A Colorimetric Sensor for the Highly Selective

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Mar 20, 2017 - Na2SO3, Na2S2O3, NaH2PO4, Na2HPO4, Na3PO4, Zn(NO3)2, Fe(NO3)3, Cu(NO3)2, CaCl2, Mg(Cl)2,. NaF and other inorganic salts were ...
sensors Article

A Colorimetric Sensor for the Highly Selective Detection of Sulfide and 1,4-Dithiothreitol Based on the In Situ Formation of Silver Nanoparticles Using Dopamine Lingzhi Zhao 1,2, *, Liu Zhao 3 , Yanqing Miao 1 , Chunye Liu 1 and Chenxiao Zhang 2 1 2 3

*

Department of Pharmacy, Xi’an Medical College, Xi’an 710021, China; [email protected] (Y.M.); [email protected] (C.L.) Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China; [email protected] Beijing Research Center of Agricultural Standards and Testing, Beijing 100097, China; [email protected] Correspondence: [email protected]; Tel.: +86-158-2958-3060

Academic Editor: W. Rudolf Seitz Received: 18 February 2017; Accepted: 16 March 2017; Published: 20 March 2017

Abstract: Hydrogen sulfide (H2 S) has attracted attention in biochemical research because it plays an important role in biosystems and has emerged as the third endogenous gaseous signaling compound along with nitric oxide (NO) and carbon monoxide (CO). Since H2 S is a kind of gaseous molecule, conventional approaches for H2 S detection are mostly based on the detection of sulfide (S2− ) for indirectly reflecting H2 S levels. Hence, there is a need for an accurate and reliable assay capable of determining sulfide in physiological systems. We report here a colorimetric, economic, and green method for sulfide anion detection using in situ formation of silver nanoparticles (AgNPs) using dopamine as a reducing and protecting agent. The changes in the AgNPs absorption response depend linearly on the concentration of Na2 S in the range from 2 to 15 µM, with a detection limit of 0.03 µM. Meanwhile, the morphological changes in AgNPs in the presence of S2− and thiol compounds were characterized by transmission electron microscopy (TEM). The as-synthetized AgNPs demonstrate high selectivity, free from interference, especially by other thiol compounds such as cysteine and glutathione. Furthermore, the colorimetric sensor developed was applied to the analysis of sulfide in fetal bovine serum and spiked serum samples with good recovery. Keywords: sulfide; 1,4-dithiothreitol; silver nanoparticles; colorimetric sensor

1. Introduction Hydrogen sulfide (H2 S), for centuries considered as a toxic gas with its characteristic odor of rotten egg, displays both acute and chronic toxicity at high concentrations [1–3]. On the other hand, recently, H2 S has emerged as the third endogenous gaseous signaling compound (gasotransmitter) along with nitric oxide (NO) and carbon monoxide (CO) [4,5]. More recent studies have demonstrated that H2 S is also produced via enzymatic reaction in the brain and several smooth muscles and can mediate a wide range of physiological processes, such as vasodilation, antioxidation, anti-apoptosis, and anti-inflammation [5–8]. The abnormal level of H2 S was closely associated with diseases such as Alzheimer’s disease, hypertension, liver cirrhosis, and Down’s syndrome [9–11]. Therefore, new methods for the accurate and sensitive detection of H2 S levels, recognized as providing valuable information on illuminating the physiological roles of H2 S in biology, are in high demand. Since H2 S is a kind of gaseous molecule, conventional approaches for H2 S detection are mostly based on the detection of sulfide for indirectly reflect H2 S levels. In aqueous media, sulfide can be Sensors 2017, 17, 626; doi:10.3390/s17030626

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found in different forms such as dissolved H2 S, bisulfide anion (HS− , pKa1 = 6.88), and sulfide anion (S2− , pKa2 = 14.15), depending on water pH [12,13]. Thus, there is great importance in developing new and practical methods for sulfide determination to reflect H2 S levels in biosystems. Several methods have been reported for the determination of sulfide such as electroanalytical and spectrometric methods, high-performance liquid chromatography, and inductively coupled plasma atomic emission spectroscopy (ICP-AES) [14–24]. However, these methods are time-consuming, involve complicated procedures, necessitate expensive and complex equipment, or require specialized skills. Therefore, the further development of highly sensitive, fast-responding, cost-effective, non-polluting, portable devices for monitoring trace levels of sulfide anion in a variety of environmental applications and biosystems is a worthwhile and challenging undertaking. Owing to the high affinity between many metal ions and sulfide (Ksp of CuS = 1.27 × 10−36 , Ksp of Ag2 S = 6.3 × 10−50 at room temperature) [25,26], many works have aimed to design a tunable difunctional probe that not only can realize the detection of metal ions but can also be tuned to construct a chemosensing ensemble with metal ions for the detection of S2− . Among these metal nanoparticles, colorimetric sensors based on gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) have attracted much attention because of their unique optical properties. For instance, AgNPs make a strong absorption band that can be observed in the visible to near-UV region of the spectrum, and the color of AgNP suspension depends on the diameter and distance of the nanoparticles. In particular, the dispersed AgNP solution is bright yellow, while the highly aggregated AgNP solution is brownish black. This characteristic leads to new competencies for chemical sensing that are both useful and extraordinarily sensitive for the detection of various species in biological, chemical, and environmental fields. Owing to their simplicity, high sensitivity, and designability, AgNPs based on plasmon resonance have made great achievements in the colorimetric assays for the detection of cysteine, metal ions, and physiologically important species [27–37]. However, the synthesis of AgNPs with distinctive sizes and shapes is usually carried out by chemical reduction with or without stabilizing agents, which is effective but uses many toxic substances, making the process potentially harmful to the environment. Therefore, environmentally friendly or green synthetic methods are in high demand. In this work, we used silver/dopamine nanoparticles to develop a one-step and colorimetric sensing method for the detection of sulfide. Herein, silver/dopamine nanoparticles were in situ synthesized by using only AgNO3 , dopamine, NaOH, and Milli-Q water and mixing them in sequence, this process that meets demands for environmentally friendliness or green synthesis. Dopamine could reduce Ag+ to AgNPs and functionalize it to form monodispersed AgNPs in alkaline conditions. It is very interesting to note that the addition of sulfide to the initially prepared dopamine-stabilized AgNP colloidal solution decreased the plasmon absorbance of AgNPs at 400 nm and turns the dispersion color from bright yellow to dark brown observed with the naked eye. This phenomenon was elucidated by the degradation of the structure of silver nanoparticles (AgNPs) due to the formation of Ag2 S by the reaction between Ag+ and S2− . A graphic representation of the sensor design and the detection strategy is displayed in Scheme 1. Since the AgNPs becomes decomplexed from dopamine, the material properties change, e.g., plasmon absorbance and the yellow color disappear. These features actually form the basis for the development of a technically simple yet effective colorimetric method for the quantitative analysis of sulfide in biosamples. Moreover, considering that thiol compounds have a strong effect on plasmon resonance of AgNPs, we also studied field transmission electron microscope (TEM) images, absorption spectra, and UV-Vis spectra of AgNPs in the presence of different thiol compounds, including L-cysteine (Cys), homocysteine (Hcy), glutathione (GSH), oxidized glutathione (GSSG), 11-mercaptoundecanoic acid (MUA), N-acetyl-L-cysteine (N-Cys), and 1,4-dithiothreitol (DTT). Among these thiol compounds, only DTT, which contained two proximal sulfhydryl groups, can crosslink monodisperse AgNPs to form aggregated AgNPs with a larger particle size and a reddish brown color, resulting in the corresponding changes in the absorbance peak. This feature actually established the development of the technically simple yet effective colorimetric method for the quantitative analysis of DTT, a safe use of this important substance in cell biology, biochemistry,

