2020 Volume 68 Issue 7 Pages 628-634
Facile and effective detection of dopamine (DA) plays a significant role in current clinical applications. Substantially, special optical nanomaterials are important for fabricating easy-to-control, cheap, selective, and portable fluorescence DA sensors with superior performance. Herein, carbon dots (CDs) prepared from melting method were applied as signal to establish a simple but effective fluorescence strategy for DA determination based on the enzymatic activity of acid phosphatase (ACP), which induces DA to form polydopamine (pDA). The formed pDA caused by the enzymatic oxidization of ACP toward DA can interact with CDs through the inner filter effect. Such behavior effectively quenched the CDs’ fluorescence. The degree of fluorescence quenching of CDs was positively correlated with the DA content. Under the optimized reaction conditions, the proposed fluorescence method exhibited a comparable analytical performance with other DA sensors with good selectivity. Furthermore, this method has been successfully applied to detect DA in DA hydrochloride injection and human serum samples. It shows that this method features potential practical application value and is expected to be used in clinical research.
Dopamine (DA) is a typical and important catecholamine neurotransmitter mainly distributed in the central nervous system of humans and other mammals.1,2) Studies have shown that DA can convey information on emotional changes.2,3) Variable DA reflects the fluctuation of human emotions, and the DA content reflects the health of the nervous system at the same time.1,2) Particularly, abnormal levels of DA in organisms are thought to be closely related to the occurrence of certain neurological diseases, such as schizophrenia, Parkinson’s disease, and Alzheimer’s disease.4–6) Moreover, the control of DA at an appropriate level is significant for normal cell activities and signalling transduction.7,8) Thus, the accurate and convenient detection of DA is a crucial and valuable work in biological areas. To date, a variety of analytical methods, such as electrochemistry,9,10) capillary electrophoresis,11,12) chemiluminescence,13) and HPLC,14,15) have been developed for DA detection. Although the abovementioned strategies offer efficient and sensitive DA sensing technologies, they face certain constraints of complex procedure, consuming time, and special equipment, thus further development is needed to achieve the practical requirements of convenient and accurate determination for DA. Compared with the analytical methods for DA detection, fluorimetry is considered as an ideal detecting technique because of its facile portability, simple signal sources, sensitivity, and easy-to-control instrumentation.16,17) However, a special and effective fluorescence method for DA with simple operating procedure is still needed.
Fluorescence spectrometry has been widely used in pharmaceuticals, life sciences, and chemistry because of its numerous advantages. In fluorescence analytical methods, several strategies are fabricated based on the mechanism of the specially induced reaction of DA to interact with fluorescent probes.16–18) Two possible routines are available for DA reaction to promote the interaction between the DA reaction products and probes.19–21) One routine is the controllable polymerization of DA to polydopamine (pDA) to modulate fluorescence signal. The other strategy is by using DA as the substrate for enzymatic catalysis. The stimulated fluorescence from DA oxidative polymerization to pDA19) and self-polymerization of DA on the fluorescent quantum dot surface20) has been proven an effective tool for DA detection. Moreover, using DA as the intermediate or substrate for special enzymes, such as tyrosinase, facile and sensitive analytical methods were designed to monitor the enzyme activity22–24) or DA concentration.21) Although the application of DA as substrate with a controlled concentration is a valuable tool for the enzyme activity monitoring, the combination of enzymatic catalysis and DA polymerization for DA sensing is rare.
Carbon fluorescent nanomaterials,25,26) such as graphene quantum dots and carbon dots (CDs), have received wide interests due to their optical property, biocompatibility, and economy. The optical property of carbon fluorescent nanomaterials can be facile-modulated through heteroelement doping and surface modification, especially for CDs.27,28) Using CDs as fluorescent probe will encourage several sensitive and selective methods for target detection. For example, by using Cr(VI)-quenched CDs as probe, DA can be detected through the redox reaction between DA and Cr(VI) to recover the fluorescence of CDs.29) DA sensing was also achieved based on the mixture of DA aptamer-labeled CDs and nanographite according to the interaction between the DA and aptamer-modified CDs.30) Although DA sensing based on carbon dots as probe illustrated acceptable performance,29–31) most of the reported methods for DA fluorescence detection require the special modification or the interaction between the probe and several active substances or composites. Thus, designing fluorescence strategies with improved operationality and efficiency for DA detection via simple mixing procedure are still demanded.
