Online Enzyme Discrimination and Determination of Substrate

Publication Date (Web): June 29, 2012 ... Fax: +86-431-85099762. ... (EMMA) method for fast online discrimination and determination of substrate enant...
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Online Enzyme Discrimination and Determination of Substrate Enantiomers Based on Electrophoretically Mediated Microanalysis Wenwen Zhao, Miaomiao Tian, Rongbin Nie, Yulin Wang, Liping Guo, and Li Yang* Faculty of Chemistry, Northeast Normal University, Changchun, Jilin, 130024, People’s Republic of China S Supporting Information *

ABSTRACT: We proposed the first application of an electrophoretically mediated microanalysis (EMMA) method for fast online discrimination and determination of substrate enantiomers, which was achieved by just one EMMA assay. Lactate dehydrogenase (LDH)-catalyzed reaction was studied to evaluate the feasibility and performance of the presented method. The L- and D-LDH chiral enzymatic reactions, which are highly stereoselective to the lactate enantiomers, were initiated successively in one capillary, and the corresponding products, nicotinamide adenine dinucleotide (NADH), were online discriminated and detected by UV absorption. Excellent linear dependence of the two NADH peak intensities on the concentration of the corresponding lactate enantiomers was obtained within a wide range of 0.1−10 mM. The limit of detection was 26 μM for D-lactate and 49 μM for L-lactate (S/N = 3). Good repeatability of online chiral discrimination was demonstrated with relative standard deviation (RSD) < 6.3% for NADH peak height and RSD < 1.5% for migration time (n = 5). Km values for L- and D-lactate were measured and were consistent with those of the off-line enzyme assays. The presented method was successfully applied to determine the L-/D-lactate in several yogurt and wine samples. Our study shows a new application of the EMMA method utilizing high stereoselectivity of enzymes for fast online chiral analysis.

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enzymatic reaction columns18 or enzyme immobilized biosensors19,20 for online enzymatic assays were proposed. Those methods require not only an efficient procedure of enzyme immobilization but also a complicated apparatus. Fast and simple online chiral discrimination using enzyme assay is still a challenging task. A very important technique in enzyme assay is capillary electrophoresis (CE), which has several advantages compared to other techniques, such as very high resolution, extremely low sample injection, very short analysis time, and so on.21 The introduction of electrophoretically mediated microanalysis (EMMA) by Bao and Regnier in 1992 makes CE not only a high-performance separation tool, but an efficient online enzyme assay method.22 In EMMA, the variability in electrophoretic mobilities among enzyme and its substrate(s) is used for both the initiation of an enzymatic reaction inside the capillary and the separation and detection of the reaction product(s), thus integration of multiple functions “injection, mixing, reaction, separation and detection” in a single capillary. During the past 2 decades, EMMA has been widely used in almost all aspects of enzyme assay and is still continuously attracting interests for the development of its new applications.23,24

nzymes are important compounds catalyzing biochemical reactions necessary for normal growth, maturation, and reproduction through the whole living world. Many studies have been carried out for enzyme assay to evaluate the enzymatic activity,1−3 enzyme kinetics,4−6 enzyme inhibition and activation,7−9 and the investigation of enzyme-mediated metabolic pathways.10,11 Among all purposes of the enzyme assays, one important aspect, of which the studies were rather sparse, is chiral discrimination and determination of the substrate enantiomers. It is well-known that enzymes, which are chiral, show remarkable chemo-, regio-, and stereoselectivities and exhibit excellent chiral specificity on the substrate, i.e., an enzyme with L-configuration (or D-configuration) can only catalyze the reaction of the L-configuration (or D-configuration) substrate. Hence, enzyme assay can be an efficient methodology for chiral discrimination. For example, enzyme assay has been used for determination of lactate enantiomers in biological fluids12−14 and food analysis.15,16 Usually two parallel enzyme assays are necessary to determine the pair of enantiomers of a substrate, each for determination of one enantiomer.16 This is a timeconsuming labor work and, more importantly, reduces the repeatability and accuracy of chiral determination. In addition, there are still some difficulties to apply enzymes for chiral analysis, for example, cross-reaction with endogenous compounds, large sample and enzymatic reagent consumptions, and long reaction time to complete the enzymatic reaction.17 To overcome some of the shortcomings of off-line enzymatic assays, several flow injection analysis systems with two different © 2012 American Chemical Society