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and biomedical applications. In conclusion, a colorimetric sensor for the highly selective detection of sulfide and DTT was developed based on the in situ formation of AgNPs using dopamine, which helps to further establish microfluidic paper-based analytical devices for point-of-use diagnostics without external power supplies or supporting equipment based on the color change of AgNPs. 2. Experimental 2.1. Reagents and Materials Dopamine, lactate, glucose, sodium ascorbate, uric acid, 3,4-dihydroxyphenylacetic acid, 5-hydroxytryptamine, and homocysteine (Hcy) were all purchased from Sigma-Aldrich (Shanghai, China). Cysteamine hydrochloride (Cym), L-cysteine (Cys), L-dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), glutathione (GSH), oxidized glutathione (GSSG), N-acetyl-Lcysteine (N-Cys) and other amino acids, adenosine 5’-triphosphate disodium salt hydrate (ATP), adenosine 5-diphosphate sodium salt (ADP), adenosine 5-monophosphate monohydrate (AMP), and sodium sulfide non-ahydrate (Na2 S·9H2 O) were obtained from the Aladdin company (Shanghai, China). Silver nitrate (AgNO3 ), NaOH, potassium pyrophosphate (PPi), Na2 SO4 , NaCl, KNO3 , Na2 SO3 , Na2 S2 O3 , NaH2 PO4 , Na2 HPO4 , Na3 PO4 , Zn(NO3 )2 , Fe(NO3 )3 , Cu(NO3 )2 , CaCl2 , Mg(Cl)2 , NaF and other inorganic salts were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). All other chemicals were at least analytical grade reagents and used without further purification. Aqueous solutions were prepared with Milli-Q water (18.2 M·Ω·cm−1 ). Unless otherwise pointed out, all experiments were carried out at room temperature. 2.2. Apparatus The absorbance and photoluminescence (PL) spectra were recorded in 1 cm quartz cells on a UV-Vis spectrophotometer (UV-2450, Shimadzu Corporation, Tokyo, Japan). Field transmission electron microscope (TEM) studies were carried out with the Tecnai G2 F20 (FEI Company, Hillsboro, OR, USA) operating at a 200 kV accelerated voltage. 2.3. Recommended Analytical Procedure Silver/dopamine nanoparticles were synthesized according to a previously reported method [38]. Twelve microliters of dopamine (4 mM) and 1 mL of ultrapure water were mixed in a 1.5 mL centrifugal tube. Then, 16 µL of NaOH (0.1 M) and 30 µL of AgNO3 (8 mM) were added to the tube in sequences. The mixture was incubated at room temperature for 15 min to obtain monodispersed and stable silver/dopamine nanoparticles. For the detection of S2− , dithiothreitol, and other analytes, the stock solution (10 mM) of various analytes was first prepared in ultrapure water and then was added into silver/dopamine nanoparticles in the tube at different final concentrations for 20 min before UV-Vis measurements and photographs were taken. 2.4. Colorimetric Sensing of S2− in Fetal Bovine Serum The samples were all prepared by dissolving Na2 S directly into Milli-Q water as a standard S2− solution. Twenty microliters of the solution at different concentrations and 400 µL of methanol were added to 180 µL of serum and vortexed thoroughly. The mixture was allowed to stand for about 10 min at room temperature to complete protein precipitation. After centrifugation at 13,000 r/min in an Eppendorf centrifuge, the solvent was evaporated to dryness, and samples were redissolved in 20 µL of Milli-Q water as spiked samples. For the colorimetric sensing of S2− in fetal bovine serum, a 20 µL sample was added to 1 mL of the aqueous dispersion of AgNPs. After being allowed to stand for 15 min, the concentrations of S2− in the fetal bovine serum were determined via UV-Vis spectroscopy.