Recently, our group discovered that the DA polymerization to pDA with the optimal alkaline condition control is a powerful tool for DA sensing.20) However, it is difficult to convert DA to pDA in neutral or other conditions, which is needed for the extended application of the DA detection in the actual physiological conditions. Thus, motivated by the transformation of DA to pDA, we proposed a means for achieving pDA from DA by using some other methods to explore a fluorescent method for DA sensing in neutral physiological condition. Herein, as shown in Chart 1, acid phosphatase (ACP) was employed as a new active enzyme to catalyze DA transformation into pDA in neutral facile conditions. pDA formation will interact and quench CDs in a particular degree in accordance with the DA concentration. The decreased fluorescence intensity of CDs is directly related to the DA concentration. Therefore, a simple, effective, sensitive, and fast fluorescence analysis method for DA determination without disruption was proposed. This method not only offers a new horizon for DA testing based on ACP catalysis but also exhibits the possibility of exploiting enzymatic performances of enzymes on an unexpected substrate for the fabrication of analytical methods or synthetic strategies.
(Color figure can be accessed in the online version.)
ACP from potato, DA, glutathione (GSH), ascorbic acid (AA), glycine (Gly), lysine (Lys), α-glucosidase, alkaline phosphatase (ALP), horseradish peroxidase (HRP), xanthine oxidase (XOD), glucose oxidase (GOD) and tyrosinase (TYR) were supplied by Sigma-Aldrich (Shanghai, China). Citric acid, polyethylene polyamine (PEPA), tyrosine, norepinephrine, levodopa and adrenaline were purchased from Aladdin Industrial Corporation (Shanghai, China). Acetone, NaCl, and glucose were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All aqueous solution used in this work were prepared in ultrapure water from a Millipore system. All reagents with analytical grade were used without any purification.
ApparatusFluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, U.S.A.). UV-visible (UV-vis) absorption spectra were measured on a UV-2450 UV-vis spectrophotometer (Shimadzu Corporation, Japan). Fourier transform (FT) IR Spectroscopy was collected using a NICOLET iS50 Infrared Spectroscopy (Thermo Fisher Scientific, U.S.A.). Transmission electron microscopy (TEM) images were performed on a FEI Talos F200S (Thermo Fisher Scientific).
Synthesis of CDsCDs were prepared according to our previous work23) with minor revisions. Briefly, the mixture containing 1.0 g citric acid (CA) and 0.3 g GSH was mixed and placed in a flask. Then, the mixture was heated at 160°C until the CA and GSH completely melted. Subsequently, 4 mL polyethylene polyamine was added to the molten mixture with continuous reaction for another 90 min at 180°C. After the resultant mixture was cooled to room temperature, acetone was added to obtain a precipitate with 10 min centrifugation at 6000 rpm. Thereafter, the precipitate was dissolved in ultrapure water and dialyzed for 48 h by using a 2000-mesh dialysis bag. Lastly, the purified CDs were stored at 4°C in the dark.
Detection of DA by Using ACP and CDsFor fluorescent DA assay, the solution containing 20 µL ACP (4 U/mL) and 15 µL CDs (25 µg/mL) (named as ACP-CDs system) was mixed in an Eppendorf tube (EP) and then mixed with various concentrations of DA (0–2000 µM). The detecting mixture was labeled the ACP-DA-CDs system in this work. Then, the ACP-DA-CDs system was diluted to 200 µL with phosphate-buffered saline (PBS) (10 mM, pH 7.0). After incubation at 30°C for 120 min, the fluorescence emission spectra of the different ACP-DA-CDs system with variable DA contents were recorded at 350 nm excitation in the wavelength range of 360–600 nm.
Selectivity of DA DetectionFluorescence monitoring of each testing system was performed according to the same procedure illustrated in “Detection of DA by using ACP and CDs.”
Considering the complexity of the substances contained in clinical samples, we selected several common substances in clinical samples for the selectivity study of this method. Two testing routines were applied. The interference (200 µM AA, Glucose and Gly, 10 µg/mL bovine serum albumin (BSA) and Lys, 50 U/L Try, ALP, TYR, urease, HRP, XOD and GOD) and 200 µM DA were applied to investigated the selectivity of this method. At first, the individual interference and the mixed interferences were correspondingly added to the ACP-CDs system to investigate the fluorescence response of the proposed method. For another routine, the abovementioned interference and the mixture of the interference were added to the ACP-DA-CDs system for the following fluorescence monitoring.
Considering the similar structure of phenylethylamine drugs that contain phenolic compound, several DA analogues (200 µM tyrosine, norepinephrine, levodopa and adrenaline) and 200 µM DA were respectively added to the ACP-CDs system to compare the fluorescence response to evaluate the anti-interfering ability of this method for DA detection.
