Selective and Sensitive Monitoring of Cerebral Antioxidants Based on

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Selective and Sensitive Monitoring of Cerebral Antioxidants Based on the Dye-Labeled DNA/Polydopamine Conjugates Shishi Ma,† Yan-Xia Qi,† Xiao-Qin Jiang,‡ Jie-Qiong Chen,† Qiao-Yu Zhou,† Guoyue Shi,† and Min Zhang*,† †

School of Chemistry and Molecular Engineering and ‡School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China S Supporting Information *

ABSTRACT: A simple and novel method for evaluating antioxidants in complex biological fluids has been developed based on the interaction of dye-labeled singlestrand DNA (ssDNA) and polydopamine (PDA). Due to the interaction between ssDNA and PDA, the fluorescence of dye-labeled ssDNA (e.g., FITC-ssDNA, as donor) can be quenched by PDA (as acceptor) to the fluorescence “off” state through Förster resonance energy transfer (FRET). However, in the presence of various antioxidants, such as glutathione (GSH), ascorbic acid (AA), cysteine (Cys), and homocysteine (Hcys), the spontaneous oxidative polymerization reaction from DA to PDA would be blocked, resulting in the freedom of FITC-ssDNA and leading to the fluorescence “on” state. The sensing system shows great sensitivity for the monitoring of antioxidants in a fluorescent “turn on” format. The new strategy also exhibits great selectivity and is free from the interferences of amino acids, metal ions and the biological species commonly existing in brain systems. Moreover, by combining the microdialysis technique, the present method has been successfully applied to monitor the dynamic changes of the striatum antioxidants in rat cerebrospinal microdialysates during the normal/ischemia/reperfusion process. This work establishes an effective platform for in vivo monitoring antioxidants in cerebral ischemia model, and promises new opportunities for the research of brain chemistry, neuroprotection, physiological, and pathological events.

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proaches were developed for the sensing of antioxidants, involving cyclic voltammetry and flow injection analysis with amperometric detection.7 Koleva et al. reported a HPLC assay for antioxidants using a methanolic solution of 2,2′-diphenyl-1picrylhydrazyl and an optimized instrumental setup.8 Compared with these analytical methods, fluorescence methods have their unique advantages due to their simplicity (e.g., mix-andread format), cost-effectiveness, stability, high sensitivity, and good reproducibility.9 Dopamine (DA) is a catecholamine neurotransmitter regulating many biological processes in the cerebral system. It can be oxidized through the oxidation of catechol to dopaminequinone under an aerobic and alkaline condition and then further oxidized and polymerized to form polydopamine (PDA, a cross-linked homopolymer) through deprotonation and intermolecular Michael addition reaction (eq 1).10

xidative stress is defined as an excessive load of reactive oxygen species (ROS), which leads to persistent or reversible damage on a cellular or systemic level.1 Oxidative stress is connected to various neurodegenerative diseases and pathological diseases, such as Parkinson’s disease, Alzheimer’s disease, cardiovascular disease, mild cognitive impairment, cancer, and so on.2 To some degree, ROS can also alter various biological processes including gene expression, proliferation, and genomic stability.3 It is revealed that antioxidants, such as glutathione (GSH), ascorbic acid (AA), cysteine (Cys) and homocysteine (Hcys), can chemically react with ROS to eliminate and inactivate the free radicals including superoxide (O2−), superoxide free radicals (ROO•), hydroxyl radical (•OH), and peroxynitrite (ONOO−).4 In view of the difficulty of measuring individual antioxidant components, determining the total antioxidant capacity, as an important index, is one of the most common strategies to assess the freeradical/antioxidant balance in biological systems.5 Many methods have been reported for detecting antioxidant capacity in body fluids, food, and so on. Cao et al. presented a spectrophotometric assay for the oxygen-radical absorbing capacity of antioxidants, in which many specifical reagents are used including β-phycoerythrin as an indicator protein, 2,2′azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator and Trolox (a water-soluble vitamin E analogue) as a control standard.6 Many sophisticated electrochemical ap© XXXX American Chemical Society

Received: August 17, 2016 Accepted: November 14, 2016 Published: November 14, 2016 A