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3. Results and Discussion 3.1. Absorption and TEM Studies of Silver/Dopamine Nanoparticles after Incubation with S2− Because of the degradation of the structure of silver nanoparticles (AgNPs) due to the formation of Ag2 S by the reaction between Ag+ and S2− , silver nanoparticles are chosen as an assay material for an effective optical determination of S2− . What is more, it is well known that AgNPs display an SPR absorption band at around a wavelength of 400 nm. Since the position of the SPR band depends on the AgNP size and the local environment, such as the pH and the salting strength, AgNP colloidal solution is very sensitive to the addition of analytes with the alteration in SPR spectra and color change in the solution, which can be easily monitored with the naked eye or with a UV-Vis absorption spectrophotometer. In particular, the dispersed AgNP colloidal solution is bright yellow, while the highly aggregated AgNP solution is red or black. From the above, AgNPs serve as an excellent material for employing and developing colorimetric sensors for the detection of physiologically important species in biosystems. Moreover, considering that dopamine has the ability to reduce Ag+ to a monodispersed AgNP colloidal solution and functionalize the formed AgNPs in an alkaline solution and at ambient temperature, silver/dopamine nanoparticles, which can obtained by a simple corresponding solution mixed in sequences and does not need complicated synthesis and modification procedures, was chosen in the development of a colorimetric sensor for the detection of S2− . To investigate the feasibility of the as-designed sensing method based on the in situ formation of an AgNP colloidal solution using dopamine as a reducing and protecting agent, we conducted UV-Vis absorption spectrum measurements of silver/dopamine nanoparticles in the presence and absence of S2− . Curve A of Figure 1A displayed a strong absorbance band centered at about 400 nm with a bright yellow color for silver/dopamine nanoparticles in the absence of S2− (Figure 1A inset: the first tube from left to right), which is the characteristic absorption profile of the as-formed AgNPs. Transmission electron microscopy (TEM) images of the as-formed AgNPs displayed a particle size about 10 nm in diameter, indicated a homogeneous size distribution, and was well dispersed (Figure 2A). The addition of S2− to the initially prepared dopamine-stabilized AgNP colloidal solution sharply decreased the plasmon absorbance of AgNPs at 400 nm and turned the dispersion color from bright yellow (the first tube) to dark brown (Figure 1A inset). Based on the phenomena mentioned above, a possible mechanism was proposed by the degradation of the structure of silver nanoparticles (AgNPs) due to the formation of Ag2 S, which consequently precipitated from the dispersion by the reaction between Ag+ and S2− according to the following equation: 4Ag + O2 + 2H2 O + 2 S2− → 2Ag2 S + 4OH− Since the solubility product constant of Ag2 S is 6.30 × 10−50 , Ag has a very high reactivity with sulfide in an aqueous solution, which in turn results in the formation of Ag2 S. In general, the process must be divided into two steps: Figure 1A inset shows the color changes in the AgNPs before and after the reaction, with different concentrations of S2− ; as can be seen, the color changed from bright yellow (the first tube) to dark brown (Figure 1A inset). The reaction time is 20 min, and the dark brown color can be explained by the fact that the AgNPs became closer to each other and aggregated to some extent after the introduction of S2− in the first 20 min. Another reason is that the as-formed dark colloidal solution must be an Ag2 S colloidal solution, which was validated by mixing equal amounts of AgNOs and Na2 S (the product must be Ag2 S), as shown in Figure S1. We can also obtain the same dark colloidal solution. This is the first step; however, when the reaction time exceeded 1 h, the AgNP colloidal solution was broken down, and the as-formed complex between AgNPs and S2− was consequently precipitated from the dispersion and, after 24 h, this reaction colloidal solution gradually became a transparent dispersion, and small black particles as well as biothiols gathered at the bottom of the tubes. This is the second step. These small black particles must be Ag2 S. We confirmed the composition of the products in Figure 2C. Ag and S had the highest content among the elements.

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Sensors 2017, 17, 626 The ratio of Ag to S is almost 3:1, which means that the ratio of Ag2 S to Ag must be 1:1. Sensors 2017, 17, 626

5 of 13 The Ag became 5 of 13 decomplexed from dopamine as material properties such as plasmon absorbance and yellowness demonstrated that the average particle sizeand of the AgNPs TEM after reaction withalso S2− demonstrated grew from 10 nm to disappeared due the changes in the size size (Figurewith 2B) demonstrated thattothe average particle of thestructure. AgNPs after reaction S2− grew from 10 nmthat to 2 − about 20–30 nm in diameter. This diminishing of the plasmon absorbance of AgNPs at 400 nm in the the average particle size of the AgNPs after reaction with S absorbance grew fromof10AgNPs nm to at about 20–30 nm about 20–30 nm in diameter. This diminishing of the plasmon 400 nm in2− the 2− presence of This S2− against the blank wasplasmon used as an analyticalofparameter for the determination of S .SThe in diameter. diminishing of the absorbance AgNPs at 400 nm in the presence of presence of S2− against the blank was used as an2−analytical parameter for the determination of S2−. The 2 − working principle of a colorimetric assay of S is schematically represented in Scheme 1. Figure 1A,B against the blank of was used as an analytical for therepresented determination of S .1.The working working principle a colorimetric assay of S2−parameter is schematically in Scheme Figure 1A,B 2−spectrum shows aof typical UV-Vis absorption of the AgNP colloidal solution1.after reaction with Sa2−, principle a colorimetric assay of S is schematically represented in Scheme Figure 1A,B shows shows a typical UV-Vis absorption spectrum of the AgNP colloidal solution after reaction with S2−, 2− , to and the relative spectrum spectrum change depended on the concentrations ofafter S2− in a rangewith fromS0.1 20 the μM, typical UV-Vis absorption of the AgNP colloidal solution reaction and 2− and the relative spectrum change depended on 2−the concentrations of S in a range from 0.1 to 20 μM, − obtaining a linear equation of A 400 = 0.6828CS − 0.0366 (R22= 0.975) in a concentration range of 2–15 relative spectrum depended the concentrations of S in a range from 0.1 to 20 µM, obtaining obtaining a linearchange equation of A400 =on 0.6828C S2− − 0.0366 (R2 = 0.975) in a concentration range of 2–15 2 = 0.975) μM· S2−equation with a detection limit of2−0.03 μM, (R spanning two aorders of magnitude. TheseµMresults − a linear of A = 0.6828C 0.0366 concentration range ofThese 2–15 · S2 − 400 limit of S0.03 μM, spanning twoin orders μM·S2− with a detection of magnitude. results 2− demonstrate that AgNPs are highly sensitive to S . In further studies, theresults effect demonstrate of physiologically with a detection of 0.03 spanning two magnitude. These that demonstrate thatlimit AgNPs are µM, highly sensitive toorders S2−. In of further studies, the effect of physiologically 2 − important species as potential interferents on the plasmon peak of AgNPs was examined and studied AgNPs are highly sensitive to S . In further studies, the effect of physiologically important species as important species as potential interferents on the plasmon peak of AgNPs was examined and studied later. interferents on the plasmon peak of AgNPs was examined and studied later. potential later.

Scheme 1. Proposed mechanism of colorimetric determination of 2S−2−, DTT, biothiols with Scheme 1. Proposed Proposed mechanism mechanism ofof colorimetric colorimetric determination determination of of S2−, , DTT, DTT, biothiols biothiols with with Scheme silver/dopamine nanoparticles. silver/dopamine nanoparticles. silver/dopamine nanoparticles.

