁺) adsorption capacity of CDs

To verify that the proton (H⁺) adsorption capacity of CDs can affect the detection of DOPA, proton adsorption experiments with the CDs were performed. Due to the CDs being full of functional groups, such as –OH, –COOH and –C=O, on the surface, we suspected that the uncontrolled hydrophilic groups on CDs may help the H⁺ closer to the surface of the CDs, which is good for enhancing the adsorption capacity.^25,26 On account of the excellent water solubility of CDs, the adsorption experiments were conducted with a dialysis method (see Experimental section for details). The amount of adsorbed HCl, Q (mg g⁻¹), was calculated by the following equation:
where C₀ and C_e are the initial and equilibrium concentrations (mg L⁻¹), respectively, V is the volume of HCl solution (mL) and W is the weight (g) of the CDs adsorbent. Fig. 4 shows the dependence on contact time of the removal of H⁺ by CDs, from which the adsorption of H⁺ was extraordinarily rapid in the first 4 min, then increased gradually with prolongation of the contact time. After 6 min of adsorption, the amount of H⁺ remained constant, which suggests that 6 min is the equilibrium time in this adsorption experiment. The amount of adsorbed H⁺ (based on the quantity of HCl) was calculated as about 12.4 mg g⁻¹ for CDs. These results indicated the CDs possess adsorption capacity for H⁺, which could promote the detection of DOPA and shorten the testing time for DOPA.

Fig. 4 Dependence on contact time of removal of H⁺ by CDs.

3.4 The properties and characterization of the hybrids

To further explore the interaction between CDs and TYR in the CDs/TYR hybrid catalysts, the two components were mixed in solution and the resulting hybrids were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Fig. 5a shows that the hybrids did not stain the SDS-PAGE gels under visible light because CDs and CDs/TYR are colorless. Fig. 5b shows that the CDs and TYR formed stable complexes. Illumination under UV light allowed visualization of the CDs within the complexes, which showed that CDs are more mobile than the TYR/CDs hybrids (Fig. 5a, lanes A and B, CDs and CDs/TYR, respectively). This effect was due to an increase in the hydrodynamic radius of the CDs upon absorption of the PL. As shown in Fig. 5c, we found that the PL spectrum of the CDs (red trace) was the same as the PL spectrum of the CDs/TYR hybrid (black trace). This leads us to believe that CDs are connected to the TYR by non-covalent bonds.

Fig. 5 SDS-PAGE of the CDs (lanes A) and CDs/TYR hybrids (lanes B) was used to separate mixtures on 20% gels. Gels were photographed under (a) visible light and (b) UV illumination. (c) The PL spectra of CDs/TYR (black trace) and CDs (red trace) in PB solution, respectively.

To prove the PL of CDs could be efficiently quenched by dopaminechrome, the fluorescence spectra of different samples were recorded. Fig. 6 shows the fluorescence spectra of CDs (black line), CDs in the presence of 0.13 mM DOPA (CDs-Dopa, red line), CDs in the presence of 20 μg mL⁻¹ TYR (CDs-TYR, blue line), and CDs in the presence of a mixture containing 20 μg mL⁻¹ TYR and 0.13 mM DOPA (CDs-TYR-Dopa, green line). As shown, the PL of the CDs was almost the same after mixing with DOPA (red line) and TYR (blue line), respectively. However, when the CDs were in the presence of a mixture containing TYR and DOPA, the fluorescence of CDs was quenched efficiently (green line). The results demonstrate that the CDs/TYR hybrids can efficiently quench the PL of CDs.

Fig. 6 The fluorescence spectra of CDs (black line), CDs in the presence of 0.13 mM DOPA (CDs-Dopa, red line), CDs in the presence of 20 μg mL⁻¹ TYR (CDs-TYR, blue line), and CDs in the presence of a mixture containing 20 μg mL⁻¹ TYR and 0.13 mM DOPA (CDs-TYR-Dopa, green line).