Received: April 29, 2012 Accepted: June 29, 2012 Published: June 29, 2012 6701

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Figure 1. (A) Schematic diagram of the modified EMMA plug−plug analysis procedure for online chiral discrimination and detection of lactate enantiomers. See text for the detail. (B) Typical electropherograms of EL−ED mode (red) and ED−EL mode (black). The NADH peak with shorter migration time was the product of the first-injected enzyme catalyzed reaction. The running buffer contained 10 mM glycylglycine (pH 8.8) and 4.7 mM NAD+. A 10 mM D-/L-lactate standard enantiomers sample and 25 U/mL L-LDH and 500 U/mL D-LDH were used for the test.

Buffer and Sample Preparation. The CE running buffer (also serving as the enzymatic reaction buffer) was 10 mM glycylglycine (pH 8.8) containing 4.7 mM β-NAD+. Stock solutions of 200 mM D-lactate or L-lactate standard were prepared in deionized water, stored at 4 °C, and diluted into desired concentrations for EMMA experiments using 10 mM glycylglycine buffer. All the solutions were filtered using an inorganic 0.22 μm membrane filter prior to use. Yogurts and red wines, bought from a local super market, were diluted with buffer by 50-fold and 10-fold, respectively. The diluted yogurts were centrifuged at 3000 rpm for 10 min. The supernatants of the yogurts and the diluted wines were filtered with 0.22 μm filters before analysis. CE Conditions. CE experiments were carried out using a CE apparatus (CL1020 Beijing Cailu Science Apparatus, China) under 22 °C cooling air. The UV signals were recorded at the wavelength of 340 nm. The separation and detection were performed with a capillary (50 μm i.d., Hebei Yongnian Optical Fiber Factory, China), which was precoated with UltraTrol LN (Target Discovery). The total and effective lengths of the capillary were 35 and 26 cm, respectively. The operated electric field strength was −429 kV/cm. Before use, a new capillary was pretreated by 0.1 M NaOH solution for 20 min, followed by flushing with deionized water, UltraTrol LN solution, and running buffer for 5 min, respectively. Between two consecutive runs, the capillary was rinsed sequentially with UltraTrol LN solution (2 min), water (2 min), and then running buffer (3 min). Modified EMMA Plug−Plug Analysis Mode for Online Enzyme Chiral Discrimination. The following chiral enzyme (L-LDH/D-LDH) catalyzed reactions were investigated in this study.

In respect of chiral discrimination using enzyme assay, EMMA can be a powerful technique due to its unique features especially for fast online analysis. However, to the best of our knowledge, there is still no report of the use of EMMA for chiral determination. Here, we present a method for fast, efficient, and accurate online enzyme chiral determination based on EMMA. We modified the plug−plug EMMA analysis mode by injecting enzyme enantiomers and the substrates successively. Due to different mobilities of the enzyme and the substrate, the chiral enzyme catalyzed reactions occur in the capillary and the products corresponding to the two enantiomers of the substrate in the sample are welldiscriminated and detected by capillary electrophoresis. In this way, we have achieved online chiral discrimination and determination with just one EMMA assay. Good repeatability of the presented method has been demonstrated, using L-/DLDH-catalyzed reactions as a test example. Finally, the presented method has been successfully used to determine L-/D-lactate in several yogurt and wine samples, and the results agreed well with those obtained by traditional off-line enzymatic method.