Figure Figure1.1.(A) (A)UV-Vis UV-Visabsorption absorptionresponses responsesofofAgNPs AgNPsininthe theabsence absence(curve (curvea)a)and andpresence presenceofofdifferent different 2−absorption Figure 1. (A) UV-Vis responses of AgNPs in the absence (curve a) and presence of different concentrations concentrationsofofS S2−(from (fromcurve curveaato tocurve curvek:k:0,0,0.1, 0.1,0.5, 0.5,1,1,2,2,3,3,5,5,7,7,10, 10,15, 15,20 20µM). μM).The Thecolor colorchanges changes 2− (from curve a to curve k: 0, 0.1, 0.5, 1, 2, 3, 5, 7, 10, 15, 20 μM). The color 22−− (from concentrations of S changes ofofAgNPs before (the first tube) and after the reaction with different concentrations of S left AgNPs before (the first tube) and after the reaction with different concentrations of S (from curve of before (the first tube) and afterare theshown reaction with different concentrations ofmin. S2− (from curve toaAgNPs right: 0, 0.5, 1, 3, 5, 7, 10, 15, 20 µM) in the inset. The reation time, 20 (B) UV-Vis to curve l: 0, 0.5, 1, 3, 5, 7, 10, 15, 20 μM) are shown in the inset. The reation time, 20 min. (B) UV− aabsorption to curve l:responses 0, 0.5, 1, 3,versus 5, 7, 10, 20concentration. μM) are shown inreation the inset. The time, 20 min. (B) UVthe15, S2the The time, 20reation min. Vis absorption responses versus S2− concentration. The reation time , 20 min. Vis absorption responses versus the S2− concentration. The reation time , 20 min.

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C

Figure 2. TEM images of of dopamine-functionalized AgNPs absence (A)the and the presence Figure 2. TEM images dopamine-functionalized AgNPs in in thethe absence (A) and presence (B) of (B) . Thereation reationtime, time, 20 20min. X-ray spectroscope (EDS) spectrum of of of 10 10 µMμM ·S2−S.2−The min. (C) (C)Energy Energydisperse disperse X-ray spectroscope (EDS) spectrum dopamine-functionalized AgNPs the presenceof of10 10 µM μM ·SS2−2.− . dopamine-functionalized AgNPs in in the presence

3.2. Absorption and TEM Studies of Silver/Dopamine Nanoparticles after Incubation with Thiol Compounds

3.2. Absorption and TEM Studies of Silver/Dopamine Nanoparticles after Incubation with Thiol Compounds It was found that thiol compounds have a strong effect on the plasmon resonance of AgNPs.

ItTherefore, was found thiol have a strong on in thethe plasmon AgNPs. we that studied thecompounds UV-Vis spectra of the AgNPeffect plasmon presenceresonance of differentofthiol Therefore, we studied the UV-Vis spectra of the AgNP plasmon in the presence of different compounds including Cys, Hcy, GSH, GSSG, MUA, N-cys, and DTT. Figure 3 shows a typical UV-thiol compounds including Cys, Hcy, GSH, GSSG, MUA, N-cys, and DTT. Figure 3 shows typical3A), UV-Vis Vis absorption spectrum of AgNPs in the presence of different concentrations of Cys a(Figure absorption spectrum of AgNPs in the presence different concentrations of Cys (Figure 3A), Hcy Hcy (Figure 3B), and GSH (Figure 3C). As can beofseen, upon the addition of these reduced biothiols, the3B), absorbance at maximum wavelength, 400seen, nm, decreased differentof degrees, as shown biothiols, in Figure the (Figure and GSH (Figure 3C). As can be upon thein addition these reduced 3. The decrease in plasmon intensity could be due to the interaction of the –SH of Cys, Hcy, and absorbance at maximum wavelength, 400 nm, decreased in different degrees, as shown in GSH Figure 3. with silver. Unlike these reducedcould biothiols, the to molecule structure of andofN-cys, whichand alsoGSH The decrease in plasmon intensity be due the interaction of MUA the –SH Cys, Hcy, had a sulfhydryl group, did not lead to the diminishing of the plasmon absorbance of AgNPs (Figure with silver. Unlike these reduced biothiols, the molecule structure of MUA and N-cys, which also had 3D,F). In addition, oxidized biothiols GSSG, which had no free sulfhydryl group, did not induce a a sulfhydryl group, did not lead to the diminishing of the plasmon absorbance of AgNPs (Figure 3D,F). decrease in the plasmon absorbance of AgNPs (Figure 3E) or in MUA. According to TEM (Figure 2A), In addition, oxidized GSSG, which had nothe freereduction sulfhydryl notdopamine induce a decrease the average sizebiothiols of AgNPs formed through of group, AgNO3did with was in theapproximately plasmon absorbance AgNPs (Figure 3E) ordistributed. in MUA. According TEM (Figure 2A), the 10 nm andofthe particles were evenly The hydroxyltogroups of dopamine average size of AgNPs reduction of and AgNO with dopamine was approximately were reported to beformed the mainthrough reducingthe groups for Ag+, oxidation product polydopamine from 3 processwere adsorbed AgNP surfaces and stabilized theofmonodispersed AgNPs. 10 nmthis andreaction the particles evenlythe distributed. The hydroxyl groups dopamine were reported However, the TEM images in Figure 5C,D further prove that, upon the addition of reduced biothiols, to be the main reducing groups for Ag+, and oxidation product polydopamine from this reaction theadsorbed size of AgNPs larger with uneven distribution. These phenomena indicate that the AgNP process the became AgNP surfaces and stabilized the monodispersed AgNPs. However, the TEM structure broke down. Furthermore, as the incubating time with reduced biothiols increased (after images in Figure 5C,D further prove that, upon the addition of reduced biothiols, the size of AgNPs about 24 h at room temperature), the AgNP colloidal solution severely broke down, and the asbecame larger with uneven distribution. These phenomena indicate that the AgNP structure broke formed complex between AgNPs and reduced biothiols was consequently precipitated from the down. Furthermore, as the incubating time with reduced biothiols increased (after about 24 h at room temperature), the AgNP colloidal solution severely broke down, and the as-formed complex between AgNPs and reduced biothiols was consequently precipitated from the dispersion, resulting in a clear and transparent dispersion, and small black particles gathered at the bottom of the tubes,