Application of the Proposed Method for DA Detection in DA Hydrochloride Injection and Human SerumFor the DA hydrochloride injection detection, 2 mL acetone was initially added to DA hydrochloride injection. Then, a certain amount of the injected injection sample was absorbed and diluted into a 50 µM DA solution with PBS. Subsequently, the diluted DA solution was tested according to the same procedure shown in “Detection of DA by using ACP and CDs.”
Approved by the Ethics Committee at the First Affiliated Hospital of Fujian Medical University (Fuzhou, China), human serum from healthy volunteers with informed consent was obtained and applied to evaluate the feasibility of this assay for DA detection in humans. The recovery of DA in human serum sample was monitored to evaluate the accuracy of the proposed method. A 10-fold diluted serum (20 µL) was added to the ACP-CDs system, which contained 20 µL ACP (4 U/mL) and 15 µL CDs (25 µg/mL). Then, variable concentrations of DA were added and tested according to the proposed strategy as illustrated in “Detection of DA by using ACP and CDs.”
The morphology and luminescent property of CDs were investigated. As shown in Fig. 1A, TEM result illustrated that the prepared CDs featured a quasi-spherical structure with a regular and monodisperse morphology. The average diameter was measured to be 2.1 nm with a lattice spacing of 0.22 nm of the characteristic (100) diffraction facet. CDs also exhibited an excitation-independent luminescence behavior in the excitation range of 310–390 nm, and the maximum excitation wavelength was 350 nm (Fig. S1). In addition, the CDs displayed the maximum emission wavelength at 450 nm with the excitation at 350 nm (Fig. 1B). Furthermore, the CDs decreased the fluorescence intensity by less than 5% under ordinal preservation in 3 months, suggesting the stable optical property of CDs.
The possible signal change in CDs caused by the introduction of DA was investigated to evaluate the feasibility of DA detection using the proposed method. As shown in Fig. 1C, with the addition of ACP or DA alone, the CDs illustrated negligible fluorescence change with a characteristic emission wavelength at approximately 450 nm. No fluorescence was observed in the mixture of ACP and DA. Compared with the pure CDs, CDs was evidently quenched in the mixture of ACP and DA. This phenomenon indicated that the coexisting ACP and DA can interact and induce the quenching of CDs. When the ACP content was stably controlled, varied DA concentrations promoted the different fluorescence responses of CDs. The degree of fluorescence quenching can well reflect the DA concentration, indicating the feasibility of this strategy for DA detection.
Inset in (A) shows the size distribution (upper) and HRTEM (under) of CDs. (Color figure can be accessed in the online version.)
The possible interaction of ACP to DA was investigated. Using FTIR, the reaction product of ACP and DA was characterized and compared with pure DA (Fig. 2A). The DA spectrum confirmed the typical molecular structure of catecholamine ammonia. For example, the absorption peaks at 3342 and 1610 cm−1 were ascribed to the stretching and bending vibrations of –NH2, respectively. In addition, the band at 1502 cm−1 denotes the absorption of the stretching vibration of aromatic C=C. By contrast, the reaction product of ACP and DA illustrated the different molecular structures observed by FTIR. The findings showed the characteristic absorption peaks at 1539 and 1650 cm−1, which are assigned to the C=N ring bond and aromatic C=C bond of indole, respectively. The wide absorption band from 3440 to 3045 cm−1 is ascribed to the stretching vibration of N–H and O–H, whereas the peak at 1180 cm−1 represents the stretching vibration from the combination of C–N and C–O. The above FTIR of the reaction product suggests that the product is the oxidized polymer of DA.20,32) Moreover, UV-vis was applied to investigate the difference between the original DA and the reaction product of ACP and DA. As shown in Fig. 2B, DA illustrated a relatively strong but characteristic absorption peak at 280 nm, which was ascribed to its phenylethylamine structure. ACP also showed a weak band at approximately 280 nm due to protein absorption. After the reaction of ACP and DA for 2 h, except for the absorption peak at 280 nm, two another peaks at 305 nm and a wide peak centered at 470 nm were observed. The band peak at 305 nm can be considered as the absorption of dopaminochrome, the early oxidation product of DA. Meanwhile, the wide absorbance band around 470 nm is ascribed to the oligomers of pDA,33) suggesting the oxidation of DA into pDA in the reacting system containing ACP. Furthermore, wide absorbance band at 470 nm will interact with the CDs and cause the inner filter effect between the emission of CDs at 450 nm and the absorbance band at 470 nm34) (Fig. 2B). Moreover, the absorbance band at 470 nm was enhanced with more DA reacting with the constant ACP (Fig. 2C), confirming the feasibility of this method for DA sensing, as shown in Fig. 1C.