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EXPERIMENTAL SECTION Chemicals. β-Nicotinamide adenine dinucleotide (βNAD+) and nicotinamide adenine dinucleotide (NADH) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). L-LDH (2.0 kU/mL) and D-LDH (20 kU/mL) (LDH, EC1.1.1.27), L-/D-lactate, and glycylglycine buffer (0.5M, pH 10.0) were purchased from Megazyme International Ireland Ltd. (Ireland). Aspartic acid, glutamic acid, tartaric acid, succinic acid, acetic acid, citric acid, and ascorbic acid were purchased from Tianjin Fine Chemicals Co., Ltd. (Tianjin, China). Malic acid, manolic acid, and pyruvic acid were purchased from Tianjin YuanHang Chemicals Co., Ltd. (Tianjin, China). Other reagents were all analytical grade and were used without further purification.

L‐lactate

(or D‐lactate) + NAD+

L‐LDH(or D‐LDH)

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ pyruvate + NADH + H+ 6702

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LDH or D-LDH injected first, respectively. Typical electropherograms are shown in Figure 1B, with EL−ED mode (red color) and ED−EL mode (black color). Two baseline-separated NADH peaks were clearly observed in each electropherogram, which are stoichiometric to the pair of lactate enantiomers. It should be mentioned that our chiral assay of lactate enantiomers was based on discrimination and detection of NADH peaks formed in L- and D-LDH-catalyzed reactions, not really chiral “separation” of lactate enantiomers. Still, term of resolution was used to describe and evaluate the performance of the assay. High resolution was obtained (11.6 for EL−ED and 11.2 for ED−EL in Figure 1B), indicating good discrimination of the substrate enantiomers. In each electropherogram, the NADH peak with shorter migration time was the product of the first-injected enzyme catalyzed reaction. In the test case, it can be seen that the intensity of the NADH peak of the D-LDH-catalyzed reaction was larger than that of the L-LDH-catalyzed reaction. As we changed the injection mode from EL−ED to ED−EL, the two NADH peaks were reversed, which serves as another support of the feasibility of our online chiral assay. In the experiments throughout the paper, the EL−ED mode was used if not mentioned otherwise. To confirm the stereoselectivity of LDH enantiomers of the presented method, we injected the plugs of L-LDH, running buffer and 10 mM D-lactate (instead of the D-/L-lactate mixture) successively, each at a height differential of 10 cm for 2 s, and then −429 kV/cm separation electric field strength was applied across the capillary. As expected no NADH peak was observed. This was also the case if D-LDH, running buffer and 10 mM L-lactate were injected. The results obviously demonstrate that LDH enantiomers provide excellent stereoselectivity, i.e., L-LDH only catalyzes the L-lactate reaction while D-LDH only catalyzes the D-lactate reaction. That is the precondition for chiral discrimination and simultaneous determination of lactate enantiomers using the presented method. CE separation of lactate enantiomers has been reported in literature using cyclodextrin derivatives as chiral selectors.25−28 Although such CE chiral separation is more direct than our EMMA method, sample stacking or preconcentration procedure is required to enhance the sensitivity of chiral analysis and the resolution was reported to be 1.02−1.58 with the separation time of 23−40 min. Our method provides a new methodology for chiral analysis of lactate enantiomers. Despite that it is not direct chiral separation, it is feasible for fast and accurate online chiral discrimination and determination of lactate enantiomers, with the entire analysis time less than 2 min and resolution larger than 11.0. Optimization of the Experimental Conditions for Online Chiral Enzyme Discrimination. Since lactate has larger electrophoretic mobility than LDH enzyme, it is possible that, in the EL−ED mode, for example, L-lactate of the lastinjected substrate plug may catch up the L-LDH enzyme plug during the separation and detection step (Figure 1A, step f). If this happens, an additional NADH peak other than the two NADH product peaks mentioned above will be produced (a schematic diagram to show how this additional peak is formed can be found in the Supporting Information Figure S-1). To avoid such an NADH peak, it is crucial to control the distance between the L-LDH plug (first-injected enzyme enantiomer) and the last-injected sample plug, which can be achieved by adjusting the electrophoresis time in the step c of the procedure. Figure 2 shows the electropherograms with different