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as shown in Figure S2. From the above, although the addition of reduced biothiols including Cys, dispersion, in acolloidal clear andsolution transparent and small black particles gathered at the Hcy, and GSH toresulting the AgNP haddispersion, similar UV-Vis absorption responses as well as S2− , bottom of the tubes, as2−shown in Figure S2. From the above, although the addition of reduced we can still discriminate S from Cys, Hcy, or GSH based on the differentiation over the reaction biothiols including Cys, Hcy, and GSH to the AgNP2colloidal solution had similar UV-Vis absorption time. In particular, the absorption responses for S − reached a constant value in only 20 min; for responses as well as S2−, we can still discriminate S2− from Cys, Hcy, or GSH based on the Cys, Hcy, or GSH, a constant value was not reached until after 24 h at room temperature. Thus, the differentiation over the reaction time. In particular, the absorption responses for S2− reached a constant 2− differentiation of the absorption kinetics can bevalue relied onnot to reached distinguish Hcy, or value in only 20 min; for Cys,spectrum Hcy, or GSH, a constant was untilSafterfrom 24 h Cys, at room 2 − GSH. temperature. The absorption spectrum change of AgNPs toward S is compared with other thiol compounds Thus, the differentiation of the absorption spectrum kinetics can be relied on to in Figure S3. distinguish S2− from Cys, Hcy, or GSH. The absorption spectrum change of AgNPs toward S2− is compared with other thiol compounds in Figure S3.

Figure 3. UV-Vis absorption responses of AgNPs in the absence (curve a) and presence of different

Figure 3. UV-Vis absorption responses of AgNPs in the absence (curve a) and presence of different concentrations of (A) Cys (from curve a to g: 0, 10, 15, 20, 30, 50, 100 μM), (B) Hcy (from curve a to g: concentrations of (A) Cys (from curve a to g: 0, 10, 15, 20, 30, 50, 100 µM), (B) Hcy (from curve a to g: 0, 0, 10, 15, 20, 30, 50, 100 μM), (C) GSH (from curve a to g: 0, 10, 15, 20, 30, 50, 100 μM), (D) GSSG(from 10, 15,curve 20, 30, 50, µM), (C)30, GSH (from curve f: 0, 10, 20,curve 30, 50, 100 µM), (D)30, GSSG(from curve a to g: 100 0, 10, 15, 20, 50, 100 μM), and a (E)toMUA (from a to f: 0, 10, 20, 50, 100 μM), a to g:and 0, (F) 10,N-cys 15, 20, 30, 50, 100 µM), (E) curve to f:changes 0, 10, 20, 30, 50,before 100 µM), (from curve a to g: 0, and 10, 15, 20,MUA 30, 50,(from 100 μM). Theacolor of AgNPs (the and (F) N-cys curve a the to g:reaction 0, 10, 15, 20,different 30, 50, 100 µM). The color AgNPs first (from tube) and after with concentrations of (A) changes Cys (fromofcurve a tobefore g: 0, 10,(the 15, first 50, 100 (B) Hcy(from curve concentrations a to g: 0, 10, 15, 20, 100(from μM), curve (C) GSH (from a to tube) 20, and30,after the μM), reaction with different of 30, (A)50, Cys a to g: 0,curve 10, 15, 20, 30, g: 0, 10, 15, 20, 30, 50, 100 μM) are shown in the corresponding inset. The reation time, 2 h. 50, 100 µM), (B) Hcy(from curve a to g: 0, 10, 15, 20, 30, 50, 100 µM), (C) GSH (from curve a to g: 0, 10, 15, 20, 30, 50, 100 µM) are shown in the corresponding inset. The reation time, 2 h.

