Development and Application of an Efficient Medium for Chromogenic

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Article Cite This: ACS Omega 2019, 4, 5459−5470

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Development and Application of an Efficient Medium for Chromogenic Catalysis of Tetramethylbenzidine with Horseradish Peroxidase Meng Li,† Haiping Su,† Yan Tu,† Yazhuo Shang,*,† Yu Liu,*,‡ Changjun Peng,† and Honglai Liu† †

Key Laboratory for Advanced Materials, School of Chemistry and Molecular Engineering and ‡State Key Laboratory of Chemical Engineering and School of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China

ACS Omega 2019.4:5459-5470. Downloaded from pubs.acs.org by 178.159.100.75 on 03/19/19. For personal use only.

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ABSTRACT: The alkylimidazolium tetrafluoroborate ionic liquids (ILs) ([Cnmim][BF4] n = 2, 4, 6, 8, 10) and anionic surfactant sodium dodecyl sulfate (SDS) were combined together to produce effective mediums for chromogenic catalysis of tetramethylbenzidine (TMB) with horseradish peroxidase (HRP) in the presence of H2O2. The chromogenic performance, kinetic behavior, and the possible influencing mechanism for the chromogenic catalysis of HRP-H2O2-TMB were discussed in detail. Therein, the roles of ionic liquids (ILs) were highlighted by the combination of experiments and theoretical calculations. The SDS/[C4mim][BF4] combination displayed superiority in chromogenic catalysis by improving both the substrate solubility and product stability to the maximum extent possible. Furthermore, SDS/[C4mim][BF4] combination showed uniqueness for TMB in improving the chromogenic performance compared with other chromogenic substrates of HRP. Inspired by the efficient chromogenic system, an enhanced enzyme-linked immunosorbent assay strategy for the detection of human immunoglobulin G was established and the sensitive colorimetric strategies for the detection of H2O2 and glucose were further developed by employing SDS/ [C4mim][BF4] combination as the medium of chromogenic catalysis of HRP-H2O2-TMB. This unique chromogenic system is endowed with multitude of potential applications in biological systems. becomes an optimum substrate of HRP.10,11 Nevertheless, some shortcomings of TMB suffering from poor water solubility and short color developing time derived from the highly unstable chromogens limit the scope of its practical application to a great degree.10,12,13 Therefore, developing a chromogenic catalysis system of HRP-H2O2-TMB with higher detection efficiency is still the goal pursued by researchers. Optimizing the properties of the mediums such as composition, component concentrations, as well as the pH has become an effective way of improving the efficiency of enzymatic catalysis during the past few decades and great efforts have been devoted to explore the effect of different mediums on enzyme activity.14 Moniruzzaman et al. tested the activity of HRP in the medium of water-in-ionic liquid (IL) microemulsions comprising an aerosol OT surfactant (sodium bis(2-ethyl-1-hexyl)) using pyrogallol as a substrate, in which the intrinsic activity of HRP and the solubility of both the substrate (pyrogallol) and the product (purpurogallin) in the HRP-catalyzed reaction were all significantly increased compared with that in the medium of isooctane.15 Kühlmeyer

1. INTRODUCTION Peroxidases are a large family of enzymes that typically catalyze the oxidation of their substrates with peroxide (H2O2 in most cases).1 Horseradish peroxidase (HRP) is the most important heme-containing peroxidase found in the roots of horseradish.2 HRP has been widely used in bioanalytical and clinical chemistry because of the relatively stable properties, cheap production, effective catalytic activity, as well as wider variety of substrates than other oxidoreductive enzymes.3−6 It is worth mentioning that the HRP has potential applications in enzymelinked immunosorbent assay (ELISA),3 electrochemical immunosensors,7 as well as other biosensors8 for diagnosis and biochemical detection because it can catalyze various aromatic amine and phenolic compounds oxidation in the presence of H2O2 to produce a chromogenic reaction. Previous study has shown that the chromogenic sensitivity varies with the chromogenic substrates used in the detection. It is reported that tetramethylbenzidine (TMB) has the highest sensitivity to HRP compared to other chromogenic substrates such as ophenylenediamine (OPD), 2,2-diazo-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 5-aminosalicylic acid (5-AS) etc.1,9 Apart from lower toxicity and nonteratogenicity,10 TMB shares the advantages of splendid affinity with the enzyme and the chromogen with high absorption coefficient, and thus © 2019 American Chemical Society

Received: November 29, 2018 Accepted: February 18, 2019 Published: March 19, 2019 5459

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of alkyl chain lengths of ILs [Cnmim][BF4] on the nature of chromogenic catalysis were investigated systematically by UV spectrum, dynamic light scattering (DLS), electrochemical analyzer, as well as transmission electron microscopy (TEM). The chromogenic performance, kinetic behavior, and the possible influencing mechanism for the chromogenic catalysis of HRP-H2O2-TMB were discussed in detail. Therein, the roles of ILs were highlighted by the combination of experiments and theoretical calculations. Moreover, the SDS/[C4mim][BF4] combinations were singled out and further optimized as the medium of chromogenic catalysis of HRP-H2O2-TMB for ELISA enhancement (detection of human immunoglobulin G (IgG)) and detection of H2O2 and glucose. We expect that the present study not only provides a unique catalytic system with multitude of potential applications in biological systems but also advances the understanding of the roles of ILs in affecting the nature of general enzymatic catalysis, which is significant for the design and optimization of enzymatic reaction mediums based on ILs.