L-LDH or D-LDH specifically catalyzes the oxidation of Llactate or D-lactate, converting the coenzyme NAD+ into NADH, which is stoichiometric to each substrate enantiomer, respectively. To achieve online chiral discrimination and determination of the pair of the lactate enantiomers, we carried out a modified EMMA plug−plug analysis mode, as schematically shown in Figure 1A. In the procedure, we first inject the plugs of L-LDH, running buffer, and lactate sample (a mixture of D-/L-lactate) successively, each at a height differential of 10 cm for 2 s (Figure 1A, step a). Before each injection the inlet end of the capillary and the electrode are dipped in water in a vial to prevent reagents from cross contamination. Because the electrophoretic mobility of the substrate is larger than that of the enzyme, the plug of lactate can catch up and mix with the plug of LDH electrophoretically when a high electric field strength of −429 kV/cm is applied (Figure 1A, step b). Therein L-lactate enantiomer in the sample is discriminated by L-LDH and is oxidized to produce NADH. After electrophoresis for 60 s to separate lactate (mixture of D-lactate and residual L-lactate), NADH (stoichiometric to L-lactate), and L-LDH (Figure 1A, step c), the high voltage is turned off, and D-LDH, running buffer, and the same lactate sample as in the step a are injected into the capillary successively (10 cm for 2 s) (Figure 1A, step d). This time, D-lactate is discriminated by D-LDH, and the product NADH which is stoichiometric to D-lactate is formed (Figure 1A, step e). Finally, six plugs, i.e., two lactate sample plugs, two product NADH plugs, and two enzyme plugs, are separated and move to the detection window (Figure 1A, step f). According the absorbance of NADH peaks, concentrations of L-and D-lactate are measured. As an adequate amount of product is formed during interleaving of enzyme and substrate plugs, no online incubation is necessary. In addition, the electroosmotic flow (EOF) is significantly reduced since the capillary is precoated with UltraTrol LN. Thus, the entire assay time is shortened to be less than 2 min. Off-Line Enzyme Assay. A test combination (L- and Dlactate assay, Megazyme International Ireland Ltd., Ireland) was used for the off-line enzyme assay to determine L- and D-lactate enantiomers. The enzymatic activities of L- and D-LDH were determined by measuring the absorbance intensities of the NADH products at 340 nm. The reaction mixture (200 μL) contained 10 mM glycylglycine (pH 8.8), 4.7 mM β-NAD+, and L- or D-lactate of different concentrations. Reactions were initiated by the addition of 10 μL of 25 U/mL L-LDH or 500 U/mL D-LDH, respectively, into the mixture. Aliquots of 10 μL were periodically removed from the reaction mixture and injected into the capillary at a height of 10 cm for 2 s for CE analysis. The CE running buffer was 10 mM glycylglycine (pH 8.8). The electric field strength for CE analysis was −429 V/ cm.

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RESULTS AND DISCUSSION Feasibility of the Online Chiral Assay Procedure. We first investigated feasibility of our modified EMMA plug−plug injection mode for online chiral discrimination. The running buffer contained 10 mM glycylglycine (pH 8.8) and 4.7 mM NAD+. A 10 mM L/D-lactate standard sample and 25 U/mL LLDH and 500 U/mL D-LDH were used for the test. In the analysis procedure illustrated in Figure 1A, we can inject either L-LDH or D-LDH first at the step a, while the other enzyme enantiomer is at the step d. For convenience of discussion, we named EL−ED mode or ED−EL mode for L6703

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Figure 2. Electropherograms with different electrophoresis times in the step c of the analysis procedure, using (a) EL−ED mode and (b) ED−EL mode. In each figure, the two NADH peaks at 60 s are corresponding to two chiral enzyme reactions, respectively. As the electrophoresis time decreases, an additional NADH peak appears between them. See text for detail. Other experimental conditions were the same as those in Figure 1B.