DOI: 10.1021/acs.analchem.6b03216 Anal. Chem. XXXX, XXX, XXX−XXX

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antioxidants, DA can be oxidized and polymerized to form PDA under an aerobic and alkaline condition. FITC-labeled ssDNA (FITC-ssDNA) can be assembled on the surface of the resultant PDA with strong affinity, and then the fluorescence of FITC-ssDNA (as donor) is quenched by PDA (as acceptor) to the fluorescence “off” state through FRET. However, in the presence of antioxidants, the oxidative polymerization of DA to produce PDA is inhibited, resulting in the freedom of FITCssDNA and leading to the fluorescence “on” state, which enables the detection of antioxidants in a fluorescent “turn on” format. Moreover, by combined with in vivo microdialysis sampling,24 we further develop a new strategy for facile, selective, and sensitive sensing the dynamic changes of cerebral antioxidants in rat brain microdialysates during the normal/ ischemia/reperfusion process.

Recently, PDA has been widely investigated for surface coating and molecular imprinting, such as PDA-coated Fe3O4 nanocomposite,11 PDA-coated silver nanoparticles,12 and PDAcoated gold nanoparticles.13 In addition, PDA possesses a broad-band absorbance in the UV−vis spectrum, which endows it with great promise as a quencher for the development of fluorescent biosensors. Although there are many nanomaterials that have been reported as quenchers, including copper oxide nanobelts,14 carbon nanotubes (CNTs),15 graphene oxide (GO),16 MoS2 nanosheets,17 WS2 nanosheets,18 and metal− organic framework (MOF).19 Only a few of those fluorescencequenching nanomaterials have the similar excellent adhesive, biodegradability, and are as hydrophilic as PDA. So far, PDA has been broadly applied as a quencher in the biological fields, especially by coupled with DNA probes. DNA can interact with PDA by hydrogen bonding.20 Moreover, single-strand DNA (ssDNA) could be assembled on the surface of PDA with strong affinity by “π-π stacking” interactions between the nucleobases of the ssDNA and the aromatic groups of PDA.21 Large surface-to-volume ratio of PDA makes it a favorable substrate for assembling ssDNA probes and quenching the dyelabeled ssDNA with broad emission wavelengths due to Förster resonance energy transfer (FRET).22 Therefore, recent years have witnessed the development of fluorescent assays for mRNA,21 DNA,22a thrombin,22a ATP,22b cancer cells,22c and so on13 based on the marriage of PDA with dye-labeled ssDNA. These assays mainly depend on the target-induced release of dye-labeled ssDNA from the surface of PDA, giving rise to a “turn-on” signal for the detection of target. But to the best of our knowledge, no study has been performed that employs the target-regulated formation of PDA to interact with dye-labeled ssDNA for biosensing. It is reported that the spontaneous oxidative polymerization reaction from DA to PDA can be effectively inhibited by the presence of antioxidants.23 Thus, antioxidants would be a potential target to regulate the formation of PDA for biosensing application by incorporated with dye-labeled ssDNA. Herein, we present a novel fluorescent “turn-on” assay for antioxidants by using the target-responsive formation of PDA coupled with dye-labeled ssDNA (Scheme 1). In the absence of

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EXPERIMENTAL SECTION Chemicals and Materials. 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), lactate (Lact), glucose, glycine (Gly), alanine (Ala), valine (Val), serine (Ser), leucine (Leu), proline (Pro), phenylalanine (Phe), isoleucine (Ile), tyrosine (Tyr), tryptophan (Trp), threonine (Thr), methionine (Met), asparagine (Asn), glutarnine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), cysteine (Cys), homocysteine (Hcys), reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbic acid (AA), dopamine hydrochloride (DA), N-ethylmaleimide (NEM), and ascorbate oxidase (AAOx) were purchased from Sigma-Aldrich (St. Louis, MO). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) and metal salts were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Tris-HCl buffer (10 mM, pH 8.5) was prepared using metalfree reagents in distilled water. FITC-labeled ssDNA (FITCssDNA: 5′-FITC-CGACCATGGCTGTAGACTGTTA-3′) was ordered from Sangon Inc. (Shanghai, China). Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into distilled water, and the pH of solution was adjusted to 7.4. Apparatus and Measurements. Fluorescence spectroscopy was measured in a microplate reader (infinite M200 pro, TECAN, Switzerland) using a black 384-well microplate (Fluotrac 200, Greiner, Germany). The excitation wavelength used was 480 nm for the emission spectra. UV−vis absorption spectroscopy of PDA was also measured in a microplate reader (infinite M200 pro, TECAN, Switzerland) using a transparent 96-well plate (Corning, U.S.A.). Transmission electron microscope (TEM) image was collected on a Transmission Electron Microscope (JEM-2100F, Japan) operated at 200 kV. The gel imaging was taken under ChemiScope 6300 instrument (Clinx Science Instruments Co., Ltd., China). Fluorescence Quenching and Antiquenching Investigation. To investigate the fluorescence quenching efficiency of the resultant PDA to FITC-ssDNA, DA with different final concentrations (0, 0.5, 2.5, 5.0, 10, and 20 mM) was mixed with FITC-ssDNA (0.1 μM) in Tris-HCl buffer (10 mM, pH 8.5). Then, the mixture was incubated with thermostatic mixing device at 400 rpm for 1 min at 25 °C. After 20 min, the mixture was measured by fluorescence spectroscopy and UV−vis absorption spectroscopy. To investigate inhibition of the PDA formation (5 mM DA used), GSH with varying concentrations (0, 20, and 40 μM) were first added into the FITC-ssDNA/buffer solution as control experiment.