et al. found that the functional stability of HRP can be increased by the addition of polyvinylsaccharides, which depended on various parameters of the medium such as the degree of polymerization, various sugars employed, ionic groups and their degree of substitution in the side chain, and polymer concentration.16 Many studies have confirmed that adjusting the reaction medium was really a simple and feasible way of improving the efficiency of enzymatic catalysis. Consequently, more and more species were introduced to the system of enzymatic catalysis. Among a number of mediums used for biochemical reactions, ionic liquids (ILs) have evolved as potentially attractive “green” and “designer” solvents due to their unique advantages compared with other conventional organic solvents or aqueous reaction mediums. So far, the activity and stability of a large number of enzymes in ILs have been evaluated.17 Machado and Saraiva investigated the enzymatic activity of HRP in alkylimidazolium-based ILs with different cation chain lengths (ethyl-, butyl-, and hexylimidazolium-based ILs) and proved that the activity of HRP in [Hexmim][Cl] was least.18 Attri et al. found that less hydrophobic ILs with small alkyl chain molecules such as triethyl ammonium acetate ([TEA][Ac]) and triethyl ammonium phosphate ([TEA][PO4]) can stabilize α-chymotrypsin, whereas more hydrophobic imidazolium and phosphonium cations carrying longer alkyl chains of ILs such as 1benzyl-3-methylimidazolium chloride ([Bzmim][Cl]) and 1benzyl-3-methylimidazolium tetrafluoroborate ([Bzmim][BF4]) act as destabilizers.19 Lu et al. reported three imidazole-based cations (including [Emim]+, [Bmim]+ and [Hmim]+) with a ranking of [Emim]+ < [Bmim]+ < [Hmim]+ in maintaining HRP structural stability by a bioelectrochemical method.20 Numerous studies have shown that the solubility, stability, as well as bioactivity of biomolecules such as proteins and DNA, particularly enzymes, can be significantly improved by introducing ILs to the native system21−26 and the hydrophobic chain length carried by ILs is the key factor affecting the enzyme performance. However, almost all of these studies focus on the activity and stability of enzymes in systems containing ILs. By far, few studies have been conducted on the detailed information of the effect of ILs on the nature of enzymatic catalysis including the interaction between ILs and substrates/products, the synergistic effect of ILs with the cosolutes in the mediums, as well as the effect of ILs on the kinetic behaviors of reactions, which not only limits the comprehensive understanding to the mechanism of ILs promoting enzymatic catalysis but also restricts the application of ILs in enzymatic catalysis systems. In our previous study, the ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF4]) and the traditional surfactant sodium dodecyl sulfate (SDS) were combined together for creating an efficient medium for chromogenic catalysis of TMB with HRP in the presence of H2O2. The results have revealed that the SDS/[Emim][BF4] (SDS/ [C2mim][BF4]) combination not only enhances the catalytic activity of HRP remarkably but also stabilizes the blue chromogen formed in the HRP oxidation of the substrate TMB compared to the conventional medium because the introduction of [Emim][BF4] ([C2mim][BF4]) can promote the formation rate of blue chromogen and the existence of SDS can stabilize the blue chromogen.27 Inspired by previous work, we introduce ILs [Cnmim][BF4] with different chain lengths (n = 2, 4, 6, 8, 10) to the chromogenic catalysis of HRP-H2O2TMB for making full use of the advantages of SDS. The effects

2. RESULTS AND DISCUSSION 2.1. Behavior of Chromogenic Catalysis in Mediums of SDS/[Cnmim][BF4] Combination. To achieve a desired chromogenic performance, we referred the results of our previous work that the optimal concentration of SDS/ [Emim][BF4] combination was 5.0 mM (2.5 mM SDS and 2.5 mM [Emim][BF4]) corresponding to the fast reaction rate and deepest coloration for the chromogenic catalysis of HRPH2O2-TMB,27 by taking the example that 2.5 mM ILs [Cnmim][BF4] of different alkyl chain lengths (n = 2, 4, 6, 8, 10) were combined with 2.5 mM SDS to obtain five different SDS/[Cnmim][BF4] combinations as the reaction mediums for the catalysis of HRP-H2O2-TMB. First, the chromogenic behaviors of the catalysis in different mediums are studied by UV−vis spectra as shown in Figure 1. Compared with the sole SDS system, the introduction of ILs not only increases the equilibrium concentration of the blue chromogens (Figure 1a),

Figure 1. (a) UV−vis spectra and (b) time-dependent UV−vis absorbance (652 nm) for chromogenic catalysis of HRP-H2O2-TMB occurred for 5 min in different mediums. (A)−(G) respectively represented the samples prepared by phosphate-buffered saline (PBS) (pH 7.4), sole SDS solution (2.5 mM), and SDS/[Cnmim][BF4] (n = 2, 4, 6, 8, 10) combinations. The concentrations for SDS/ [Cnmim][BF4] combinations are 2.5 mM SDS and 2.5 mM [Cnmim][BF4]. The average formation rates of the blue chromogens within 100 s are equal to the slopes of the portion marked by a dash line in (b). The corresponding color images of (A)−(G) are on the top. 5460

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but also improves the formation rate of the blue chromogens (Figure 1b). Moreover, the compounded solutions seem more competent in stabilizing the blue chromogens because without any green change in color they can be detected as can be seen in sole SDS solutions of 5.0 and 10 mM (F and G in Figure S1), which characterizes the coexistence of blue chromogen and yellow end-product in equilibrium.28 In different SDS/ [Cnmim][BF4] combinations, the efficiency of the HRP-H2O2TMB catalysis varies with the chain lengths of [Cnmim][BF4] in the medium. Obviously, [Cnmim][BF4] can promote catalysis in different degrees. For ease of statement, SDS/ [Cnmim][BF4] is abbreviated as SDS/Cn later, the catalytic efficiency can be indicated by the product yields and the average formation rate within initial 100 s (vav) (Figure S2), thus, the capacity of the mediums for improving the catalytic efficiency rank the order of SDS/C4 > SDS/C6 > SDS/C2 > SDS/C8 > SDS/C10 as shown in Figures 1 and S2. This is an unforeseen result, the higher catalytic efficiency would have been the system containing longer-chain ILs that has much stronger hydrophobic interaction with the HRP,19,20 whereas the distinct suppression of catalysis virtually occurred in systems with SDS/C8 and SDS/C10 combinations. The unexpected results may suggest that the interaction between HRP and the ILs is not the unique factor affecting the efficiency of the studied catalysis, other factors such as the interaction between mediums and substrates or products as well as the changes of kinetic behaviors of the reaction caused by mediums, may also be the key factors affecting the nature of catalysis that cannot be ignored. 2.2. Catalytic Activity of HRP in Different Mediums. To investigate the detailed information of the effects of the mediums on the nature of the HRP-H2O2-TMB catalysis, first, the effect of the mediums on the structure of HRP was studied by electrochemical methods due to the excellent electrochemical activity of HRP.29−31 The redox signal at glassy carbon electrode (GCE) comes from the redox process between ferrous iron (Fe(II)) and ferric iron (Fe(III)) on the catalytic center heme covered by proteins of HRP,32 which can reflect the effect of the electrolyte on the structure of HRP that correlated with the catalytic activity of HRP. Thus, the cyclic voltammograms (CVs) of HRP in different mediums were employed as indicators to characterize the effect of mediums on the catalytic activity of HRP. In the case of desorption of HRP from GCE during the working process of the electrode, only the first cycle of the CV curve was recorded. As shown in Figure 2a, the oxidation peak currents correspond to the oxidation process of Fe(II) to Fe(III), whereas the reductive peak currents reflect the reduction process of Fe(III) to Fe(II). Compared with the distinct reductive peak currents, the oxidation peak currents of all samples are too weak to be observed, suggesting that most heme in the solution maintain their original active state of Fe(III), few Fe(II) formed by the breakage of the Fe−N coordination bond in the studied mediums, which implies that the catalytic activity of HRP barely decreases in the studied mediums. However, distinct reduction peak currents in different mediums presented here indicate that the structure of HRP varies with the chain length of ILs in SDS/ [Cnmim][BF4] combinations, higher currents correspond to more exposed heme caused by stronger interaction between HRP and ILs. Thus, in terms of reduction peak currents in Figure 2a, the activity of HRP displayed in different mediums is ranked as the following order: SDS/C2 > SDS/C4 > SDS/C6

Figure 2. (a) Cyclic voltammograms of HRP in (A) PBS, (B) SDS solution (2.5 mM), (C−G) SDS/[Cnmim][BF4] combination (2.5 mM SDS/2.5 mM [Cnmim][BF4], n = 2, 4, 6, 8, 10). Scan rate: 0.05 V/s. (b) Average distance between HRP and ionic liquid. Coordinate of HRP is represented by the heme Fe, whereas the coordinate of ionic liquid is represented by the mass center of imidazole.