As is well known, in the present system (TYR-Dopa), the dopamine quinone produced from the TYR-catalyzed oxidation will be converted by a rapid spontaneous auto-oxidation to dopaminechrome, which has a strong absorption at 470 nm (UV-vis spectrum of dopaminechrome, Fig. 7a).²⁷ Fig. 7b reveals that, with excitation at 370 nm, the CDs show an emission band from 400 nm to 650 nm and the strongest emission at about 460 nm. Simultaneously, the absorbance peak of dopaminechrome is from 350 to 650 nm and the strongest absorption is at 470 nm. Therefore, the PL of CDs can be quenched efficiently by DOPA in the presence of TYR. Moreover, during the DOPA oxidation catalyzed by TYR, as shown in Fig. 7c, 2H⁺ and 2e⁻ should be involved, which may also promote the PL quenching of CDs (CDs are excellent as both electron donors and electron acceptors under visible light, see Fig. 3, and they also have proton (H⁺) adsorption capacity, see Fig. 4).

Fig. 7 (a) UV-vis absorption spectrum of dopaminechrome (inset is the digital photographs of dopaminechrome). (b) The superposed graphs of dopaminechrome UV spectrum and CDs PL spectrum. (c) Schematic illustration from DOPA to dopaminechrome during the detection process.

It should be noted that, in the present hybrid probe system, many assay conditions would affect the detection results. To search for optimal detection conditions for DOPA, different parameters, such as enzymatic factors, temperature, pH value and incubation time, have been investigated. Firstly, the different concentrations of enzyme were selected, and found that when the concentration of TYR was 20 μg mL⁻¹, the quenching effects reached the maximum as shown in Fig. 8a. The optimal pH, temperature and response time for the PL quenching efficiency in the presence of 0.13 mM DOPA were also discussed. Fig. 8b shows when the pH was changed from 5.0 to 8.0, the ratio I₀/I was different, and the optimum pH was 6.8. Due to the quenched fluorescence intensity of CDs being best at pH 6.8, pH 6.8 was chosen as the favorable pH for the detection of DOPA. Fig. 8c reveals that the quenching effect reaches a maximum at 35 °C; thus, the optimal incubation temperature was 35 °C. Fig. 8d reveals that when the incubation time reached 40 min, the signal response attained a stable value; thus, the optimal incubation time was 40 min. Therefore, all further detection experiments were performed at 35 °C, pH 6.8 and an incubation time of 40 min.

Fig. 8 The effects of different TYR concentration (a), pH (b), temperature (c) and incubation time (d) on the quenched efficiency of the CDs in the presence of 0.13 mM DOPA and 20 μg mL⁻¹ TYR.

In the experiments, under the optimal detection conditions (35 °C, pH 6.8, and incubation time of 40 min), the quantitative detection of DOPA was carried out. In the present system, when the DOPA is detected by a solution containing CDs and 20 μg mL⁻¹ TYR, the PL intensity of CDs was difference between the absence and presence of analytes and changes linearly with different concentrations; therefore, the plot of DOPA exhibits good linearity. Fig. 9 shows the PL of CDs was quenched under concentrations of DOPA from 1.318 × 10⁻⁴ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹. The ratio I₀/I (I₀ and I are the PL intensities of CDs in the absence and presence of analytes, respectively) was proportional to the DOPA concentration. Fig. 9a and c show that the PL of CDs was quenched under high concentrations of DOPA, the linear regression equation was I₀/I = 1.089 + 2653.11C_DOPA (R² = 0.995), and the detection range was from 1.318 × 10⁻⁴ mol L⁻¹ to 6.591 × 10⁻⁶ mol L⁻¹. As shown in Fig. 9b and d, the PL of CDs was quenched under low concentrations of DOPA and the other regression equation was I₀/I = 1.066 + 5756.07C_DOPA (R² = 0.975), and its detection range was from 8.239 × 10⁻⁶ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹. Therefore, the detection range of DOPA is 1.318 × 10⁻⁴ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹ and the detection limit of DOPA is 6.0 × 10⁻⁸ mol L⁻¹. The experimental data revealed that the minimum detectable concentration of DOPA (2.06 × 10⁻⁷ mol L⁻¹) is lower than that determined for the CdTe QD gel (5.0 × 10⁻⁵ mol L⁻¹).¹¹ The lower detection limit and wide detection range proved that the CDs are excellent candidates for fabrication of a PL biosensor for a variety of phenolic compounds.

Fig. 9 (a and b) The normalized fluorescence spectra of CDs containing 20 μg mL⁻¹ TYR upon addition of different concentration of DOPA from top to bottom. (c and d) The linear response of the quenching efficiency I₀/I of the CDs vs. concentrations of DOPA.