Scheme 1. Schematic Illustration of the Mechanism of Fluorescent Sensing of Cerebral Antioxidants in Rat Brain Microdialysates

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Analytical Chemistry Detection of Antioxidants. GSH aqueous solution with increasing concentrations (50 nM−10 μM) were mixed with FITC-ssDNA (0.1 μM) in a set of tubes, then DA (5 mM) was added to the mixture, adjusting the total volume to 100 μL with Tris-HCl buffer (10 mM, pH 8.5) and further incubated with a thermostatic mixing device at 400 rpm for 1 min at 25 °C. After 20 min, the mixture was measured by fluorescence spectroscopy at an excitation wavelength of 480 nm. The fluorescence emission spectra and fluorescence intensities at 522 nm were obtained. For other antioxidants, such as AA, Cys, and Hcys, the same analysis has also been carried out. For the determination of the level of striatum antioxidants in rat brain microdialysates, the samples of microdialysates (5 μL) from different times were added to the sensing system (total volume: 100 μL) and after incubated for 20 min, the fluorescence intensities were measured. Measurement of GSH by the DTNB Method. The measurement of GSH using the DTNB method was performed according to reported literature.25 The solution of DTNB (0.4 mg/mL) and different concentrations of GSH were dissolving in 0.1 M sodium phosphate (pH 8.0). A total of 50 μL of the DTNB solution was added to 250 μL of different concentrations of GSH at room temperature, and the UV absorbance of each solution was measured from 300 to 600 nm. The same procedure was also used for the determination of GSH in artificial cerebrospinal fluid. Animals and Surgery. All surgeries involving animals were conducted with approval of the Animal Ethics Committee in East China Normal University, China. In vivo microdialysis experiments were performed as reported previously,25 and detailed experimental information can be found in Supporting Information.

Figure 1. (A) Fluorescence emission spectra of FITC-ssDNA incubated with different concentrations of DA (0, 0.5, 2.5, 5, 10, and 20 mM) after 20 min, λex = 480 nm and λem = 512−650 nm. (B) Quenching efficiency of FITC-ssDNA upon the addition of different concentrations of DA (0, 0.5, 2.5, 5, 10, and 20 mM) for 20 min. (C) Fluorescence emission spectra of FITC-ssDNA (0.1 μM) upon incubation with DA (5 mM) in Tris-HCl buffer (10 mM, pH 8.5) from 0 to 60 min. Samples were collected for testing per 5 min intervals. (D) Time-course study on the formation of PDA from 5 mM DA via UV−vis absorption monitoring from 0 to 60 min. Samples were collected for testing per 5 min intervals.