> SDS/C8 > SDS/C10 > SDS > PBS. Obviously, the weaker activity of HRP in PBS should attribute to the deep burial of the electroactive center of native HRP in PBS. To the system containing SDS, the reduction current increases slightly, which may derive from the relatively weaker hydrophobic interaction between SDS and amino acid moieties of HRP. However, the introduction of ILs makes the reduction currents increase sharply, which may attribute to the conjunction between the imidazolium cation and the carboxyl of HRP by the relatively stronger electrostatic interaction. The stronger electrostatic interaction not only makes heme more exposed to the solution but also provides more chances for the cationic imidazole head group binding on HRP to adsorb on the GCE at the negative potential, which accelerates the direct electron transfer between the HRP and the electrode. Thus, HRP exhibits a higher catalytic activity in the mediums containing ILs. Additionally, CV curves in Figure 2a-C−G show clearly that the reduction currents decrease with the increase of the alkyl chain length (n) of ILs. Generally speaking, longer-chain ILs in the SDS/[Cnmim][BF4] combinations have stronger interfacial activity, which can be inferred from the results listed in Table 1, in which the critical micelle concentration (CMC) and the Table 1. Colloidal Properties of Different Mediums mediums SDS SDS/C2 SDS/C4 SDS/C6 SDS/C8 SDS/C10

CMC (mM)a 2.50 2.02 1.50 0.80 0.25 0.06

± ± ± ± ± ±

0.32 0.16 0.17 0.02 0.23 0.13

γ (mN/m)a

d̅b,cT (nm)

± ± ± ± ± ±

17.84 28.22 37.84 68.06 112.42 134.08

39.26 35.28 34.21 31.64 27.24 25.40

3.14 1.18 0.42 1.13 1.17 1.06

ζ potential (mV)a,cT −58.0 −77.7 −83.1 −93.2 −40.8 −31.2

± ± ± ± ± ±

7.4 6.2 3.5 6.0 2.2 0.2

Mean value ± standard deviation (n = 3). bAverage diameter (d̅) of aggregates obtained from hydrodynamic diameter distribution. cThe data were obtained by using 2.5 mM SDS solution and 2.5 mM SDS/ 2.5 mM [Cnmim][BF4] (n = 2, 4, 6, 8, 10) combinations respectively. a

surface tension of SDS/[Cnmim][BF4] combinations both decrease significantly with the increase of the alkyl chain length of ILs. Obviously, ILs with longer alkyl chain are apt to form mixed micelles with SDS molecules. In contrast, ILs with a shorter alkyl chain, such as [C2mim][BF4], tend to exist in a mono molecule in aqueous solution, which shows that the ILs and HRP easily undergo electrostatic interactions. Correspondingly, HRP has stronger currents in the mediums 5461

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Figure 3. TEM images of species in 2.5 mM SDS solution (a) and 2.5 mM SDS/2.5 mM [Cnmim][BF4] (n = 2, 4, 6, 8, 10) combinations ((b)−(f) corresponding to the sample containing ILs with chain length from 2 to 10 respectively).

mediums. As can be seen, only a little of micelles with a diameter of about 10 nm appear in the sole SDS solution. However, the number and the diameter of micelles increase significantly with the introduction of ILs, which coincide with the results obtained by DLS measurements, as shown in Table 1. Reasonably, the CMC of SDS in PBS is about 2.5 mM, at which the spherical micelles of SDS start to form. With the addition of [Cnmim][BF4], the number of aggregates in the mediums increase obviously with the increase of aggregate sizes gradually, which provides the foundation for the solubilization of species in the reaction system. Without doubt, the introduction of ILs alters the charges carried by the aggregates in the mediums that is vital to stabilization of positively charged blue chromogens. Table 1 displays the ζ potentials of different systems. The surficial negative charges of the aggregates increase gradually with the increase of the chain length (n) of [Cnmim][BF4] for the combinations of SDS/ [Cnmim][BF4] (n = 2−6) but decrease sharply when the chain length (n) of [Cnmim][BF4] increase to 8, 10. Obviously, the dual effects of the co-solvent and co-surfactant of [Cnmim][BF4] not only affect the properties of solvent but also the size and the surficial charges of the micelles formed in the system by participating in the formation of the micelles. Consequently, the introduction of ILs can regulate the properties of colloids, further affect the kinetic behavior of the catalysis of HRPH2O2-TMB. 2.3.2. Kinetic Assay of Catalysis. It has been proved that the conformation fluctuations of HRP induced by reaction environments can greatly influence the kinetic behaviors and even change the kinetic mechanism of the reaction by means of a single-molecule kinetic model in very recent studies.36,37 To investigate the effect of different mediums on the kinetic behaviors of HRP-H2O2-TMB catalysis, the kinetic assays of the studied catalysis were carried out in SDS/[Cnmim][BF4] combinations. Figure 4 provides the Michaelis−Menten curves of catalysis of HRP-H2O2-TMB in different mediums, which are obtained by the fitted Michaelis equation (eq 1). The calculated apparent kinetic parameters are summarized in

containing ILs with shorter chains. Furthermore, the stereohindrance effect of ILs also affects the interaction between HRP with ILs. The result of quantum chemical calculation (Figure 2b) demonstrates that the average distance between HRP and ILs increases with the increase of the alkyl chain length of ILs, which confirms that ILs with longer alkyl chain tend to be far away from heme due to the increase of the stereo-hindrance effect and thus limits the increase of the catalytic activity of HRP significantly. It is worth mentioning that the cathode potentials shift negatively in the samples containing ILs, from original ∼−0.3 V moved to ∼−0.5 V. This can be ascribed to the weakness of the process of proton coupled electron transfer (PCET) due to the conjunction between HRP and ILs at the sixth coordination position in the heme iron.33 PCET is a process of electron transfer accompanied by proton transfer.34 For many heme-proteins that undergo PCET, the hydrogen bond between the water molecule and heme can not only provide an important interface for PCET but also leads to heme protonation, and enhanced protonation leads to a positive shift of formal potentials of heme proteins.35 On the contrary, the decrease of protonation may lead to a negative shift of formal potentials of these heme proteins. However, the PCET process can be tuned by the electrostatic interaction between the iron center and the charged sites in the vicinity of the heme group.34 In this case, the electrostatic combination between ILs and HRP occupies the location of the hydrogen bond that diminishes the heme protonation, and further causes a negative shift of formal potentials. 2.3. Kinetic Behavior and Mechanism of Catalysis in Mediums of SDS/[Cnmim][BF4] Combinations. 2.3.1. Colloidal Properties of Different Mediums. The mediums of SDS/[Cnmim][BF4] combinations increase the activity of the HRP doom to affect the kinetic behavior of the catalysis of HRP-H2O2-TMB, which should be related to the colloidal properties of the mediums closely. Thereby, the species in different mediums were characterized systematically. Figure 3 provides the TEM images of species formed in different 5462

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Figure 5. Binding states and dissociation energy (Ed) of heme group with the imidazole ring of His170 on native HRP when surrounded without ILs (a) and a [C4mim] group (b).