The conversion reaction mechanism from dopamine to polydopamine by acid phosphatase was investigated. Firstly, the common inhibitors, like NaF and sodium tartrate, that may inhibit ACP to catalyze its common substrate like p-Nitrophenyl phosphate, were applied to investigate whether they play inhibition effect on the enzyme activity of ACP with DA as substrate. The possible inhibition process was carried out using Uv-vis to evaluate the production of ACP and DA with variable concentrations of NaF or sodium L-tartrate, as shown in Fig. S2. The absorbance behavior was kept whether how many NaF or sodium L-tartrate was added, suggesting that the common inhibitors played no inhibition on the catalytic activity of ACP to DA. This result implied that ACP played a different catalytic role on DA polymerization. In general, AA is a common reducing agent for reacting and exhausting oxidants, such as reactive oxygen species. Thus, AA was introduced to the reaction system of ACP and DA, and CDs was used as signal probe. The quenching degree of the reaction of ACP and DA toward CDs was evidently weakened by AA, confirming that the conversion mechanism from DA to pDA by acid phosphatase was based on the happen of oxidation and polymerization reaction. However, it is not clear why ACP can oxidize and polymerize DA into PDA under neutral condition, which needs further study. In a word, the above results confirmed that DA detection by enzymatic oxidation of ACP and CDs as probe defers to the following processes. First, ACP induces the enzymatic oxidation of DA to form pDA oligomers. Then, the pDA oligomers quench the CDs through the inner filter effect.
(Color figure can be accessed in the online version.)
Several reacting conditions of this method were optimized to achieve the improved testing performance. The fluorescence changed degree ((F0−F)/F0) was employed to monitor the regularity of the varied testing conditions. F0 states the initial fluorescence intensity of CDs, whereas F represents the fluorescence intensity of CDs in the ACP-DA-CDs system.
This experiment involved an enzymatic catalytic reaction. The activity of the introduced enzyme played an important role in the detection performance for DA. In addition, the reaction temperature and reaction time considerably affected the enzyme’s activity. Therefore, the reaction temperature and reaction time were optimized to obtain the optimum experimental conditions for DA detection. We mainly optimized the kinetic reaction process of ACP activity at 25, 30, 37, and 45°C. The results are shown in Fig. S3. With the prolonged reaction period, the fluorescence quenching efficiency of CDs increased quickly at the initial stage and gently from 2 h. Meanwhile, with the temperature increased, (F0−F)/F0 illustrated a faint change under the investigated temperatures, suggesting the weak effect of temperature to ACP activity. Considering the practical application conditions, physiological conditions, and the control of reaction time to achieve facile operation, 120 min and 30°C were selected as the optimal reaction time and reaction temperature for this method for DA detection, respectively.
In this experiment, DA was oxidized and polymerized to form pDA under the conditions of ACP enzymatic catalysis. Considering the self-polymerization of DA under alkaline conditions20,35) and the working environment of ACP, the optimal pH of the reacting solution was mainly carried out under acidic conditions. Figure S4A shows that the fluorescence intensity of pure CDs decreased with the increase in pH, indicating the pH-sensitive property of CDs. Moreover, the ACP-DA-CDs system underwent fluorescence quenching at varied pH conditions, suggesting the enzymatic catalysis of ACP toward DA. Figure S4B shows the increasing fluorescence quenching efficiency of the reaction system with the increase in pH. The maximum fluorescence quenching efficiency was obtained at pH 7.0. Consequently, PBS at pH 7.0 was selected as the optimal system for DA detection.
Analytical Performance of This Method for DAUnder the optimal experimental conditions, the fluorescence intensity of CDs at different DA concentrations was monitored by fixing the ACP concentration. Figure 3A shows the relation of the fluorescence quenching process of CDs (ordinate) and DA concentration (abscissa), which was applied to establish a standard curve. Notably, pDA formation increased in accordance with the increase in DA, and the fluorescence intensity of CDs gradually decreased as the DA concentration increased. The fluorescence quenching efficiency gradually increased and then reached a plateau in accordance with the increased content of DA, as shown in Fig. 3B. The formation of plateau may be due to the exhaustion of ACP with the excess DA in the testing system. Furthermore, the fluorescence quenching efficiency ((F0−F)/F0) of CDs showed a good linear relationship with the concentration of DA (CDA) in the range of 3.0–20.0 µM. The linear fitting equation is Y = 0.007 CDA + 0.033, R2 = 0.9991, and the limit of detection (LOD) was calculated to be 1.0 µM based on S/N = 3.
