Target-Induced Horseradish Peroxidase Deactivation for Multicolor

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Target-Induced Horseradish Peroxidase Deactivation for Multicolor Colorimetric Assay of Hydrogen Sulfide in Rat Brain Microdialysis Zhonghui Chen, Chaoqun Chen, Huawei Huang, Fang Luo, Longhua Guo, Lan Zhang, Zhenyu Lin, and Guonan Chen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Analytical Chemistry

Target-Induced Horseradish Peroxidase Deactivation for Multicolor Colorimetric Assay of Hydrogen Sulfide in Rat Brain Microdialysis

Zhonghui Chen†, Chaoqun Chen†, Huawei Huang†, Fang Luo†‡, Longhua Guo†*, Lan Zhang†*, Zhenyu Lin†*, Guonan Chen

†Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China ‡College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian 350116, China

*Corresponding author: E-mail: [email protected] (L. Guo); [email protected] (L. Zhang); [email protected] (Z. Lin) Tel. & Fax.: +86 591 22866135

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ABSTRACT Hydrogen sulfide (H2S) is important for normal neural functions, which involves in protecting neurons from oxidative stress and neuronal transmission modulation in brain. The detection of H2S is significant for revealing its role in the diagnosis of various disease. In this study, a novel multicolor colorimetric assay based on the etching of gold nanorods (Au NRs) is proposed to evaluate H2S level with the naked eye. This measurement relies on the catalytic oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) via horseradish peroxidase (HRP) to produce TMB2+, which could etch the Au NRs quickly and accompany with a distinct color change. The vivid colors can be easily distinguished with the naked eye without any sophisticated instruments. The presence of H2S can cause the deactivation of HRP, which affects the amount of TMB2+ produced and consequently affects the color changing of the system. Based on this mechanism, a simple but sensitive multicolor colorimetric assay is developed for H2S detection with a linear range of 0.05 ~ 50 µM. The proposed method is demonstrated for monitoring extracellular H2S in rat brain coupled with microdialysate.


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INTRODUCTION Hydrogen sulfide (H2S), along with nitric oxide and carbon monoxide, as a gasotransmitter molecule in living biosystems has drawn much attention recently.1-3 As an indispensable component of signaling pathways, H2S involves in several physiological functions under physiological conditions, including vasodilation, angiogenesis, oxygen sensing, apoptosis, inflammation, and neuromodulation.4-6 In addition, endogenous H2S level is related to many diseases, such as Down syndrome, Alzheimer’s disease, diabetes, and liver cirrhosis. Considering the far-ranging impacts of H2S homeostasis on human disease, many sensitive methods,

such

as

colorimetric,7-13

electrochemistry,14-20

electrochemiluminescent,21

fluorescence spectroscopy,22-30 chemiluminescent,31 and surface-enhanced raman scattering,32 have been developed for H2S detection. Colorimetric assay is one of the most popular techniques for target detection because it is easy to be recognized by naked eye or quantified by ultraviolet-visible spectrometer (UV-vis). The classical colorimetric detection method for H2S only present single color changing from light to deep.7 As human eyes are insensitive to optical density variations of the same color, and the method can be only used for qualitative detection owing to the one-color change in response to different concentrations of H2S and a spectrometer is need for quantify. If one assay shows vivid color variations upon the addition of H2S, it can greatly improve the application of H2S colorimetric assay since naked eye are sensitive to different color change.

Recently, noble metal nanomaterials based colorimetric sensing have attracted great interest owing to their excellent physical and chemical properties. Noble metal nanomaterials, such as gold nanoparticles (Au NPs),33 gold nanorods (Au NRs),34-38 gold nanobipyramids (Au NBPs),39 show great potential for the development of different kinds of chemical and biological sensing depend on their highly controllable shape and tunable localized surface plasmon resonance (LSPR) peak from the visible to near-infrared wavelength range. Among these noble metal nanomaterials, Au NRs show great promising applications as the signal transducer in the design of multicolor colorimetric sensing. Au NRs have two separate surface plasmon resonances (SPR) bands, which corresponding to transverse and longitudinal peak,



respectively. Different optical signals can be acquired in a wide range of wavelength by simply adjusting the aspect ratio. On the basic of this character, our group developed a novel multicolor biosensor through the etching of gold nanorods (Au NRs).35-37 The biosensor relied on the catalytic oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) via horseradish peroxidase (HRP) to produce TMB2+, which could etch the Au NRs quickly and accompanied with a distinctly color change of the system. This strategy has been applied to detect many disease markers, such as carcino-embryonic antigen, prostate specific antigen. The procedure of the proposed multicolor colorimetric method is simplified by mixing of the required compounds, which can operate by untrained person easily and apply to evaluation in the field.