Figure 4. Michaelis−Menten plots for HRP with TMB (a) and H2O2 (b) in 2.5 mM SDS solution and 2.5 mM SDS/2.5 mM [Cnmim][BF4] (n = 2, 4, 6, 8, 10) combination. The error bars represent the standard error derived from three repeated measurements.

invasion of ILs. Besides, Ed increases at different levels when [Cnmim] groups with different chain lengths approach heme (Figure S3). The increase of Ed means the formation of a more stable structure, indicating that the heme is surrounded by [Cnmim], which is beneficial for improving the activity of HRP. However, the invasion of [Cnmim] groups maybe an obstacle to the binding between the substrate and HRP. Consequently, weaker affinity between substrates and HRP corresponding to higher Km values appeared. vmax and kcat are the two significant indictors of catalytic efficiency of the catalytic reaction. Clearly, vmax and kcat in Table 2 tend to increase with the chain length of [Cnmim] [BF4] (n = 2, 4), then further increase the chain length of [Cnmim][BF4] (n = 6, 8, 10), vmax and kcat decrease gradually, which agrees well with the results obtained in Section 2.1. 2.3.3. Mechanism of Catalysis of HRP-H2O2-TMB in SDS/ [Cnmim][BF4] Combination. The kinetic mechanism of HRPH2O2-TMB in SDS/[C2mim][BF4] combination has been illustrated as a four-stepwise reaction in the previous work.27 The catalytic mechanism can be briefly described as follows. First, HRP combines with H2O2 to generate a molecule of H2O and a high oxidation state intermediate HRP I. Second, the obtained HRP I can oxidize a molecule of TMB to form a cation radical TMB•+ and the intermediate HRP II. Third, HRP II is reduced to HRP by oxidizing the other TMB molecule, and a new cation radical TMB•+ is formed simultaneously. Two active cation radicals TMB•+ will then polymerize to form the diamine/diimine charge transfer complex (blue chromogen) eventually, which can exist in SDS/[C2mim][BF4] combination stably. However, the roles of SDS/[C2mim][BF4] involved in the mechanism have not been revealed profoundly. On the basis of more comprehensive investigation and discussion as above, the nature of the catalysis of HRP-H2O2-TMB in SDS/[Cnmim][BF4] combination is proposed. In the present chromogenic system, the activity of HRP, the solubility of substrates, the stabilization of products, as well as the kinetics of the catalysis are all quite dependent on the chain length of [Cnmim][BF4] in the mediums. For the systems containing [Cnmim][BF4] (n = 2, 4), there are more free [Cnmim]+ (n = 2, 4) cations in the aqueous solution that can activate HRP molecules by electrostatic interaction between ILs and HRP, which promotes the catalysis of HRP-H2O2TMB in a certain degree. At the same time, the existence of [Cnmim][BF4] (n = 2, 4) increases the density and size of the micelles in the mediums, which provide more chances for encapsulating the hydrophobic TMB. Furthermore, more negatively charged micelles in the SDS/[Cnmim][BF4] (n = 2, 4) combinations can greatly stabilize the positively charged

Table 2. It can be seen that the Km values obtained in the mediums containing SDS are much lower than that in PBS, Table 2. Apparent Kinetic Parameters for Catalysis of HRPH2O2-TMB in Different Mediums medium

substrate

buffer solutionb

TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2

SDS SDS/C2 SDS/C4 SDS/C6 SDS/C8 SDS/C10

Km (μM)a 434 3700 41.8 16.4 61.6 35.7 61.7 33.7 59.0 35.5 93.2 39.7 127.7 40.8

± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 200 3.2 1.3 5.4 7.1 3.1 8.2 4.9 6.1 8.2 12.1 21.9 12.8

vmax (μM min)a

kcat (103/s)

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.088 0.076 0.265 0.249 0.437 0.429 0.493 0.484 0.446 0.457 0.327 0.280 0.290 0.251

6.00 5.23 18.16 17.05 29.92 29.35 33.74 33.13 30.49 31.28 22.40 19.17 19.81 17.20

0.53 0.37 0.29 0.31 0.62 1.39 0.43 1.91 0.63 1.28 0.56 1.43 1.08 1.31

a Mean value ± standard deviation (n = 3). bThe apparent kinetic parameters of catalysis of HRP-H2O2-TMB in buffer solution are referred from other work.38

suggesting that HRP has a higher binding affinity to the substrates in the studied mediums compared with that in buffer solution. According to our previous study, this should be attributed to the immobilization of HRP on the surface of negatively charged micelles that encapsulate the hydrophobic TMB.27 Upon further analysis it was found that the Km values obtained in SDS/[Cnmim][BF4] combination are slightly higher than that in sole SDS solution. The most possible reason for this is the increased steric hindrance caused by the invasion of ILs around heme, which may hinder the combination between HRP and substrates. It should be pointed out that the weakness of the combination between HRP and substrates cannot impair the activity of HRP, which has been confirmed by means of molecular simulation. Essentially, the five-coordinated heme iron atom bonds to the imidazole ring of His170 by the Fe−N coordination bond on native HRP (Figure 5a), the dissociation energy (Ed) between heme and His170 is 20.53 kcal/mol, which increases to 27.85 kJ/mol when a [C4mim] group accesses (Figure 5b), accordingly, the conformation of heme changes upon the 5463

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Figure 6. Optimization for the concentration ((a) and (b)) and the pH (c) of SDS/[C4mim][BF4] combination as mediums for catalysis of HRPH2O2-TMB. The different ratios of molar concentration of SDS/[C4mim][BF4] are presented in (a) and (b).