Inset of B was the linear calibration equation. The detection system contained 0.4 U/mL ACP and 1.875 µg/mL CDs in 200 µL PBS. (Color figure can be accessed in the online version.)
In actual detection, the presence of interfering substances may play a negative role in the quantitative monitoring of DA. Thus, the possible influence of common interferences on this method was investigated to evaluate the specificity. As shown in Fig. 4, compared with the same detection process of DA, interfering substances, such as ascorbic acid, glucose, glycine, tyrosinase, and so on, exerted negligible effects on the fluorescence changes in the mixture of CDs and ACP. Furthermore, the mixture of the common disturbing substances containing AA exhibited only about 1/7 fluorescence response degree to the mixture of CDs and ACP compared with the fluorescence response of the ACP-DA-CDs system (Fig. 4A). Meanwhile, except for ascorbic acid, no significant effect was observed on the fluorescence response of the ACP-DA-CDs system with coexisting single interference. The mixture of interference and DA showed a slightly decreased response in the DA detection compared with the ACP-DA-CDs system (Fig. 4B). Furthermore, the possible interference effect of several drugs containing phenolic compound, such as tyrosine, norepinephrine, levodopa, adrenaline were applied to the testing system of ACP-CDs. The comparison of the response of the different drugs containing phenolic compound was shown in Fig. S5. It showed that except the relatively high response of adrenaline (1/3 response of the testing result of DA), other drugs containing phenolic compound were ignorable response on ACP-CDs, suggesting the high selectivity of this method for DA from the analogues that also belong to phenylethylamine drugs. These results indicated that the proposed method exhibited an acceptable selectivity for DA detection, and this property was possibly related to the highly selective catalysis of ACP to DA to form pDA.
(Color figure can be accessed in the online version.)
DA hydrochloride injection was used as the actual sample to evaluate the reliability and real life application potential of the proposed method in drug quality control. DA concentration was detected and applied to calculate the labeling amount of the injection on the basis of the standard curve shown in Fig. 3B. The labeled amount of DA hydrochloride injection was tested to be 96.8%, and the relative standard deviation (RSD) was 1.1% (n = 3), meeting the Chinese pharmacopoeia (Edition 2015) regulation on the labeling amount of DA (93.0–107.0%). The recovery of DA in the diluted serum was tested to investigate the accuracy and applicability of this method for the detection of DA in serum systems. As shown in Table 1, the recoveries of DA in the human serum samples were in the range of 96.1–109.3%, and the RSD ranged from 0.08 to 1.35%. These results suggest the application possibility of the proposed method for DA detection in actual blood samples and clinical diagnosis. Moreover, the analytical performance of the proposed method for DA was compared with that of reported DA fluorescence sensors based on varied nanomaterials (Table S1). The results show that the novel method exhibits a comparable performance in terms of linear range, LOD, and real-life application and illustrates the advantage of easy-to-control operation and low cost.
| Serum samples | Added (µM) | Measured (µM) | Recovery (%) | RSD (%) (n = 3) |
|---|---|---|---|---|
| 1 | 3 | 3.08 | 102.5 | 0.75 |
| 2 | 6 | 6.04 | 100.6 | 0.57 |
| 3 | 12 | 11.53 | 96.1 | 1.35 |
| 4 | 15 | 15.32 | 102.1 | 1.01 |
| 5 | 18 | 19.68 | 109.3 | 0.08 |
In summary, a fluorescence sensor was fabricated by employing CDs as a simple fluorescence signal source. In addition, the enzymatic capability of ACP toward DA was utilized to form pDA for DA sensing. Using DA as a new substrate, ACP showed an outstanding enzymatic catalysis performance toward DA to form pDA. The formed pDA, which is controlled by the added certain concentration of DA, will quench the fluorescence response of CDs. Thus, the quenching degree can sensitively quantify the DA concentrations with acceptable anti-interference capability. In addition, the easy-to-control operation was ascribed to the simple mixing procedure. This work also illustrates a new horizon in which the exploration of certain reactions based on special enzyme and its new kind of substrate is proposed to be an effective analytical method for specific targets.
The authors gratefully acknowledge for financial support from Joint Funds for the Innovation of Science and Technology, Fujian Province (2017Y9121), the National Science Foundation of Fujian Province (2017J01328, 2017J01532), Wu Jieping Medical Foundation (320.6750.19090-45) and Start Fund for Scientific Research, Fujian Medical University (2017XQ1054).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