In this study, we demonstrate a technologically simple but sensitive and selective method for direct multicolor colorimetric sensing of H2S. The present of H2S can cause the deactivation of HRP and hence further affects the amount of TMB2+ produced. Different deactivation degrees of HRP produced different amount of TMB2+ that can etch Au NRs, which is accompanied by the vivid colors changing, and this phenomenon can be discern by the naked eye easily. The proposed colorimetric assay has been applied to analyze H2S in the rat brain successfully.

EXPERIMENTAL SECTION Material and Reagent Sodium sulfide (Na2S·9H2O, 98%), HRP, TMB liquid substrate system (containing 3,3′,5,5′ tetramethylbenzidine and peroxide solution), ascorbic acid (AA) and sodium bromide (NaBH4) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cetyltrimethyl ammonium bromide (CTAB), hydrochloric acid (HCl) and chloroauric acid tetrahydrate (HAuCl4·4H2O) were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Artificial cerebrospinal fluid (aCSF) was prepared by dissolving 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 ultrapure water, and the pH of the solution was adjusted to the range of pH 7.2 ~ 7.4. All polystyrene 96-well signle-break strip plates were


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purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). All other reagents were of analytical grade without further purification. Ultrapure water was from Direct-Q3 UV system (Millpore, 18.2 MΩ·cm).

Instruments Transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, FEI, USA) was used to study the different oxidation stage of the Au NRs. UV-vis absorption spectra were recorded with Microplate spectromphotometer (Multiskan GO, Thermo Scientific, USA). All photos were taken by digital camera (EOS 600D, Canon, Japan).

Synthesis of Au NRs Au NRs were prepared according to early reported literature.34,36,37 Briefly, the seed solution was prepared by adding a freshly prepared, ice-cold NaBH4 solution (0.01 M, 0.6 mL) to a mixed solution containing HAuCl4 (5 mM, 5 mL) and CTAB (0.2 M, 5 mL) in a 15 mL glass tube under vigorous stirring for 2 min. The seed solution turned to brownish yellow and kept at room temperature for 30 min. To prepare the growth solution, AA (0.1 M, 5.5 mL) was added to a well-mixed solution containing HAuCl4 (0.01 M, 5 mL), AgNO3 (0.01 M, 0.6 mL), and CTAB (0.1 M, 50 mL). The mixture solution was diluted to 100 mL using deionized water. Subsequently, 200 µL of the seed solution was injected into the growth solution. The resultant mixture was vigorously stirred for 30 s and then centrifuged at 30 oC for 12 h. Finally, Au NRs were purified through centrifugation twice (8000 rpm).

Multicolor Colorimetric Protocol for H2S Detection Various concentrations of H2S standard were initially prepared using Na2S as the source and aCSF as the sample matrix. H2S standard solution (or microdialysates, 120 µL) was added to the tube bottom, and HRP solution (15 µL) was pipetted onto the internal side of the cap of the tube. Then the above tube was sealed immediately and incubated at room temperature. Subsequently, HRP solution (10 µL) was transferred into the 96-well plates, followed by the addition of TMB substrate solution (100 µL). HCl (5 M, 20 µL) was then added to the plates to end the enzymatic reaction. The solution turned from blue to yellow, suggesting TMB2+



was produced. Finally, Au NRs was added to the solution with gentle mixing for 3 min. Such solutions were monitored by microplate spectrophotometer or distinguished with the naked eye.