of [C4mim][BF4], effectively suppressing the catalytic activity of HRP.39 In particular, when [C4mim][BF4] concentration is over 7.5 mM, the formation rate of the blue chromogen is nearly zero due to a stronger electrostatic interaction between the imidazolium cation and the carboxyl on HRP that leads to HRP inactivation. Accordingly, the structure of HRP should be changed, which can be verified by the variation of UV absorbance of HRP at 403 nm obtained in different mediums as is shown in Figure S4. The UV absorbance of HRP in PBS and guanidine hydrochloride solution (enzyme inactivating agent) corresponds to the structure of native HRP and the structure of deactivated HRP, respectively. Obviously, the UV absorbance of HRP in mediums containing 7.5 and 12.5 mM [C4mim][BF4] are much lower than that in PBS, but the values approximate to the absorbance obtained in enzyme inactivating agent guanidine hydrochloride solution, which confirms that HRP is fully deactivated in the mediums containing 7.5 and 12.5 mM [C4mim][BF4]. Obviously, 2.5 mM [C4mim][BF4] matches well with 2.5 mM SDS. Furthermore, the optimal concentration of SDS should be determined. Likely, the concentration of [C4mim][BF4] remains constant at 2.5 mM and increases the SDS concentration in the mediums as Figure 6b shows, the blue chromogens tend to decompose quickly in the mediums without SDS (in PBS and 2.5 mM [C4mim][BF4] solution), whereas the equilibrium concentration of the blue chromogen increases with the increase of the SDS concentration when the SDS concentration is below 2.5 mM. Once the SDS concentration exceeds 2.5 mM, the catalytic efficiency appears to decrease due to the decrease of the activity of HRP.27 Consequently, 2.5 mM SDS/2.5 mM [C4mim][BF4] can be regarded as the superior combination for the catalysis of HRPH2O2-TMB. Under the optimal combination of 2.5 mM SDS/2.5 mM [C4mim][BF4], the appropriate pH of the system is determined as Figure 6c demonstrates. It can be seen that the equilibrium concentration of the blue chromogen increases with the increase of pH of mediums until the pH reaches 7.4. This may result from the TMB solubility increase with the pH as is found in the experiments, which benefits to improve the catalytic efficiency of the chromogenic catalysis. Then further increases the pH to 8.0, less blue chromogens are formed, suggesting that high pH inhibited enzyme activity.40 Thus, the preferable pH is 7.4 for 2.5 mM SDS/2.5 mM [C4mim] [BF4] combination for the catalysis of HRP-H2O2-TMB. Given that most chromogenic assays are usually carried out at room temperature, moreover, the reaction temperature of 25

blue chromogens, under the three favorable effects, SDS/ [Cnmim][BF4] (n = 2, 4) combinations perform better in promoting catalysis. As for the systems containing longer-chain [Cnmim][BF4] (n = 6, 8, 10), because majority of [Cnmim]+ (n = 6, 8, 10) cations participate in the formation of mixed micelles with SDS molecules, on the one hand, minority of free [Cnmim]+ (n = 6, 8, 10) cations are left in the aqueous solution to activate HRP, on the other hand, the decrease of the negative charges of mixed micelles weaken the stabilization of the positively charged blue chromogens. The above adverse effects may eventually lead to a gradual decrease of the catalytic efficiency of HRP-H2O2-TMB with the increase of the chain length of [Cnmim][BF4] (n = 6, 8, 10) in the medium. Among the studied mediums, SDS/[C4mim][BF4] combination can produce much denser and more negatively charged micelles for encapsulating TMB and combining positively charged blue chromogen, which endows the combination with the best performance in improving the catalytic efficiency of HRP-H2O2-TMB. It is undeniable that surfactant SDS mainly expert at stabilizing the blue chromogens and [C4mim][BF4] contributes to activate HRP but is incapable of stabilizing the blue chromogen.27 Additionally, the diffusion coefficient (DS) and diffusion velocity constant (k) of both TMB and H2O2 were calculated theoretically (Table S2). It is found that substrate diffusion is not the rate-limiting step for the studied system (for details, see Sections 3 and 4 in the Supporting Information). Therefore, the decrease of the catalytic efficiency of HRP-H2O2-TMB in the mediums containing [Cnmim][BF4] (n = 6, 8, 10) is mainly attributed to weaker activation for HRP and the decreased stabilization of blue chromogen instead of the slightly decreased substrate diffusion. 2.4. Optimization for the Medium of SDS/[C4mim][BF4]. As a preferable candidate, SDS/[C4mim][BF4] combination exhibited superiority in promoting the efficiency of catalysis. To better apply the chromogenic system of HRPH2O2-TMB, SDS/[C4mim][BF4] combination is singled out and the applicable conditions are optimized. For the studied system, the molar concentrations of SDS and [C4mim][BF4] in the medium as well as pH of the medium are influential factors for the enzymatic catalysis. We optimize the concentration of the components by fixing one component concentration and changing the other concentration. First, the SDS concentration is fixed at 2.5 mM and then increases the [C4mim][BF4] concentration in the medium as Figure 6a shows, the formation rate of the blue chromogen increases first and then largely decreases when the [C4mim][BF4] concentration is over 2.5 mM, which may attribute to the high concentration 5464

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°C can achieve a satisfied chromogenic performance. Thus, the reaction temperature is determined as 25 °C. 2.5. Substrate Specificity in the Medium of SDS/ [C4mim][BF4] Combination. It is proverbial that one important factor determining the sensitivity of ELISA is the sensitivity of the final chromogenic reaction stage.41 For the sake of setting a practical basis for an enhanced ELISA strategy based on the unique chromogenic medium, five common chromogenic substrates of HRP were selected for comparison. As the proper reaction conditions and test conditions vary with the substrates of HRP, the corresponding experimental conditions for OPD, ABTS, o-dianisidine dihydrochloride (ODA), and 5-AS are adopted according to references (Table S1).9,13,42 Figure 7a demonstrates the maximum optical density

in PBS (pH 5.0)), the formation rate of the chromogen of OPD is slower than that in other systems at the initial stage of the reaction. As for system D (the catalysis of HRP-H2O2ABTS in PBS (pH 4.0)), it is observed that the OD value of chromogen of ABTS at 414 nm increases slowly after the reaction of 15 min, which may result from the nonenzymatic oxidation of ABTS that may cause a false positive results in ELISA. 42 Furthermore, the later three catalysis need termination with 2 M H2SO4 after the reaction of 15 min. Obviously, the chromogenic system of the catalysis of HRPH2O2-TMB in SDS/[C4mim][BF4] combination completely prevails in providing an assay format for enhanced ELISA. 2.6. Application of the Chromogenic System Based on SDS/[C4mim][BF4] Medium. 2.6.1. Enhanced ELISA Using the Chromogenic System Based on SDS/[C4mim][BF4] Medium. The established chromogenic system based on SDS/ [C4mim][BF4] combination was applied to detect the human IgG (the anti-IgG-HRP as the second antibody). Figure 8

Figure 7. (a) Maximum OD values (ODmax) of chromogens of different substrates (TMB, OPD, ABTS, ODA, and 5-AS, respectively) in SDS/[C4mim][BF4] combination and PBS after the reaction for 30 min. Inset is the corresponding color images for the five substrates in SDS/[C4mim][BF4] (top) and PBS (bottom). (b) The kinetic process in four different chromogenic systems with higher ODmax (in Figure 6a). Systems A, B, C, and D represents the chromogenic process of TMB in 2.5 mM SDS/2.5 mM [C4mim][BF4] combination (pH 7.4), TMB in PBS (pH 5.0), OPD in PBS (pH 5.0), and ABTS in PBS (pH 4.0) respectively.