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Sensors 2017, 17,DTT, 626 which had two proximal sulfhydryl groups, was chosen to examine its 8response of 13 In addition, 2017,with 17, 626 AgNPs. Figure 4 shows the absorption spectrum of AgNPs in the presence 8 of 13 after Sensors reaction of In addition, DTT, which had two proximal sulfhydryl groups, was chosen to examine its different concentrations of DTT. As can be seen, when DTT was present in the detection system, the In addition, DTT, which had twoFigure proximal sulfhydryl groups, was chosenoftoAgNPs examine its response after reaction with AgNPs. 4 shows the absorption spectrum in the AgNP colloidal solution would change from bright yellow to reddish brown of inin 5 the mindetection (Figure response after reaction with AgNPs. 4 can shows the absorption AgNPs in the 4A presence of different concentrations of Figure DTT. As be seen, when DTT spectrum was present inset),presence implying the formation of aggregated Meanwhile, the was absorbance peak of AgNP of different concentrations ofwould DTT. AgNPs. As can be seen,bright whenyellow DTT present in the detection system, the AgNP colloidal solution change from to reddish brown in the 5 min colloidal solution at maximum wavelength 400 nm decreased andMeanwhile, was red-shifted from 400 420 system, the inset), AgNP colloidal the solution would fromAgNPs. bright yellow to reddish brown inpeak 5tomin (Figure 4A implying formation of change aggregated the absorbance of nm. 4A inset), implying the aggregated400 AgNPs. Meanwhile, thewas absorbance peak of and These(Figure responses could be ascribed to theofcrosslinking interaction between thered-shifted –SH of DTT the AgNP colloidal solution atformation maximum wavelength nm decreased and from the AgNP solution at maximum wavelength nmcan decreased andmonodispersed was red-shifted from 400In toparticular, 420 colloidal nm. These responses could be ascribed to the crosslinking interaction between the –SH of to AgNPs. two proximal sulfhydryl groups of400 DTT crosslink AgNPs 400 to 420 nm. These responses could be ascribed to the crosslinking interaction between the –SH of DTT and AgNPs. In particular, two proximal groupsin ofthe DTTcorresponding can crosslink monodispersed form aggregated AgNPs with a larger particlesulfhydryl size, resulting changes in color DTT andtoAgNPs. InThe particular, two proximal sulfhydryl groups of DTT can crosslink monodispersed AgNPs form aggregated AgNPs with a larger particle size, resulting in the corresponding changes and absorbance peak. aforementioned reaction mechanism is demonstrated in the TEM image. AgNPs form aggregated AgNPs a larger particle size,mechanism resulting inisthe corresponding changes in colortoand absorbance peak. The with aforementioned reaction demonstrated in the TEM Figure 5A,B further proves that the size of the AgNPs changed and formed larger particles with in color Figure and absorbance peak. The aforementioned mechanism demonstrated in the TEM image. 5A,B further proves that the size of reaction the AgNPs changedisand formed larger particles uneven distribution after the addition of DTT. Different from the aforementioned reduced biothiols, image. Figure 5A,B further proves that the size of the AgNPs changed and formed larger particles with uneven distribution after the addition of DTT. Different from the aforementioned reduced DTT with can significantly damageafter the the structure of of AgNPs and causeand the the formation of largerreduced particles in uneven addition DTT. Different aforementioned biothiols, DTTdistribution can significantly damage the structure of AgNPsfrom cause the formation of larger 5 minbiothiols, with obvious color changes, Cys, andCys, GSH needed more than 24 h tothan produce slight DTT can significantly damage the Hcy, structure of AgNPs andGSH cause the formation of 24 larger particles in 5 min with obvious while color changes, while Hcy, and needed more h to color particles changes. These characteristic spectrums of AgNPs in the presence of DTT actually form the 5 mincolor withchanges. obvious These color changes, while spectrums Cys, Hcy, and GSH needed than of 24 DTT h tobasis produce in slight characteristic of AgNPs in themore presence produce slight color changes. characteristic of AgNPs in the presence offorDTT for the development ofbasis an effective, direct, colorimetric method for the detection of DTT. Figure 4 actually form the for theThese development of anspectrums effective, direct, colorimetric method the actually the Figure basisresponses for the development of colorimetric method fordifferent the detection of DTT. 4 shows typical absorption responses of AgNPs the absence (Curve A) shows typicalform absorption of AgNPs in an theeffective, absencedirect, (Curve A)inand presence of detection ofofDTT. Figure 4 concentrations shows typical absorption responses AgNPs in theintensity absence (Curve A) and presence of different of DTT, intensity and the relative absorption at 400 nm concentrations DTT, and the relative absorption at of 400 nm depended on concentrations and presence of different concentrations of DTT, and the relative absorption intensity at 400 nm depended on concentrations of DTT in a range from 1 to 50 μM DTT, obtaining a linear equation of of DTT in a range from 1 to 50 µM DTT, obtaining a linear equation of A400 = 0.9159 − 0.0157C DTT depended on−concentrations DTT in from 1 torange 50 μM obtaining a linear equation of A400 = 0.9159 0.0157CDTT (R2 of = 0.997) in aa range concentration of DTT, 3–40 μM DTT with a detection limit 2 (R = 0.997) in a concentration 2range of 3–40 µM DTT with a detection limit of 0.3 µM.

A = 0.9159 of4000.3 μM. − 0.0157CDTT (R = 0.997) in a concentration range of 3–40 μM DTT with a detection limit of 0.3 μM.

B B

(A) UV-Vis absorption responses AgNPsin in the absence a) a) and presence of different FigureFigure 4. (A)4.UV-Vis absorption responses ofofAgNPs absence(curve (curve and presence of different Figure 4. (A)ofUV-Vis absorption responses of0,1, AgNPs absence (curve a) and presence of different concentrations of DTT (from curve a toj: j:0, 1,3,3, 5, 5,in 10,the 15, 20, 40, μM). The color changes of of concentrations DTT (from curve a to 10, 15, 20,30, 30, 40,5050 µM). The color changes concentrations DTT (from curve a to the 0,reaction 1, 3, 5, 10, 15,different 20, 30, 40, 50 μM). Theofcolor changes of AgNPs before (the first tube) and after with concentrations captopril (from AgNPs before (the of first tube) and after thej: reaction with different concentrations of captopril (from AgNPs and theare reaction different concentrations of captopril (from curve abefore to i: 0, (the 1, 3, first 5, 10,tube) 20, 30, 40,after 50 μM) shownwith in the corresponding inset. The reation time, 20 curve a to i: 0, 1, 3, 5, 10, 20, 30, 40, 50 µM) are shown in the corresponding inset. The reation time, curve a toUV-Vis i: 0, 1, 3, 5, 10, 20, 30, 40, 50 μM) are the shown the corresponding inset. The reation time, 20 min. (B) absorption responses versus DTTinconcentration. 20 min. (B) UV-Vis absorption responses versus the DTT concentration. min. (B) UV-Vis absorption responses versus the DTT concentration.

B B

A A

Figure 5. Cont.

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D

Figure 5. TEM image of dopamine-functionalized AgNPs in the presence of (A) and (B) 20 μM DTT,

Figure 5. TEM image of dopamine-functionalized AgNPs in the presence of (A) and (B) 20 µM DTT, and (C) 50 μM Cys, (D) 50 μM GSH. The reation time for DTT, 20 min; The reation time for Cys and and (C) 50 µM Cys, (D) 50 µM GSH. The reation time for DTT, 20 min; The reation time for Cys and GSH, 2 h. GSH, 2 h.

3.3. Interference Study

3.3. Interference Study

To investigate the selectivity of silver/dopamine nanoparticles, potential interferences such as