In Vivo Microdialysis Animal surgery and in vivo microdialysis were carried out according to the reported procedures.21 All experimental procedures were approved by the Ethics Committee of Experimental Animals of Fujian Medical University. Briefly, adult male Sprague-Dawley (SD) rats (250-300 g) purchased from Experimental Animal Center of Fujian Medical University (Fuzhou, China) were served as subjects. The rats were housed on a light-dark schedule (12:12 h) with food and water ad libitum. The rats were anesthetized with 2 ~ 3% isoflurane by animal anesthesia ventilator system (RWD Life Science Co. Ltd, Shenzhen, China) and positioned onto a stereotaxic frame (RWD Life Science Co. Ltd, Shenzhen, China). During the surgery and anesthetic, the temperature of animal body was maintained at 37 oC using a heating pad (RWD Life Science Co. Ltd, Shenzhen, China). The microdialysis guide cannulas (Eicom, Kyoto, Japan) were carefully implanted in different brain regions according to standard stereotaxic procedures. The guide cannula was fixed with three skull screws and dental cements. Insertion of guide cannulas will cause tissue trauma, which could influence the result of microdialysis experiments. So, after being embedded the micordialysis guide cannulas, the animals are normally allowed to recover for at least 24 h before in vivo microdialysis.40 Then, a microdialysis probe (50 kD, 3 mm) was implanted into the rat region to perfuse aCSF solution at 1.0 µL/min. After the aCSF solution continuously perfused (at least 90 min) for equilibration, the catheter was directly delivered into the cryogenic sample collector (Eicom, Kyoto, Japan) for continuous collecting of microdialysate for 120 min. Finally, 120 µL microdialysis was added to the tube and the next procedure was the same as the abovementioned process for H2S detection.

RESULTS AND DISCUSSION Principle of the Proposed Multicolor Colormetric Assay


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Scheme 1 shows the principle of the designed multicolor colorimetric method for H2S detection. A droplet of HRP solution was coated onto the inside tube cap by surface tension firstly. As H2S gas can easily volatilize from the samples and then be trapped by the droplet in the tube cap, making HRP lost its catalytic activity. Compared with HRP, the deactivated HRP had lower affinity for TMB liquid substrate, which has been verified by steady-state kinetic parameters (Figure S1 in supporting information). The result is similar to that reported.41-43 HRP with different deactivation degrees were transferred into the 96-well plates, followed by the addition of TMB liquid substrate. The TMB0 (colorless) was oxidized into TMB+ (blue) by H2O2 in presence of different deactivation degrees of HRP. HCl was then added to end the enzymatic reaction, TMB+ was turning into TMB2+ (yellow). The concentration of TMB2+ produced was gradually reduced with the increase of H2S concentration. Finally, an appropriate amount of CTAB-capped Au NRs was added to the TMB2+ solution with sufficient mixing. Au NRs were selectively etched to different levels by TMB2+, causing the vivid color changing of the system and which can be discerned with the naked eye easily; this outcome can be used to represent the concentrations of H2S. On the basis of this principle, a pretty straightforward multicolor colorimetric assay can be developed for H2S detection.

The preferred position for Scheme 1

Control assays were performed to demonstrate the feasibility of the proposed system. Figure 1A is a typical UV-vis spectrum for Au NRs, two extinction peaks exhibit at 739 and 515 nm (curve a), corresponding to the longitudinal plasmon band and transverse plasmon band, respectively. Upon the introduction of TMB liquid substrate to Au NRs solution, the longitudinal LSPR peak did not change and the color of the solution was the same as that of the Au NRs, indicating that only TMB liquid substrate cannot successfully etch Au NRs. If HRP has been deactivated by the different concentration of H2S firstly, then added into the microwell plates, which induced the blue shifts longitudinal plasmon band of Au NRs (curve b, c, and d). And the solution displayed different color, indicating the successful etching of Au NRs. The etching process was further confirmed by TEM. The corresponding TEM images were shown in Figure 1B. The length, average diameter, and aspect ratio of original Au NRs



were ~57 nm, ~13 nm, and ~4.38, respectively (Figure 1B(a)). The length of Au NRs was gradually reduced while the diameter remained almost unaltered (Figure 1B(b, c, and d)). These phenomena confirm the feasibility of the principle.