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observed, indicating that PDA was gradually formed. In addition, a TEM image of the as-prepared PDA showed that the diameter was around 200−250 nm (Figure S2). As mentioned above, antioxidants, as active reducing agents, can be used to inhibit the oxidative polymerization of DA to form PDA. GSH is one of the most abundant low-molecular antioxidants.26 The thiol group of GSH would donate a reducing equivalent (H+ + e−) to unstable molecules, such as ROS (hydroxyl radical or dissolved oxygen and so on) and freeradical intermediates, inhibiting the formation of PDA from DA.23 In the present work, GSH was utilized as a model of antioxidants to test its feasibility for regulating the formation of PDA and further developing a novel bioassay based on the interaction of dye-labeled ssDNA and PDA. The fluorescence emission spectra and UV−vis absorption spectroscopy of the mixture of FITC-ssDNA and DA (FITCssDNA/DA) upon incubated respectively with 0, 20, and 40 μM GSH were shown in Figure 2A,B. In addition, the photographs of FITC-ssDNA, FITC-ssDNA/DA, and FITCssDNA/DA+GSH (20 and 40 μM, respectively) solutions were recorded under natural light, following a 60 min period with per 10 min intervals (Figure 2C). In the absence of GSH, DA can be oxidized and polymerized in Tris-HCl buffer (10 mM, pH 8.5) to form PDA, accompanied by a color change of solution from colorless to dark gray (Figure 2C, column 2), and the absorbance at 400 nm increased gradually (Figure 2B, black line). Along with the formation of PDA, the fluorescence of FITC-ssDNA continuously decreased due to its binding with PDA and the quenching effect from PDA (Figure 2A, black line). The binding of FITC-ssDNA with PDA was also verified via electrophoretic mobility shift assay (EMSA; Figure 2D).

RESULTS AND DISCUSSION Regulating the Fluorescence of FITC-ssDNA by PDA and Antioxidants. DA can be oxidized and polymerized to form PDA under an aerobic and alkaline condition. Then, FITC-ssDNA can be assembled on the surface of the resultant PDA with strong affinity. PDA shows a broad-band absorbance in the UV−vis spectrum, so it would quench the fluorescence of FITC-ssDNA due to FRET (Figure S1). To investigate its quenching effect toward FITC-ssDNA, different concentrations of DA (0−20 mM) were mixed with FITC-ssDNA (0.1 μM) in Tris-HCl buffer (10 mM, pH 8.5) through a thermostatic mixing device (400 rpm shaking for 1 min at 25 °C). Then after being incubated for 20 min, the fluorescence intensities of the mixture were measured (Figure 1A), and the results showed that the resultant PDA, oxidized and polymerized from DA, can readily quench the fluorescence of FITC-ssDNA. As shown in Figure 1B, the fluorescence intensity of FITC-ssDNA at 522 nm was nearly completely quenched upon being incubated with 5 mM DA for 20 min, and the quenching efficiency reached an equilibrium at 95.72%. Thus, 5 mM DA was considered as an optimal concentration to form PDA for quenching the fluorescence of FITC-ssDNA (0.1 μM). Moreover, the fluorescence responses of 0.1 μM FITC-ssDNA incubated with 5 mM DA were monitored at varying times (Figure 1C). The fluorescence intensity decreased with increasing reaction time and reached an equilibrium at 20 min. Therefore, 20 min was chosen as the optimal reaction time. The formation process of PDA from 5 mM DA was also monitored by UV−vis spectroscopy. As shown in Figure 1D, a continuous increase of the absorbance at 400 nm as a function of the reaction time was C

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solution mixed with 40 μM GSH would not change within 60 min (Figure 2A,B, red line; Figure 2C, column 4). EMSA was also used to test the sample of FITC-ssDNA/DA solution incubated with 40 μM GSH for 20 min. As depicted in Figure 2D, a band appeared in lane 3 similar to that of in lane 1, the result indicated that the oxidative polymerization reaction from DA to PDA was inhibited by GSH, resulting in the freedom of FITC-ssDNA in the mixture. From the above results, we can come to the conclusion that the fluorescence of FITC-ssDNA can be regulated by PDA and antioxidants, which promises great potential for developing a fluorescent assay for antioxidants. Assays for Antioxidants Using the FITC-ssDNA/DA Solution. In this study, the mixture of FITC-ssDNA (0.1 μM) and DA (5 mM; i.e., FITC-ssDNA/DA solution) was used as a sensing system to detect antioxidants. To evaluate the sensitivity, different concentrations of antioxidants were added into the sensing system. As shown in Figure 3A, with

Figure 2. Time-dependent changes of (A) fluorescence at 522 nm and (B) absorbance at 400 nm in the mixture of FITC-ssDNA/DA upon incubated respectively with 0, 20, and 40 μM GSH. Five mM DA and 0.1 μM FITC-ssDNA in Tris-HCl buffer (10 mM, pH 8.5) were used. Measurement was collected per 5 min intervals. (C) Photographs of DNA, DNA/DA, DNA/DA + 20 μM GSH, and DNA/DA + 40 μM GSH solutions showing the different color changes under natural light following a 60 min period with per 10 min intervals (0.1 μM FITCssDNA and 5 mM DA were used). (D) Verification of DNA binding with PDA via electrophoretic mobility shift assay (EMSA). Lane 1, DNA; lane 2, DNA/DA; lane 3, DNA/DA + 40 μM GSH; the samples were incubated for PDA formation and antioxidant redox reaction for 20 min and then loaded into the gel for EMSA.