Figure 8. Results of ELISA test for human IgG detection and the corresponding color images (the concentration of IgG per well is 0, 0.1, 0.5, 1.5, 3.0, 6.0, 12.0, 24.0 mg/mL, respectively). (a) Comparison of the detection performance of the catalysis of HRPH2O2-TMB in 2.5 mM SDS/2.5 mM [C4mim][BF4] combination (pH 7.4) (enhanced ELISA) with that in PBS (pH 5.0) (conventional method). (b) The linear calibration plots for IgG detection. Error bars represent the standard deviation of three repeated measurements.

(OD) values (ODmax) of different chromogens in SDS/ [C4mim][BF4] combination and PBS after the reaction for 30 min. Compared with the control experiment of the chromogenic catalysis in PBS, it is obvious that SDS/ [C4mim][BF4] only promotes the catalysis of HRP to TMB, whereas inhibits the catalysis of HRP to other four substrates in different degrees, suggesting the uniqueness of HRP-H2O2TMB catalysis in SDS/[C4mim][BF4] combination. Additionally, the chromogenic sensitivity can be evaluated by the ODmax, the ODmax of the chromogenic catalysis of HRP to TMB in SDS/[C4mim][BF4] combination far surpasses that of other systems. Furthermore, four chromogenic systems with higher ODmax including the chromogenic catalysis of TMB in SDS/ [C4mim][BF4]combination, OPD, TMB, and ABTS in PBS, respectively, (in Figure 6a) are selected to monitor the kinetic process, the results are shown in Figure 7b. Remarkably, the catalysis of HRP-H2O2-TMB in SDS/[C4mim][BF4] combination (system A) shows higher efficiency indicated by shorter equilibrium time and higher sensitivity. To system A, the OD value of the chromogen can remain stable after the reaction reaches equilibrium and the operation procedure is more convenient without termination by stop solution. However, for system B (the catalysis of HRP-H2O2-TMB in PBS (pH 5.0)), the OD value at 450 nm decreases slightly after the reaction for 15 min, which means that a bit of yellow chromogen of TMB will decompose. In system C (the catalysis of HRP-H2O2-OPD

provides the results of human IgG detection performed in different ELISA tests. Higher ELISA signals are observed in SDS/[C4mim][BF4] combination than that in the conventional sandwich ELISA system, as shown in Figure 8, although the enhanced strategy is insufficient in improving the limit of detection (LOD) compared with the conventional method (detection limit for both of the methods are 0.75 mg/mL). It may imply that TMB can be transformed into remarkable chromogen only if there are enough HRP molecules in the mediums. In linear calibration plots for IgG detection (Figure 8b), it is found that the OD value of chromogen for an enhanced strategy clearly surpasses that of the conventional method in the concentration ranging from 0.75 to 25 mg/mL, exhibiting the superiority of the enhanced ELISA. Reasonably, the amplification of the enhanced ELISA strategy is the consequence of applying the novel chromogenic systems by taking SDS/[C4mim][BF4] combination as medium instead of PBS for the catalysis of HRP-H2O2-TMB. At the same time, the advantage for enhanced ELISA strategy for detecting IgG also embodies in its time effectiveness. Only 5 min is needed to reach a measurable 5465

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and even saturated signal (depending on the target concentration) for the enhanced ELISA, whereas the conventional ELISA often requires at least 30 min to develop the color. The extended assay time is recognized as one of the major handicaps nowadays of the ELISA assay.43 Therefore, the improved strategy prevails as the better option with respect to the conventional method due to higher sensitivity, time effectiveness, and more simplified procedure. 2.6.2. Detection of H2O2 and Glucose Using the Chromogenic System Based on SDS/[C4mim][BF4] Medium. As is known that a number of mimetic peroxidases have been synthesized and are applied to design high sensitive biosensors.45 In particular, most of mimetic peroxidases are employed as catalysts for the chromogenic catalysis of TMB/ H2O2, which can achieve colorimetric determination of H2O2 and glucose.46 As H2O2 is the main product of glucose oxidation by glucose oxidase (GOx) in the presence of oxygen, thus, glucose detection could be implemented by coupling GOx with HRP to achieve the cascade reaction in the mediums of SDS/[C4mim][BF4] as illustrated in Scheme 1. According Scheme 1. Schematic Illustration H2O2 and Glucose Detection Using the Chromogenic System Based on SDS/ [C4mim][BF4] Medium

Figure 9. UV−vis absorbance and the corresponding color images for H2O2 detection (a) and glucose detection (b). The linear calibration curves of H2O2 (c) and glucose (d). Error bars represent the standard deviation for three measurements.

to Scheme 1, the colorimetric strategies for the detection of H2O2 and glucose were further developed based on the promoted chromogenic system of the catalysis of HRP-H2O2TMB in the medium of SDS/[C4mim][BF4] combination. Figure 9a provides the variation of UV−vis absorbance with the H2O2 concentration as well as the corresponding color images of the samples. The UV−vis absorbance of the blue chromogen increases with the H2O2 concentration of 0.2− 1000 μM, the presented low detection limit (LOD) of 0.2 μM and the linear range of concentration from 0.5 to 100 μM (Figure 9c) are comparable to other methods (Table S3).47−51 For glucose detection, the absorbance of the blue chromogen increases with the variation of the concentration of glucose from 0.5 to 1000 μM (Figure 9b). Furthermore, the linear calibration plots of glucose detection (Figure 9d) displays that the LOD is 1.0 μM and the linearity is at 1.0−100 μM. Compared with other colorimetric strategies for glucose detection, such as the systems based on Fe3O4 magnetic nanoparticles (NPs),44 positively charged AuNPs48 as listed in Table S3, the studied method exhibits higher sensitivity accompanied with a lower LOD of glucose. The above detection method also exhibits high selectivity for the colorimetric detection of glucose. The selectivity described here was tested with glucose and other carbohydrates including sucrose, galactose, and fructose with the procedure identical to the glucose detection. As shown in Figure 10, although the concentrations of other carbohydrate substances are 10 times higher than that of glucose, apparently higher detection signals are observed in the samples of glucose. Moreover, the color

Figure 10. Selectivity for glucose detection in the medium of SDS/ [C4mim][BF4] combination. Except that the concentration of glucose is 500 μM, all the other carbohydrates including sucrose, galactose, and fructose are 5 mM. The insert provides the color images for glucose, sucrose, galactose, and fructose from the left to right.

difference can be distinguished by the naked eye easily. Thus, the colorimetric assay is highly selective for glucose detection.