electrophoresis times using EL−ED mode (Figure 2a) and ED−EL mode (Figure 2b). It can be seen that in both modes a third NADH peak appears if the electrophoresis time in the step c is decreased to a certain value. In EL−ED mode, this additional NADH peak appears at the electrophoresis time less than 30 s, while in ED−EL mode, it appears at the electrophoresis time less than 20 s. The different electrophoresis times at appearance of the third NADH peak indicate that the electrophoretic mobility of L-LDH is different from that of D-LDH. The third NADH peak is much broader and asymmetric compared to the other two NADH peaks, which might be caused by unavoidable adsorption of enzyme onto the inner surface of the capillary. As the electrophoresis time is further decreased, the NADH peaks are overlapped with each other. Considering the analysis time and the separation efficiency as well, the electrophoresis time of 60 s in the step c was chosen for all the following experiments. Several other key factors that may affect the online chiral discrimination were also investigated. Different buffers, including phosphate, Tris/HCl, and glycylglycine, were studied and optimized to improve both CE separation efficiency and detection sensitivity. The 0.5 and 2 mM standard lactate samples were selected for test. The peak heights of NADH product for both L- and D-lactate using glycylglycine (10 mM, pH 8.8) were about 2 times higher than those using Tris/HCl (10 mM, pH 8.8) and about 4 times higher than those using phosphate (10 mM, pH 8.8). Thus, 10 mM glycylglycine buffer with its optimum pH value at 8.8 was used as electrophoretic buffer and enzymatic reaction buffer of all the experiments. Figure 3 shows the effect of the concentration of coenzyme NAD+ on the NADH peak intensity in the range of 1.0−8.0 mM. It can be seen that, as the concentration of the coenzyme is increased, the NADH peak intensity first increases and then gradually decreases, for both L-LDH- and D-LDH-catalyzed reactions. This indicated that, in our analysis procedure, either high or low concentration of NAD+ can decrease the activity of the LDH enzymatic reaction for determination of the lactate enantiomers. The optimized NAD+ concentration was obtained at 4.7 mM. The effects of the concentrations of LDH enantiomers on the chiral analysis of the lactate sample were studied, in the range of 6.25−100 U/mL for L-LDH and 125 U/mL to 1 kU/ mL for D-LDH. A 10 mM D-/L-lactate sample was used as test sample. The results are shown in Figure 4. It is not surprising to see the NADH peak intensity increases as the concentration of the LDH enzyme is increased. Taking consideration of both

Figure 3. Effect of the concentration of coenzyme NAD+ on the NADH peak intensities in the range of 1.0−8.0 mM, for L-LDH- and D-LDH-catalyzed reactions. Other conditions were the same as those in Figure 1B.

Figure 4. Effects of the concentrations of LDH enantiomers on the chiral analysis of the lactate sample, in the range of 6.25−100 U/mL for L-LDH and 125 U/mL to 1 kU/mL for D-LDH. The concentration of coenzyme NAD+ was 4.7 mM. Other conditions were the same as those in Figure 3.

detection sensitivity and the experimental cost, the concentrations of L-LDH and D-LDH were chosen to be 25 and 500 U/mL, respectively. Performance of the Online Chiral Discrimination. Under the optimum experimental conditions, the performance of online chiral enzyme discrimination using our modified EMMA analysis procedure was evaluated. The intensity of the 6704

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is alike the indirect approach of chiral analysis. The method avoids the choice of a suitable chiral selector and will be suitable for chiral analysis of those compounds which can be acted upon by enzyme and generate detectable product, such as by NADdependent enzymes. Last but not least, the method extends the application of EMMA technology, not only in online chiral discrimination but also in simultaneous investigation of chiral enzyme catalyzed reactions. Application of the Presented Method for Online Chiral Discrimination of Food Samples. To examine the selectivity, 10 substances (aspartic acid, glutamic acid, tartaric acid, malic acid, manolic acid, pyruvic acid, succinic acid, acetic acid, citric acid, and ascorbic acid), which might exist in yogurt or wine, were investigated for possible interferences. The concentrations of the tested substances (50 mM tartaric acid and malic acid, 3 mM manolic acid, pyruvic acid, and ascorbic acid, 10 mM succinic acid and acetic acid, 15 mM citric acid and aspartic acid) were higher than those commonly declared in wine and yogurt product.26,32−35 Assuming for wine analysis, the tested samples were diluted 1:10. Potential interferences were individually injected and analyzed by the presented method under the optimum conditions. No response was detected for all those substances. Using the presented EMMA method, L- and D-lactate in three brands of yogurts and two brands of wines were determined, as shown in Table 1. The ratios between D- and L-lactate in the