other amino acids, cations, and physiologically importantpotential species under physiological To investigate thevarious selectivity ofanions, silver/dopamine nanoparticles, interferences such as werevarious studied.cations, Each kind of species examined in parallel under the same conditions as otherconditions amino acids, anions, andwas physiologically important species under physiological 2− and thiol compounds. As shown in Figure 6, the separate addition of other amino acids those of S conditions were studied. Each kind of species was examined in parallel under the same conditions as and physiologically important species exceeding the upper limit of the physiological concentration those of S2− and thiol compounds. As shown in Figure 6, the separate addition of other amino acids range to the AgNP colloidal solution caused minor absorption spectrum changes in the AgNPs at 400 and physiologically important species exceeding the upper limit of the physiological concentration nm but no color change. As compared with those of the aggregation triggered by S2− and DTT, these rangeresults to thesubstantially AgNP colloidal solution caused absorption spectrum changes in the AgNPs at suggest that these aminominor acids and physiologically important species did not 2− and DTT, 400 nm but no color change. As compared with those of the aggregation triggered by S 2− interfere with the S and DTT sensing. these results substantially suggest that that thesesilver/dopamine amino acids and physiologically important species Ma et al. [36] has demonstrated nanoparticles have high selectivity and did 2−2+and not interfere with the SCu DTT sensitivity towards based on sensing. the coordination ability between Cu2+ and the nitrogen and oxygen atoms without interference other metal ions, indicating interference Cu2+ in and Ma et of al.dopamine [36] has demonstrated that from silver/dopamine nanoparticles have highfrom selectivity 2+ based detection 2+ once our work for the of sulfide and DTT. Herein,Cu we again examined sensitivity towards Cuselective on the coordination ability between and the nitrogen andthe oxygen 3+, Fe2+, Zn2+, Al3+, absorption responses of AgNPs toward 12 other kinds of conventional metal ions: Fe atoms of dopamine without interference from other metal ions, indicating interference from Cu2+ in Cr3+, Cd3, Co2+, Mg2+, Na+, K+, Ca2+, and Cu2+. The results suggest that these cations caused minor our work for the selective detection of sulfide and DTT. Herein, we once again examined the absorption absorption responses changes, except for Cu2+ Fe3+, Fe2+, Cd3+, and Co2+, which were considered as the responses of AgNPs toward 12 other kinds of conventional metal ions: Fe3+ , Fe2+ , Zn2+ , Al3+ , Cr3+ , Cd3 , main interfering species. However, we can chelate and shelter the interference cations by the addition 2+ , Na+ , K+ , Ca2+ , and Cu2+ . The results suggest that these cations caused minor absorption Co2+ ,ofMg ethylenediaminetetraacetic acid (EDTA). As shown in the red columns of Figure 7, if EDTA 2+ Fe3+ , Fe2+ , Cd3+ , and Co2+ , which were considered as the main responses changes, except for Cu coexisted with these cations, the AgNP colloidal solution remained yellow (Figure 1B), and there is interfering species. However, we canbecause chelatethese and interference shelter the ions interference cations the addition of no change in absorption response were chelated by by EDTA, which prevented the binding reaction of these cations and dopamine. inset of 7 showed the color ethylenediaminetetraacetic acid (EDTA). As shown in the red The columns ofFigure Figure 7, if EDTA coexisted 2+ in the absence and presence of EDTA. As can be for silver/dopamine nanoparticles and Cu with change these cations, the AgNP colloidal solution remained yellow (Figure 1B), and there is no change 2+, AgNPs displayed a bright yellow color just as in the absence seen, when EDTA coexisted with Cu in absorption response because these interference ions were chelated by EDTA, which prevented the 2+. Thus, by using the chelating reagent EDTA, the colorimetric sensor based on AgNPs has of Cu binding reaction of these cations and dopamine. The inset of Figure 7 showed the color change for excellent selectivity to sulfide and DTT over other coexisting metal ions. silver/dopamine nanoparticles and Cu2+ in the+ absence and presence of EDTA. As can be seen, when Considering that it is quite simple for Ag to form the precipitates from the dispersion when2+ the EDTA coexisted with Cu2+ , AgNPs displayed a bright yellow color just as in the absence of Cu . Thus, reaction between Ag+ and many anions occurs, such as Cl− for AgCl and SO42− for Ag2SO4,, the by using the chelating reagent EDTA,S2−the colorimetric sensor based on hastheir excellent selectivity selectivity of AgNPs for sensing and DTT was thus evaluated by AgNPs comparing absorption to sulfide and DTT over other coexisting metal ions. − − − − 2− response changes toward those of 15 other kinds of conventional anions, Cl , F , Br , I , SO4 , SO32−, + −, CO 2−, ATP, Considering that is 3quite simple for Ag from the dispersion ClO4−, NO3−, CH 3CO2it AMP, ADP, PPi, to andform PO43−,the withprecipitates different concentrations at the + − As shown Figure S4, anions these anions induced plasmon whenphysiological the reactionlevel. between Ag inand many occurs, such negligible as Cl for AgCl absorbance and SO4 2− for 2− and DTT changes AgNPs at 400 implying stronger Scoordination effect between dopamine and Ag2 SO ofnm, AgNPs for asensing was thus evaluated by AgNPs comparing 4 „ theinselectivity + than the associated interaction between Ag and the tested anions. The aforementioned results their absorption response changes toward those of 15 other kinds of conventional anions, Cl− , F− , Br− , I− , SO4 2− , SO3 2− , ClO4 − , NO3 − , CH3 CO2 − , CO3 2− , ATP, AMP, ADP, PPi, and PO4 3− , with different concentrations at the physiological level. As shown in Figure S4, these anions induced negligible plasmon absorbance changes in AgNPs at 400 nm, implying a stronger coordination effect between dopamine and AgNPs than the associated interaction between Ag+ and the tested anions.

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S2 −

The aforementioned results sufficiently suggest that AgNPs have a high selectivity for and DTT, sufficiently suggest that AgNPs AgNPs have aa samples. high selectivity selectivity for for SS2−2− and and DTT, DTT, allowing allowing potential potential sufficiently suggest that have high allowing potential applications in complex applications in in complex complex samples. samples. applications

Figure 6. 6. (A) (A) The UV-Vis absorption absorption responses a) Figure The color color changes changes and and UV-Vis UV-Vis absorption responses of of AgNPs AgNPs in in the the absence absence (curve (curve a) and presence of different species (from curve b to t: tryptophan, histidine, proline, alanine, lysine, and presence of different species (from curve b to t: tryptophan, tryptophan, histidine, proline, alanine, lysine, phenylalanine,leucine, leucine, threonine, arginine, aspartic acid, glycine, glycine, valine, serine, methionine, glutamine, phenylalanine, leucine, threonine, arginine, aspartic acid, valine, serine, glutamine, threonine, arginine, aspartic acid, glycine, valine, serine, glutamine, 2−, 20 μM ). (B) The color changes and UV-Vis 2− methionine, tyrosine, 40 μM; μM; 0.01% BSA and tyrosine, 40 µM; 0.01% 40 BSA and0.01% S2− , 20BSA µM ). (B) SS The color absorption methionine, tyrosine, and , 20 μMchanges ). (B) and The UV-Vis color changes andresponses UV-Vis absorption responses of AgNPs AgNPsa)in inand thepresence absence (curve (curve a) and and presence ofcurve different species (from curve of AgNPs inresponses the absence (curve of different species (from b tospecies l: glucose, glutamic absorption of the absence a) presence of different (from curve b to l: glucose, glutamic acid, 5-hydroxytryptamine, uric acid, lactate, ATP, norepinephrine, acid, 5-hydroxytryptamine, uric acid, lactate, ATP, norepinephrine, hypoxanthine, H O , and sodium b to l: glucose, glutamic acid, 5-hydroxytryptamine, uric acid, lactate, ATP, 2norepinephrine, 2 2− and ascorbate, 20 µM and S2−sodium , 20 µMascorbate, ). The reation time for 20 min.time The for reation time for hypoxanthine, H22O O and sodium ascorbate, 20 μM μM and ,20 μM ).).DTT, The reation reation time for SS2−2− and and DTT, hypoxanthine, H 22,, and 20 and SS2−2−S,20 μM The DTT, other species, 2 h. time 20 min. min. The reation reation time for for other other species, species, 22 h. h. 20 The