3. CONCLUSIONS In summary, the perfect combination of the traditional anionic surfactant SDS and ionic liquids [Cnmim][BF4] produced an effective medium for chromogenic catalysis of TMB with HRP in the presence of H2O2. The alkyl chain lengths of [Cnmim][BF4] in SDS/[Cnmim][BF4] combination had significant effects on the activity of HRP, the solubility of substrates, the stabilization of products, as well as the kinetics of the catalysis of HRP-H2O2-TMB, the difference of the catalytic efficiency was the consequence of the multiple combined effects. In the studied five SDS/[Cnmim][BF4] (n = 2, 4, 6, 8, 10) combinations, the efficiency of catalysis increased first and then decreased with the increase of the chain lengths of [Cnmim][BF4] in the mediums, therein, the SDS/[C4mim][BF4] combination displayed extraordinary superiority in improving the catalytic efficiency compared 5466

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4.2. Electrochemical Measurements. The GCE was polished and cleaned before it can be used. Prior to measurement, the HRP (1 g/L) was incubated in different mediums (PBS, 2.5 mM SDS, 2.5 mM SDS/2.5 mM [Cnmim][BF4] (n = 2, 4, 6, 8, 10)) for 1 h, then, these HRP solutions were deaerated with high purity nitrogen sufficiently, sequentially accumulated at a negative potential in different mediums under constant bubbling of nitrogen for 15 min. Then, only the first cycle of the cyclic voltammograms of HRP in different mediums was recorded. The above measurements were carried out at 25 ± 1 °C. 4.3. Kinetic Assays. The steady-state kinetic assays of HRP-H2O2-TMB were further conducted in the mediums containing SDS (2.5 mM) and SDS/[Cnmim][BF4] (2.5 mM SDS, 2.5 mM [Cnmim][BF4]) combination by fixing the HRP concentration at 0.05 μg/mL and varying concentrations of TMB (30, 50, 100, 250, 500, 800, 1000 μM) when the H2O2 concentration was fixed at 1 mM or varying concentrations of H2O2 (5, 10, 20, 50, 100, 250, 500, 1000 μM) when TMB was fixed at 200 μM. The apparent kinetic parameters were calculated based on the Michaelis−Menten equation as follows (eq 1), where, v0 is the initial velocity, vmax is the maximum initial velocity, [S] is the concentration of the substrate, and Km is the Michaelis constant that is usually identified as an indicator of enzyme affinity to substrates, whose value just related to the pH, temperature, and ionic strength of surroundings, but irrelevant to the concentration of substrates or enzymes for the specific enzymatic reaction, a lower Km value means a stronger affinity and vice versa.36,45 v0 was obtained by tracking the variation of absorbance with the time at 652 nm (ε652 = 3.9 × 104 L/mol cm).

with others. Additionally, SDS/[C4mim][BF4] combination showed uniqueness for HRP-H2O2-TMB catalysis compared with other chromogenic catalysis. Inspired by the efficient chromogenic system, an enhanced ELISA strategy for the detection of human IgG was established and the sensitive colorimetric strategies for the detection of H2O2 and glucose were further developed by employing SDS/[C4mim][BF4] combination as medium of chromogenic catalysis of HRPH2O2-TMB. This work not only developed a unique chromogenic system with multitude of potential applications in biological detection, but also provided detailed information for comprehensive understanding to the mechanism of ILs promoting enzymatic catalysis, which was of significance for the application of ILs in enzymatic catalysis systems.

4. EXPERIMENTAL SECTION 4.1. Materials and Instrument. 3,3′,5,5′-Tetramethlybenzidene (TMB) (98%), 2,2-azinobis(3-ethylbenzothiazoline6-sulfonic acid) (ABTS) (98%), o-dianisidine dihydrochloride (ODA) (98.5%), 5-aminosalicylic acid (5-AS) (≥99%), sodium dodecyl sulfate (SDS), sucrose (99%), galactose (98%), and fructose (99%) were purchased from Aladdin Industrial Co., Ltd. o-Phenylenediamine (OPD) was provided by Adamas Reagent Co., Ltd. Horseradish peroxidase (HRP, EC1.11.1.17, 190 unit/mg) and glucose oxidase (Gox) were purchased from Tokyo Chemical Industry Co., Ltd. and stored in a refrigerator at 4 °C. The ILs containing the same anion are 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim][BF 4 ]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6mim][BF4]), 1-octyl-3-methylimidazolium tetrafluoroborate ([C8mim][BF4]), and 1-decyl-3-methylimidazolium tetrafluoroborate ([C10mim][BF4]), the aforementioned ILs (99%) were provided by Monils Chemical Engineering Science & Technology (Shanghai) Co. Ltd. and used without further purification. Dimethyl sulfoxide-d6 (99%) was purchased from J&K Chemical Co., Ltd. Sodium dihydrogen phosphate dehydrate, sodium phosphate dibasic dodecahydrate, citric acid, and 30% hydrogen peroxide solution (H2O2) were purchased from Shanghai Lingfeng Chemical reagent Co., Ltd. The human IgG ELISA kit was obtained from Jining Shiye (Shanghai) Co. Ltd. Ultrapure water with a resistivity of 18.2 MΩ cm from Millipore Simplicity water purification system was used for preparing buffer solutions of different pH values. All other chemicals were of analytical reagent grade and used without further purification. UV-2550 UV−vis spectrophotometer (Shimadzu UV-2550, Japan) and a 1 mL measurement cell were used for the absorption spectrum and time-dependent spectrum. Kinetic experiments were carried out on a Biotek microplate reader. CMC of SDS was measured by the dynamic contact angle measuring instrument and tensiometer DCAT 11 (DataPhysics, GER). Cyclic voltammetry experiment was performed on a CHI 630C electrochemical analyzer (Shanghai Chenhua Co., China). The three-electrode system was composed of a working electrode (GCE), a platinum wire counter electrode, and a silver chloride reference electrode. The diameters and ζ potential of aggregations in the mediums were determined by dynamic light scattering (DLS) (Zem4228, Malvern Instruments). Transmission electron microscopy (TEM) images were obtained on a JEM-2100 (JEOL, Japan) instrument at an accelerating voltage of 200 kV. Viscosities of different mediums were measured by Brookfield viscometer (DV-II).

v0 =

vmax [S] K m + [S]

(1)

The catalytic number (kcat) indicates the catalytic efficiency for a specific enzymatic catalysis under saturated concentration of substrates, which can be calculated by eq 2, where [E] is the concentration of enzyme, the maximum initial velocity (vmax) can be obtained by eq 1. kcat =

vmax [E]

(2)