The preferred position for Figure 1

Optimization of the Reaction Conditions To acquire the best performance for H2S detection, the effect from the experimental conditions, such as CTAB concentration, HCl concentration, TMB2+ etching time, pH of H2S trapped buffer, HRP concentration, and trapped reaction time, were investigated in detail. ∆λ represents the longitudinal plasmon band peak shift of Au NRs, which is chosen as the indicator of the morphological change of the Au NRs. The CTAB concentration is the major factor for TMB2+ etching process, which has been optimized firstly. Figure 2A shows that ∆λ increased with the increment of CTAB concentration and reached a platform at 0.12 M. So, 0.12 M of CTAB was selected for the following study. HCl in this study was used to end the enzymatic reaction and increase the etching rate. The concentration of HCl was optimized as well. Figure 2B reveals that ∆λ increased rapidly and then remained unchanged when its concentration exceeded 0.3 M. So, 0.3 M of HCl was chosen as the optimal concentration. Figure 2C shows the effect of reaction time of TMB2+ etching one ∆λ. The value of ∆λ increased gradually firstly, and then reached a platform at 2.5 min. So, 2.5 min was chosen for the subsequent study. In this work, we utilized HRP to trap the H2S from the solution by static headspace strategy and the colorimetric signals were originated from the deactivation degrees of HRP. As the pH value of the buffer affect H2S trapping process and HRP catalytic activity greatly, so which was optimized too. As shown in Figure 2D, if the reaction buffer was acidic or alkaline, the activity of HRP both received inhibition and the most appropriate pH value was 6.5 (black curve). The reaction buffer was also concerned with the effects of H2S trapping process (red curve). ∆λ increased firstly and then decreased as the enhancement of the pH of the buffer. The most appropriate pH value was in the range of 6.5 ~ 7.0. So, pH 6.5 was chosen for the following work. The concentration of HRP has great influence on the performance of the


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proposed assay also. As shown in Figure 2E, the value of ∆λ increased slowly in the case that the concentrations of HRP were below 0.15 µg/mL and increased gradually when the concentrations of HRP were over 0.15 µg/mL. Therefore, 0.15 µg/mL of HRP was used to trapping H2S in this work. In addition, trapped reaction time can directly affect the deactivation degree of HRP. The colorimetric signal reduced with the increment of reaction time and remained unchanged at 3 min (Figure 2F). Longer reaction time did not cause noticeable colorimetric signal change. Hence, the reaction time was set as 3 min in this study.


Calibration Curve and Selectivity for H2S Determination Under the optimal conditions, the dynamic measurement range and the sensitivity were investigated. We used microplate reader to quantitatively detect the concentrations of standard H2S on the basis of LSPR change from 400 to 850 nm. The longitudinal plasmon band absorbance intensity showed blue shift with the decrease of the H2S concentration (Figure 3A). As presented in Figure 3B, the calibration curve had a good linear relationship with the H2S concentration in the range of 0.0500-50.0 µM and the regression equation was as follows ∆λ = -37.1 log C + 104

(1)

where ∆λ represents the longitudinal plasmon band peak shift of Au NRs, and C is the concentration of H2S. The regression coefficient was 0.997 and the limit of detection was estimated to be 0.0190 µM (S/N = 3). Furthermore, vivid colors (pink, purple, blue, green, gray and reddish brown) corresponding to varying concentrations of H2S could be readily distinguished with the naked eye at a glance. As the solution color has a direct relationship with the concentration of H2S, it offers a convenient way for semi-quantitative detection of H2S.


The selectivity for H2S detection is a significant challenge because the brain environment is very complicated. As shown in Figure 4, the selectivity of the proposed method was evaluated



by calculating ∆λ values, and the employed potential interferences included anions (Cl-, CO32-, SO42-, NO3-, Br-, F-), metal ions (Na+, K+, Mg2+, Ca2+) and biological species (GSH, Cys, AA, DA, UA, Glu). The shift of ∆λ was significantly decreased only when the target H2S added. This assay shows high selectivity owing to H2S can be easily volatilized form samples when the solution adjusts to acidity and the use of headspace trapped strategy. These results indicate that the developed multicolor colorimetric method can selectively detect H2S in the rat brain without the interference of coexisting species, even other sulfur-containing species.