Figure 7. 7. UV-Vis absorption responses A of AgNPs AgNPs in in the the absence absence (black (black bars) bars) and and presence presence of of 7. UV-Vis UV-Vis absorption absorption responses responses A A400 400 of of AgNPs 400 Figure 3+ ,2+ 2+ 2+ 2+2+ 2+ , S 2− , Al3+ , 3+ 2+ 2+ 2+ 2− 3+ 3+ 2− 3+ various metal ions containing 50 µM EDTA. The concentration of Fe Fe , Zn , Cu various metal ions ions containing containing 50 50 μM μM EDTA. EDTA. The The concentration concentration of of Fe Fe ,, Fe Fe ,, Zn Zn ,, Cu Cu ,, SS ,, Al Al ,, 20 20 μM; μM; various 3+ Cd3 , Co2+2+ 2+ ,2+ + K+ , Ca2+ , 50 µM; Photographs from curve left to right: 3, Co 2+, µM; 20 Mg Cr3+3+µM; Cd3Cr ,5 μM; μM; Mg Mg,5 Na++,, K K++,, Ca Ca2+Na 50,μM; μM; Photographs Photographs from from curve curve left left to to right: right: AgNPs AgNPs with with 80 80 Cr ,, Cd , Co2+,2+,5 , Na ,, 50 2+ AgNPs with 80 µMwith EDTA, AgNPs with 20μM µMEDTA, Cu2+ and 80 µM with 20 µM Cufor μM EDTA, EDTA, AgNPs with 20 μM μM Cu2+2+ and and 80 80 μM EDTA, AgNPs withEDTA, 20 μM μMAgNPs Cu2+2+.. The The reation time for. μM AgNPs 20 Cu AgNPs with 20 Cu reation time The reation metal ions, 22time h. for metal ions, 2 h. metal ions, h. 22−− in Fetal Bovine Serum 3.4. The The Determination Determination of of SS2− in Fetal Fetal Bovine Bovine Serum Serum 3.4. in

To To validate validate the the performance performance of of the the developed developed colorimetric colorimetric detection detection method method in real samples, we as-synthetized AgNPs AgNPs to to directly serum. then applied applied as-synthetized directly detect detect the the sulfide sulfide concentration concentration in in fetal fetal bovine bovine serum. serum. then The The treated treated serum serum as as described described in in the the experimental experimental section section was was added added into into the the aqueous aqueous dispersion dispersion of AgNPs. The average average sulfide sulfide concentration concentration in in three three replicate replicate samples samples was was about about 16.3 16.3 μM, µM, AgNPs. The μM, which which For the was consistent consistent with with other other reported reported results results about about sulfide sulfide concentrations concentrations in in serum serum [39,40]. [39,40]. For the was [39,40]. 2− 2− spiked-recovery assays, assays, the the results results revealed revealed that that the the recoveries recoveries of of the the added added SS with with the the known known spiked-recovery concentration 5, 5, 10, 10, 20, 20, 30, 30, and and 50 50 μM μM were were 97%, 97%, 99%, 99%, 103%, 103%, 98%, 98%, and and 99%, 99%, respectively, respectively, which which was was concentration

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spiked-recovery assays, the results revealed that the recoveries of the added S2− with the known concentration 5, 10, 20, 30, and 50 µM were 97%, 99%, 103%, 98%, and 99%, respectively, which was acceptable, indicating that it can be used to monitor sulfide in real biological samples including bioassays, nanotechnology, and clinical diagnostics. 4. Conclusions A facile, highly selective and reliable new method for the determination of sulfide and 1,4-dithiothreitol (DTT) are described herein. The method is based on the in situ formation of silver nanoparticles using dopamine as a reducing and stabilization agent, which showed corresponding changes in the color and the absorbance peak toward sulfide and 1,4-dithiothreitol (DTT). Field transmission electron microscope (TEM) images further proved that the size and the morphology of AgNPs changed, and formed larger particles with uneven distribution after the addition of sulfide and DTT. In conclusion, this measurement based on the in situ formation of AgNPs simplifies the operation and is completely economical and eco-friendly. Benefiting from the unique optical properties of AgNPs, the determination of sulfide and DTT can be accomplished in 10 min and we can achieve the determination of sulfide and DTT with the naked eye, which is greatly suitable for the analysis of sulfide and DTT in fieldwork. The sensor utilizing as-formed AgNPs is promising for the further establishment of microfluidic paper-based analytical devices for point-of-use diagnostics, without external power supplies or supporting equipment based on the color change of AgNPs. Supplementary Materials: The following are available online at www.mdpi.com/1424-8220/17/3/626/s1, Figures S1–S4. Acknowledgments: This work is financially supported by NSF of China (Grant Nos. 21305109 for L. Zhao, 81202492 for C. Liu, and 91332101 for C. Zhang), by the PhD Startup Foundation of Xi’an Medical University of China (No. 2012DOC09) and by the Scientific Research Plan Projects Foundation of Shaanxi Science and Technology Department of China (Nos. 2014JQ2073, 2014K02-11-01). Author Contributions: Lingzhi Zhao developed the sensors, carried out the sulfide and 1,4-dithiothreitol detection measurements, and also wrote the manuscript. Liu Zhao, Yanqing Miao, Chunye Liu and Chenxiao Zhang participated in the data interpretation. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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