4.4. Sandwich Enzyme-Based Immunoassay. The human IgG ELISA kit was removed from the refrigerated environment and then equilibrated at room temperature for 30 min before use. Initially, polystyrene 96-well microtiter plates were coated with 100 μL of capture IgG antibodies (diluted 40 times with coating buffer) in the course of incubation at 4 °C overnight. The unbound capture IgG antibodies were washed away three times with 300 μL washing buffer. Then the wells were incubated with 300 μL bovine serum albumin (BSA) solution as blocking buffer for 2 h at 37 °C to minimize nonspecific adsorption of the antigen. After excess BSA was washed away three times with 300 μL washing buffer, different concentrations of 50 μL human IgG solution were pipetted into the wells and incubated for 1 h at 37 °C and followed by addition of 100 μL human anti-IgG-HRP for 1 h incubation at 37 °C. Unbound human IgG and human anti-IgG-HRP were washed five times with 300 μL washing buffer. For the enhanced ELISA, 200 μL SDS/[C4mim][BF4] combination containing 0.3 mM substrate solution and 2 mM H2O2 was added to each well and reacted for 5 min at 37 °C in dark 5467

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conditions. Recorded the OD values at 652 nm of the samples for determining the human IgG concentration. For the conventional ELISA (controlled experiment), 150 μL buffer solution (pH 5.0) containing the same amount of substrate as above was added to each well and reacted for 30 min at 37 °C in dark. Then, added 50 μL H2SO4 (2 M) to stop the reaction and record the OD values at 450 nm of the samples. 4.5. Colorimetric Assay of H2O2 and Glucose. First, different volumes of H2O2 were added into the mediums containing SDS/[C4mim][BF4] combinations to obtain mixtures with different H2O2 concentrations (0.05−1000 μM). Then TMB stock solution (0.2 M) and HRP solution (0.1 mg/mL) were added orderly into the mixtures to obtain reaction solutions containing 200 μM TMB and 0.05 μg/mL HRP. Then the sample was mixed uniformly and reacted for 5 min, and the absorbance was recorded at 652 nm. Glucose detection was conducted as follows: (a) 1 mg/mL GOx and different concentrations of glucose (0.5−1000 μM) were incubated in SDS/[C4mim][BF4] combinations at 37 °C for 30 min. (b) Then TMB and HRP were added orderly into the mixtures to obtain reaction solutions containing 200 μM TMB and 0.05 μg/mL HRP. Then the sample was mixed uniformly and reacted for 5 min, and the absorbance was recorded at 652 nm. In the selectivity experiments, the procedure was the same as above, except that glucose (1 mM) was replaced by galactose (5 mM), sucrose (5 mM), or fructose (5 mM), respectively. 4.6. Theoretical Calculations. The average distance between HRP and ionic liquid molecules was obtained by molecular dynamics simulations. The simulation was implemented by the Forcite module of the Material Studio software. The NVT ensemble was employed. One [Cnmim][BF4] and one HRP molecule were inserted into a simulation box with a size of 8.7 nm × 8.7 nm × 8.7 nm. The temperature was 298 K, which was fixed by the velocity scale method. The universal force field was used for parameterizing the nonelectrostatic interactions and the charge of atoms were calculated by the QEq method. The total simulation time was 200 ns with a time step of 1 fs and the conformation of the system was sampled every 5 ps. The distance between the [Cnmim] and the HRP was defined by the distance between the center of the five-ring of the [Cnmim] and the Fe atom of HRP. The binding states and dissociation energy (Ed) of the heme group with an imidazole ring of His170 on native HRP when surrounded without ILs and an ionic liquid molecule was calculated by quantum density functional theory, which was performed in the Dmol3 module of the Material Studio software. The PW91 method was used for modeling the exchange correlation functional, the core−electron interaction was calculated by the effective core potentials, the OBS method was used to consider the dispersion effect, and the DNP 3.5 basis set was used for numerical treatment of the onebody wave function. The Kohn−Sham equation was solved by the self-consistent interaction, which was considered to converge when the energy change is lower than 2.625 × 10−3 kJ/mol. The conformation of the system was considered to converge when the energy change, maximum force, and maximum displacement were lower than 2.625 × 10−2 kJ/mol, 0.0872 nN, and 5 × 10−4 nm, respectively.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03345. Experiments of optimization of SDS/[C4mim][BF4] mediums and the substrate specificity tests in SDS/ [C4mim][BF4] combinations (including Table S1); the viscosity measurements of different mediums and the derivation of the rate-limiting step (including Table S2); the UV−vis spectra in sole SDS solutions (Figure S1); the mean formation rate of the blue chromogens within the initial 100 s in different mediums (Figure S2); the theoretical calculation results of the dissociation energy (Ed) (Figure S3); the UV absorbance of HRP in different mediums (Figure S4), and the comparison of various approaches for the detection of H2O2 and glucose (Table S3) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.S.). *E-mail: [email protected] (Y.L.). ORCID

Yazhuo Shang: 0000-0003-1598-4711 Yu Liu: 0000-0003-1936-0228 Author Contributions

This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Project Nos. 21476072 and 21776070), the Fundamental Research Funds for the Central Universities and the National Basic Research Program of China (973 Program), 2015CB251401.

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REFERENCES

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DOI: 10.1021/acsomega.8b03345 ACS Omega 2019, 4, 5459−5470

ACS Omega

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Peroxidase Mimetic, and Its Application to the Colorimetric Determination of Hydrogen Peroxide. Microchim. Acta 2018, 185, 417. (47) Zhao, K.; Gu, W.; Zheng, S.; Zhang, C.; Xian, Y. SDS-MoS2 Nanoparticles as Highly-Efficient Peroxidase Mimetics for Colorimetric Detection of H2O2 and Glucose. Talanta 2015, 141, 47−52. (48) Jv, Y.; Li, B.; Cao, R. Positively-Charged Gold Nanoparticles as Peroxidiase Mimic and Their Application in Hydrogen Peroxide and Glucose Detection. Chem. Commun. 2010, 46, 8017−8019. (49) Peng, J.; Weng, J. Enhanced Peroxidase-Like Activity of MoS2/ Graphene Oxide Hybrid with Light Irradiation for Glucose Detection. Biosens. Bioelectron. 2017, 89, 652−658. (50) Chen, L.; Sun, K.; Li, P.; Fan, X.; Sun, J.; Ai, S. DNA-Enhanced Peroxidase-Like Activity of Layered Double Hydroxide Nanosheets and Applications in H2O2 and Glucose Sensing. Nanoscale 2013, 5, 10982−10988. (51) Qiao, F.; Qi, Q.; Wang, Z.; Xu, K.; Ai, S. Mnse-Loaded G-C3N4 Nanocomposite with Synergistic Peroxidase-Like Catalysis: Synthesis and Application toward Colorimetric Biosensing of H2O2 and Glucose. Sens. Actuators, B 2016, 229, 379−386.

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DOI: 10.1021/acsomega.8b03345 ACS Omega 2019, 4, 5459−5470