NADH peak shows good linear dependence on the concentration of the corresponding lactate enantiomer within a wide range of 0.1−10 mM. The calibration curves of L-lactate and D-lactate were y = 0.424x + 1.337 (R2 = 0.9903) and y = 0.715x + 0.557 (R2 = 0.9961), respectively. Good repeatability of online chiral discrimination was demonstrated with RSD < 6.3% for NADH peak height and RSD < 1.5% for migration time (n = 5). The limit of detection of L-lactate was obtained to be 49 μM (S/N = 3), and that of D-lactate was 26 μM (S/N = 3). The limit of detection of lactate was reported in a wide range of 4−200 μM using CE or HPLC techniques.26−29 Very recently, the limit of detection was lowered to submicromolar region (0.125 μM for L-lactate and 0.5 μM for D-lactate), by the concentration of lactate enantiomers by supported liquid extraction followed by chiral chromatography coupled to tandem mass spectrometry.30 With regard to the presented methodology, the limit of detection may be further improved if laser-induced fluorescence (LIF) detection is applied instead of UV detection and/or a dual-enzymatic method31 is introduced in the modified EMMA procedure. The Km values for L-lactate and D-lactate were obtained from nonlinear fitting of the Michaelis−Menten diagrams, as shown in Figure 5. Each point in the figure was the averaged result of

Table 1. Comparison of the Values Determined for D- and LLactate in Yogurt and Wine Samples Using Online EMMA and Traditional Off-Line Enzymatic Methods (n = 3) food yogurt (A)

lactic acid LD-

yogurt (B)

LD-

yogurt (C)

Figure 5. Michaelis−Menten diagram of L-LDH (red triangles) and DLDH (blue circles). Each data point in the figure was the result of the average of three measurements. Other conditions were the same as those described in Figure 4.

LD-

red wine (A)

LD-

red wine (B)

LD-

three runs. The peak height of product NADH, which is proportional to the initial velocity of enzyme reaction, was measured at different concentrations of the lactate. By nonlinear regression of the Michaelis−Menten diagrams, the Km values for D-lactate and L-lactate were determined to be 3.9 ± 0.5 and 4.2 ± 0.6 mM, respectively. The Km values were in good agreement with those obtained using off-line enzyme assays, which were 3.6 ± 0.4 mM (for D-lactate) and 4.0 ± 0.5 mM (for L-lactate), indicating that the presented method did not affect the enzymatic activity and kinetics. The presented method combines the unique features of the EMMA method and the high stereoselectivity of enzymes, as well as the high-performance CE technique, thus having several advantages for online chiral discrimination. First, by modifying the plug−plug analysis mode of EMMA, we can simultaneously online discriminate and detect the pair of substrate enantiomers with just one EMMA assay. Second, due to the feature of fast online enzyme analysis of the EMMA method, the chiral discrimination can be achieved with high accuracy in less than 2 min. Third, the method utilizes the high stereoselectivity of enzymes for the purpose of online chiral discrimination, which

EMMA (mM) 58.0 5.4 77.5 6.3 69.5 2.3 24.6 5.0 7.6 1.6

± ± ± ± ± ± ± ± ± ±

2.8 0.3 3.6 0.4 3.2 0.2 1.5 0.3 0.4 0.1

off-line enzymatic method (mM) 61.3 5.3 79.9 6.5 68.0 2.0 22.4 5.1 6.8 1.4

± ± ± ± ± ± ± ± ± ±

3.1 0.3 2.9 0.3 3.5 0.2 1.8 0.2 0.4 0.1

three yogurts obtained from different manufacturers varied, which might result from the properties of the lactic bacteria that take part in lactate fermentation, since the three manufacturers use different types of lactic bacteria. Also shown in the table are the results of off-line enzyme assay. The results obtained using the presented method are consistent with those obtained by traditional off-line enzyme assay.