Figure 3. (A) Fluorescence emission spectra of the sensing system in the presence of different concentrations of GSH (0, 0.05, 0.1, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 10 μM). (B) Plot of (F − F0)/F0 as a function of the increasing concentrations of GSH, where F0 and F are the fluorescence intensity (at 522 nm) of the sensing system in the absence and the presence of GSH.

The results showed that the formed FITC-ssDNA/PDA conjugates cannot run in lane 2 and the fluorescence of FITC-ssDNA was quenched (FITC-ssDNA as a control in lane 1). However, upon FITC-ssDNA/DA solution mixed with GSH, the formation of PDA would be inhibited. The fluorescence intensity (at 522 nm) of FITC-ssDNA/DA solution decreased in a delayed manner (about 20 min) with the addition of 20 μM GSH, and remained stable by adding 40 μM GSH (Figure 2A, green and red lines, respectively). Likewise, the absorbance of FITC-ssDNA/DA solution at 400 nm increased in a delayed manner (about 20 min) upon incubated with 20 μM GSH and stayed the same for 1 h in the presence of 40 μM GSH (Figure 2B, green and red lines, respectively). At the same time, due to the donation of an electron, GSH itself would become reactive and readily react with another reactive GSH to form glutathione disulfide (GSSG) through thiol−disulfide exchange.23 After all the GSH turned to be GSSG, the oxidative polymerization reaction from DA to PDA began to accelerate so that the color of FITCssDNA/DA solution mixed with 20 μM GSH would turn dark gray after 20 min (Figure 2C, column 3), which can also be consistent with the above results in Figure 2A,B. If the concentration of GSH was enough high (e.g., 40 μM), donating a reducing equivalent from GSH would be predominant in this competitive reaction process, and the oxidative polymerization reaction from DA to PDA would be very slow. Thus, the fluorescence, absorbance, and color of FITC-ssDNA/DA

the addition of an increasing concentration of GSH (0−10 μM), the fluorescence of the sensing system was increased gradually. From Figure 3B, it can be seen that the fluorescent intensity at 522 nm is sensitive to the concentration of GSH, where the fitting range is from 50 nM to 10 μM with a linear equation: Y = 0.7577X + 0.2557 (regression coefficient R2 = 0.9973), where Y is the fluorescence ratio ((F − F0)/F0) and X is the concentration of GSH. The limit of detection was estimated to be 16.8 nM based on three times signal-to-noise level of the blank sample (3σ), which is much lower than the concentration of cerebral GSH in rat brain reported previously.27 Considering the abundance as intracellular antioxidants in living brain, AA, Cys, and Hcys were also studied using the sensing system (Figure S3), and the results were similar to that of GSH detection. So the present method could be used for fluorescent monitoring of cerebral antioxidants. In order to test the feasibility of our proposed method in real applications, we evaluated its fluorescent response toward GSH in the artificial cerebrospinal fluid. The spiked concentrations of GSH in different artificial cerebrospinal fluids were measured by our proposed method and the standard Ellman’s assay (i.e., DTNB method, see Supporting Information, Figures S4 and S5). The principle of DTNB method is based on the fact that thiol compounds can react with DTNB and the absorbance of the D