In order to further evaluate the specificity of the developed biosensor for DOPA, many kinds of metal ions, anions, amino acids, carbohydrates, as well as phenol and cresol, were selected. Fig. 10 shows that for glucose, levulose, lactose and maltose, the I₀/I value shows a slight influence on DOPA detection. For phenol and cresol, the results also show a small influence on DOPA detection, even at 10 times higher concentration than that of DOPA, indicating the high specificity. However, the amino acids, typical metal ions and some anions showed minimal effects on DOPA determination. These experimental results show that the CDs can serve as new fluorescence probes for reliable and highly selective DOPA monitoring.

Fig. 10 The effects of guest molecules (10 mM) to 0.13 mM DOPA on the PL of CDs containing 20 μg mL⁻¹ TYR.

The DOPA concentration in urine samples of healthy subjects is between 100 and 400 mg per 24 h.⁴ Hence, with a detection limit of 60 nM, this sensor could be applied for detecting DOPA in urine, and the present sensor system may be applied for DOPA analysis in biological assays. Notably, this detection method based on CDs could be directly used in real situations due to its high sensitivity and selectivity. In our further study, we tested five human urine samples by this developed method. The human urine samples were provided by the Peking Union Medical College Hospital. All measurements were performed in PB, pH = 6.8, and incubated at 35 °C for 40 min. Samples 1–5 are the five different human urine samples with different DOPA concentrations. Moreover, the same five human urine samples were also tested by the clinical detection method (HPLC) and fluorescence detection method. We found that the results obtained by the three methods matched well, as shown in Fig. 11a.

Fig. 11 (a) The comparison of detection of DOPA in urine using the fluorescence probe (CDs/TYR hybrid and clinical sample) methods and HPLC method. (b) The typical time course of normalized fluorescence intensity of the CDs/TYR hybrid (same sample) for the detection of DOPA. (c) The lifetime of the CDs/TYR hybrid as fluorescence probe at 4 °C. (d) The lifetime of the CDs/TYR hybrid as fluorescence probe at room temperature.

To further verify that the hybrid fluorescent probes are highly stable, we carried out a series of stability experiments for the hybrid fluorescent probes. Fig. 11b shows that after 70 min the fluorescence intensity of the tested sample was essentially unchanged. Fig. 11c shows that the probes were stored at 4 °C and after 90 days still accurately test the samples. As shown in Fig. 11d, when the probes were stored at room temperature, after 3 days they can also still test the urine samples, and the fluorescence intensity of the tested sample was somewhat changed. These results revealed that the hybrid fluorescent probes are highly stable, and the simple, quick and inexpensive method could be successfully applied for detecting DOPA in urine samples. Here we want to further point out that, compared with other methods, our method not only has excellent repeatability, but also has a wider linear range in most cases and high accuracy. If the PL quantum yields of CDs increase, a lower detection limit and wider detection range will be obtained. Moreover, more novel biosensors based on the assembly of CDs with redox enzymes could be expected for the highly sensitive detection of other biomolecules and medicaments.

4. Conclusion

The CDs were successfully obtained by electrolysis a NaOH and polyethylene glycol (PEG-200) mixed solution for 6 h. The CDs combined with TYR as a fluorescent biosensor can efficiently detect and analyze the concentration of DOPA. This kind of hybrid, low-cost fluorescent probe is highly efficient, sensitive, stable, and precise for the detection of DOPA. The detection limit of DOPA is 6.0 × 10⁻⁸ mol L⁻¹ and the detection range is very wide from 1.318 × 10⁻⁴ mol L⁻¹ to 2.06 × 10⁻⁷ mol L⁻¹. More importantly, the present fluorescent probe did not require enzyme immobilization and modification, and the test results could be read as soon as the probe–sample incubation was completed. Furthermore, in the present system, the test results are comparable to those of the present clinical fluorescence and HPLC methods, and the results show the three methods were matched well. Our results indicated that the CDs offer great potential for the development of various enzyme-based biosensors and portable sensing devices.

Acknowledgements

This work is supported by the National Basic Research Program of China (973 Program) (2012CB825800, 2013CB932702), the National Natural Science Foundation of China (51132006), the Specialized Research Fund for the Doctoral Program of Higher Education (20123201110018), a Suzhou Planning Project of Science and Technology (ZXG2012028), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06163b

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