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CONCLUSION In conclusion, we presented the first application of EMMA for fast online discrimination and determination of the substrate enantiomers. By injecting the enzyme enantiomers and the substrate into the capillary successively, the two reactions catalyzed by the enzyme enantiomers are initialized in the same capillary and the corresponding products which are stoichiometric to the two substrate enantiomers are discriminated and detected by capillary electrophoresis. Thus, due to the property of high stereoselectivity of enzymes, online chiral analysis is achieved without the choice of any chiral selectors. Using the 6705

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(18) Mori, H. Anal. Lett. 1999, 32, 1301−1312. (19) Nanjo, Y.; Yano, T.; Hayashi, R.; Yao, T. Anal. Sci. 2006, 22, 1135−1138. (20) Girotti, S.; Muratori, M.; Fini, F.; Ferri, E. N.; Carrea, G.; Koran, M.; Rauch, P. Eur. Food Res. Technol. 2000, 210, 216−219. (21) Geiger, M.; Hogerton, A. L.; Bowser, M. T. Anal. Chem. 2011, 84, 577−596. (22) Bao, J. M.; Regnier, F. E. J. Chromatogr. 1992, 608, 217−224. (23) Fan, Y.; Scriba, G. K. E. Electrophoresis 2010, 31, 3874−3880. (24) Hai, X.; Yang, B.-F.; van Schepdael, A. Electrophoresis 2012, 33, 211−227. (25) Kodama, S.; Yamamoto, A.; Matsunaga, A. Analyst 1999, 124, 55−59. (26) Kodama, S.; Yamamoto, A.; Matsunaga, A.; Soga, T.; Minoura, K. J. Chromatogr., A 2000, 875, 371−377. (27) Saavedra, L.; Barbas, C. J. Chromatogr., B 2002, 766, 235−242. (28) Tan, L.; Wang, Y.; Liu, X.; Ju, H.; Li, J. J. Chromatogr., B 2005, 814, 393−398. (29) Cevasco, G.; Piatek, A. M.; Scapolla, C.; Thea, S. J. Chromatogr., A 2011, 1218, 787−792. (30) Henry, H.; Conus, N. M.; Steenhout, P.; Béguin, A.; Boulat, O. Biomed. Chromatogr. 2012, 26, 425−428. (31) Shapiro, F.; Silanikove, N. Food Chem. 2010, 119, 829−833. (32) Katrlik, J.; Pizzariello, A.; Mastihuba, V.; Svorc, J.; Stred’ansky, M.; Miertus, S. Anal. Chim. Acta 1999, 379, 193−200. (33) Ligor, M.; Jarmalaviciene, R.; Szumski, M.; Maruska, A.; Buszewski, B. J. Sep. Sci. 2008, 31, 2707−2713. (34) Masar, M.; Poliakova, K.; Dankova, M.; Kaniansky, D.; Stanislawki, B. J. Sep. Sci. 2005, 28, 905−914. (35) Shapiro, F.; Silanikove, N. Food Chem. 2011, 129, 608−613.

presented method, we show that discrimination and detection of the lactate enantiomers can be achieved online within 2 min by just one EMMA assay, which ensures the repeatability and accuracy of the chiral analysis using enzyme reactions. In this work, the limit of detection of L-lactate or D-lactate was obtained to be as low as 49 or 26 μM (S/N = 3), the RSD was less than 1.5% for migration time (less than 6.3% for NADH peak height) (n = 5), and the Km values for L-lactate and Dlactate as well as the L-/D-lactate determination of several yogurt and wine samples were in good agreement with off-line enzyme assay. Our study shows that the presented method has several well-recognized advantages, such as high separation efficiency, short analysis time, small sample and chemicals consumption and thus will have a great potential for online discrimination and determination of substrate enantiomers and chiral enzyme assay.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-431-85099762. 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 under Grants 21175018 and 20805005. REFERENCES

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dx.doi.org/10.1021/ac301125j | Anal. Chem. 2012, 84, 6701−6706