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Analytical Chemistry resultant yellow-colored product can be used to quantify the thiol.25 Recovery of added known amount GSH to the samples was in general larger than 95%, and our proposed method has similar results for GSH detection as that of the DTNB method (Table S1). According to the statistic calculation by a t test (a = 0.1; calculation of t value was shown in Supporting Information), the concentrations of GSH determined by the present method were in good agreement with those obtained by the DTNB method (t = 0.69 for 2 μM, t = 1.92 for 4 μM, t = 1.22 for 8 μM). These results indicated that the present method has a promise in practical applications with great accuracy and reliability. To ensure the sensing system is optimized and stable, some control experiments were further performed to investigate the dynamic reaction process of GSH-oxidant reaction, oxidant-DA reaction, and PDA formation (Figure S6). As revealed in Figure S6, when DA, GSH, and DTNB were mixed together at the same time, the absorbance of the mixture at 412 nm (corresponding to NTB2−, the yellow-colored product from DNTB-thiol reaction) decreased within 20 min and then increased. The reason is that during the first 20 min, some part of GSH reacted with DTNB to obtain the yellowcolored product at 412 nm (Figure S6A). At the same time, DA was oxidized to form PDA which has an absorbance at 400 nm, but the absorbance values are not the same as the NTB2− and a few of GSH would react with oxygen to inhibit the PDA with the absorbance of the mixture at 412 nm decreasing (Figure S6B). Once all the GSH were consumed after 20 min, the spontaneous oxidative polymerization of DA to form PDA would be the main reaction, and the formed PDA has an absorbance at around 400 nm, which can also increase the absorbance of the mixture at 412 nm (Figure S6C,D). Selectivity is a challenge to investigate the sensing of antioxidants in the complex biological samples. The selectivity of our proposed method was evaluated by monitoring (F − F0)/F0 of the sensing system induced by other potential interferences, including amino acids (Val, Ser, Met, Pro, Ile, Thr, Arg, Phe, His, Gly, Lys, Asn, Trp, Gln, Tyr, Leu, Ala, Glu, and Asp), metal ions (Fe3+, Fe2+, Ag+, Cu+, Co2+, Ni2+, Zn2+, Al3+, Cu2+, Na+, Ca2+, K+, and Mg2+), and biological species (H2O2, 5-HT, Lact, DOPAC, GSSG, and glucose) that might coexist in the brain systems. As shown in Figure 4A,B, such potential interferences had little effect on the fluorescence of the sensing system. Meanwhile, competition tests were also investigated, and negligible changes were observed when the amino acids or metal ions or biological species were added into the sensing system in the presence of GSH (Figure 4C,D). All these results demonstrated that our presented method has good selectivity. Determination of the Level of Antioxidants in Rat Brain Microdialysates. To verify our method for neurochemical monitoring during cerebral normal/ischemia/reperfusion process, the content of antioxidants in the rat brain microdialysates was continuously monitored. The samples of rat brain microdialysates from different times were added to the sensing system and after being incubated for 20 min, then the fluorescence intensities were measured. As mentioned above, the fluorescence responses of the sensing system toward AA, Cys, and Hcys are similar to that of GSH. In this respect, for easy analysis, the antioxidant capacity of rat brain was approximately determined using the GSH calibration curve from Figure 3B as a standard, and the results were presented as GSH-equivalent antioxidant capacity (GEAC), as shown in Figure 5. Therefore, the relative concentration of total

Figure 4. (A, C) Selectivity and (B, D) competition experiments for our proposed method toward physiological interferences. (A) Selectivity of amino acids and biological species against GSH: 1, H2O2 (0.5 μM); 2, 5-HT (45 μM); 3, lactate (1.5 mM); 4, DOPAC (45 μM); 5, GSSG (45 μM); 6, glucose (15 mM); 7, Val; 8, Ser; 9, Met; 10, Pro; 11, Ile; 12, Thr; 13, Arg; 14, Phe; 15, His; 16, Gly; 17, Lys; 18, Asn; 19, Trp; 20, Gln; 21, Tyr; 22, Leu; 23, Ala; 24, Glu; 25, Asp; 26, none; and 27, GSH. All the concentrations of amino acids are 45 μM, and GSH is 10 μM. (C) Competition experiment upon the addition of GSH (10 μM) with the coexistence of the aforementioned amino acids and biological species. (B) Selectivity of metal ions against GSH: 1, Fe3+; 2, Fe2+; 3, Ag+; 4, Cu+; 5, Co2+; 6, Ni2+; 7, Zn2+; 8, Al3+; 9, Cu2+; 10, Na+; 11, Ca2+; 12, K+; 13, Mg2+; 14, none; and 15, GSH. Concentration of metal ions and GSH is 10 μM, except K+, Na+, Mg2+, and Ca2+, which are 3 mM. (D) Competition experiments upon the addition of GSH (10 μM) with the coexistence of the aforementioned metal ions.