Visual Detection of H2S in Rat Brain To demonstrate the validity of our established method, in vivo microdialysate was carried out in different regions of rat brain, including hippocampus, striatum, and cortex. Obviously, different concentrations of H2S were clearly observed in different brain regions via the developed method. These results were consistent with the reported methods.44,45 To evaluate the accuracy of the proposed method, the values detected by the H2S content assay kit were used as reference to examine the accuracy of proposed assay. Then, the recovery studies were performed using Na2S as the donor (containing 1 mM EDTA). The recovery rates ranged from 93.4% to 95.3%. The determined results were summarized in Table 1. These results suggest that our developed multicolor colorimetric assay has great potential application for in vivo detection of H2S.

The preferred position for Table 1

CONCLUSIONS In summary, we have successfully developed a novel multicolor colorimetric assay for highly selective and sensitive monitoring H2S. The present of H2S can induce HRP deactivation and reduce the amount of TMB2+ produced, which affects the etching degree of Au NRs and vivid color changing of the solution can be discerned with the naked eye easily. Our work


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demonstrated that the proposed multicolor colorimetric assay could be applied in the reliable measurement of H2S in microdialysate of different rat regions. This multicolor H2S sensor displays advantages of speediness, simplicity, visualization as well as low cost, which is not only suitable for laboratory analysis, but also suitable for in field fast measurement.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Steady-State Kinetic Parameters for the Deactivation of HRP; Figure S1; Table S1.

AUTHOR INFORAMTION ORCID Fang Luo: 0000-0001-7495-450X Longhua Guo: 0000-0003-0706-0973 Zhenyu Lin: 0000-0001-7890-6812 Corresponding Author E-mail: [email protected] (L. Guo) E-mail: [email protected] (L. Zhang) E-mail: [email protected] (Z. Lin) Tel. & Fax.: +86 591 22866135

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Science Foundation of China (No. 21575025, 21575027, 21675028 and 21775026), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), the Natural Science Foundation of Fujian Province (No. 2018J05018) and the Foundation for Scholars of Fuzhou University (No. XRC-1671).



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Scheme 1.

Scheme 1. Schematic illustration of the multicolor colorimetric biosensing for H2S detection.



Figure 1.

Figure 1. (A) The UV-vis spectra change of Au NRs under different conditions. a: Au NRs; b: H2S (50.0 µM) + HRP + H2O2 + TMB + Au NRs; c: H2S (5.0 µM) + HRP + H2O2 + TMB + Au NRs; d: H2S (0.5 µM) + HRP + H2O2 + TMB + Au NRs. The inset picture shows the colors of the corresponding solutions. (B)Typical TEM images of Au NRs before and after etching with different concentration of H2S introduced. (a) Original Au NRs; (b) 50.0 µM, (c) 5.0 µM and (d) 0.5 µM H2S present.


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Figure 2.

Figure 2. The effect of different reaction conditions on ∆λ. (A) CTAB concentration, (B) HCl concentration, (C) etching time, (D) pH of the HRP buffer (curve a: without H2S; curve b: with H2S), (E) HRP concentration, and (F) H2S enrichment reaction time. Error bars represent the standard error derived from three repeated measurements.



Figure 3.

Figure 3. (A) The UV-vis spectra change of Au NRs in the oxidation etching process with different concentrations of H2S. The concentrations are as follows: a ~ j: 0.0100, 0.0500, 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 80.0, and 100 µM; (B) The LSPR shift of Au NRs as a function of H2S concentration; (C) Color change of the plasmonic sensors with the increase of H2S concentration.


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Figure 4.

Figure 4. Selectivity of the proposed multicolor sensor for H2S detection. Error bars represent the standard error derived from three repeated measurement.



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Table 1. H2S determined by the developed method in the rat brain microdialysis (n = 3).

Detected Brain region (µM)

Detected by H2S

Relative error

content assay kit

of the results

(mean SD, µM)

(%)

Solution color

Spiked

Found (mean SD, µM)

(µM)

and recovery rate (%)

Striatum

9.6 ± 1.3

10.2 ± 1.9

-5.9

25

32.3 ± 2.0 (93.4)

Hippocampus

16.6 ± 2.7

17.5 ± 1.7

-5.1

25

39.3 ± 2.3 (94.5)

Cortex

2.5 ± 1.5

No Found

-

25

26.2 ± 2.2 (95.3)


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