Figure 5. Time-course study on the relative level of total antioxidants in the microdialysates from the striatum of rat brain under different physiological conditions: (I) normal, (II) ischemia, and (III) reperfusion. All the data were expressed as mean ± SD of three experiments.

antioxidants was estimated to be 42.75 ± 1.52 μM during the surgery of preischemia at around 30 min. The level of microdialysate antioxidants had no obvious changes in the first 10 min and kept slowly decreasing to 31.16 ± 2.05 μM at 40 min of cerebral ischemia. However, the level increased rapidly and restored to be 172.16% (73.60 ± 1.43 μM) of the basal level after 15 min of reperfusion. Then the level of antioxidants decreased gradually to normal level after 60 min of reperfusion. Considering the similar changes in fluorescent properties of the sensing system toward various antioxidants (such as AA, thiol compounds), masking agents were used to achieve the goal for simultaneously monitoring these antioxidants in rat brain microdialysates. It is reported that AA can be oxidized by ascorbate oxidase (AAOx). Thus, upon the brain microdialysate E

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Analytical Chemistry challenged with AAOx, the remaining antioxidants could be total thiol compounds and other antioxidants.28 Likewise, Nethylmaleimide (NEM), a thiol-blocking reagent, can be used to block thiol compounds in the brain microdialysate. When added NEM into the brain microdialysate, the remaining antioxidants could be AA and other antioxidants.29 Furthermore, when added both AAOx and NEM into the brain microdialysate, the remaining antioxidants could be other antioxidants. Then, by pretreatment with AAOx (40 U·mL−1) and NEM (500 μM), respectively, the relative levels of striatum AA, thiol compounds, and other antioxidants in rat brain microdialysates during the cerebral normal/ischemia/reperfusion process were also determined (Figures S7−9). The change trends were similar to the total antioxidants, which can be consistent with the previous reports.30 It is shown that ischemia causes a depletion of glucose and oxygen in brain, and the decrease of antioxidants are likely due to the reduced transportation of antioxidants into the brain because of the reduction of cerebral blood flow.31 The increase of striatum antioxidants during the reperfusion process is considered to be a comprehensive and complicated result of a series of neuropathological processes including anoxia depolarization, glutamate release and reuptake, accumulation of oxidative free radicals, and even neural cell necrosis.32 After 60 min of reperfusion, the antioxidants gradually returned to normal levels. These data demonstrated that monitoring the level of antioxidants can be used for fundamentally indicating the complicated neurochemical changes during cerebral normal/ ischemia/reperfusion processes and would be helpful for a potential sensing platform for determination of antioxidants in the brain systems.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Min Zhang: 0000-0001-5108-1275 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21277048, 21405047, 21675053, and 21635003), the China Postdoctoral Science Foundation (2014M550224 and 2016T90348), and the “Chenguang Program” of Shanghai Education Development Foundation and Shanghai Municipal Education Commission (14CG22).

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CONCLUSION In summary, we have demonstrated a new strategy based on the interaction of dye-labeled ssDNA and PDA for the evaluation of antioxidants in complex biological fluids. This study not only successfully provides an effective, selective, and reliable fluorescent method for detection of cerebral antioxidants in living brains integrated with in vivo microdialysis which is closely related to physiological and pathological events of brain, but also continuously monitored the dynamic changes of the striatum antioxidants during cerebral normal/ischemia/reperfusion process. The simplicity in operation and instrumentation of our proposed method makes it more convenient and more broadly applicable in biochemical investigations and brain chemistry.

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our method and the standard method; The t-test and the statistical comparison; Time-course study on the relative level of AA, thiol compounds, and other antioxidants compounds in the microdialysates (PDF).

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03216. Additional information as noted in text. Microdialysis experiments in vivo; Excitation and emission spectra of FITC-ssDNA and UV−vis absorption spectra of the PDA; A TEM image of PDA; Fluorescence emission of the FITC-ssDNA/DA solution in the presence of different concentration of AA, Cys, and Hcys, respectively; The principle of the DTNB method; UV spectra of the DNTB method for the detection of GSH; Selectivity analysis of amino acids and biological species against GSH using the DNTB method; Comparison of F

DOI: 10.1021/acs.analchem.6b03216 Anal. Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.analchem.6b03216 Anal. Chem. XXXX, XXX, XXX−XXX