A single-step in-situ acetylcholinesterase-mediated alginate

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A single-step in-situ acetylcholinesterase-mediated alginate hydrogelation for enzyme encapsulation in CE Jiqing Yang, Xiaotong Hu, Jia Xu, Xin Liu, and Li Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05353 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018

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Analytical Chemistry

A single-step in-situ acetylcholinesterase-mediated alginate hydrogelation for enzyme encapsulation in CE

Jiqing Yanga, Xiaotong Hua, Jia Xu, Xin Liu, Li Yang*

Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, P.R. China

* Corresponding author: Prof. Li Yang

Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, P.R. China

E-mail: [email protected]

Tel: +86-431-85099762; Fax: +86-431-85099762

a

These authors contributed equally.

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in-situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with “egg-box” structure is triggered inside a capillary with releasing of Ca2+ by changing the pH of the sol solution, which is accomplished in-situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate on-line enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for on-line CE assays with immobilized enzymes, which can be widely applied in bioanalysis.


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Miniaturized bio-enzyme reactors have been developed rapidly, which are widely applied from specialty fine chemicals, drugs, biotherapeutics, and biofuels to various bioanalytical applications, such as in genomics, proteomics metabolomics, and glycomics. Among these reactors, miniaturized immobilized enzyme reactors (IMERs) involve the immobilization of enzymes in a microchannel or capillary to perform heterogeneous biocatalysis. These reactors present essential advantages, such as enhanced enzyme stability, reduced enzyme cost and feasibility of reusing enzymes without any complex purification procedure to isolate from post-reaction mixtures, thus are very attractive in various fields1-4. As an efficient microfluidic separation technique, capillary electrophoresis (CE) has emerged as a powerful approach for enzymatic reactors without enzyme immobilization5 or fabricating miniaturized IMERs6. In CE-integrated IMERs, after the enzymatic reactions, product(s) and remaining reactants are electrophoretically separated and detected downstream of the same capillary for in-line quantitative determination of enzyme activity. This process greatly reduces sample manipulation and analysis time and is expected to fulfill the increasing demands of miniaturized bioanalytical systems6-8. To date, CE-integrated IMERs have been used in almost all aspects of enzyme assays9-11, such as evaluation of the enzymatic activity and kinetics, screening of inhibitor, investigation of enzyme-mediated metabolic pathways, and proteome analysis. Several strategies have been developed to fabricate CE-integrated IMERs. For open-tubular CE-integrated IMERs, enzymes are directly immobilized to the inner wall of a capillary by physical adsorption or covalent binding12-14. Despite the simplicity of the fabrication of open-tubular CE-integrated IMERs, these reactors have low enzyme loading capacity because of



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the relatively small surface-to-volume ratio, which is a critical issue when assaying low-activity enzymes. Alternatively, enzymes can be immobilized on the monolithic porous solid15-18 or packed particles19-20 in a capillary. Such monolithic or packed CE-integrated IMERs can significantly increase the surface-to-volume ratio, enhancing the enzyme loading capacity. However, immobilization would be rather difficult to handle because it generally requires complex steps and manipulations of fabrication conditions. Hydrogels are biomaterials formed by the self-assembly of small molecules to gel solution and have been extensively used in biopharmaceutical and biomedical applications because of their excellent properties21-25. Hydrogels are efficient matrices for encapsulation of proteins. The use of hydrogels is a promising approach to fabricate CE-integrated IMERs, because the resulting enzyme loading capacity could be comparable to that using monolithic or packing technique but with remarkably simplified fabrication procedure26. Ideal materials for hydrogel support for CE-integrated

IMERs

should

be

hydrophilic,

inert

toward

enzymes,

biocompatible,

environment-friendly, and not expensive. Alginate, a seaweed extract composed of chains of alternating

α-L-guluronic

acid

(G)

and

β-D-mannuronic

acid

(M)

residues,

is

a

biocompatible/degradable, nontoxic, and nonimmunogenic linear polysaccharide copolymer. Alginate can produce ionic cross-links by interacting with divalent cations (e.g., Ca2+), forming an “egg-box”-structure alginate-hydrogel that is pH- and thermal-stable and has high encapsulation efficiency27-29. The Ca–alginate matrix is a well-known immobilization method that has been widely used in biomedical, pharmaceutical, and proteomic applications30-31. Thus, the Ca–alginate matrix is an ideal candidate for simple and efficient immobilization of enzymes in the fabrication of CE-integrated IMERs.


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In this study, we report, for the first time, an easy approach for the fabrication of CE-integrated IMERs based on in-situ acetylcholinesterase (AChE)-mediated alginate hydrogelation. As schematically shown in Scheme 1, alginate hydrogelation is triggered by the release of Ca2+ from the reaction of calcium carbonate (CaCO3) and acetic acid. The latter is the product of AChE-catalyzed hydrolysis of acetylcholine. The principle of similar enzyme-mediated gelation has been described previously32, which is also viewed as the internal setting method for hydrogelation33-34, i.e., the cross-linking cations release into the alginate solution from the dispersed insoluble precursor initiated by changing of pH. In our study, the AChE-mediated hydrogelation occurs in situ in the capillary, and as the alginate network grows, enzymes, such as AChE and any other enzyme injected together [e.g., xanthine oxidase (XO) in our study], are encapsulated. This approach results in single-step formation of CE-integrated IMERs for both enzymes in the same capillary. Our study provides a new method for efficient and homogenous enzyme encapsulation by in-situ alginate hydrogelation in the capillary. To the best of our knowledge, this study is the first report on the use of enzyme-mediated hydrogelation to fabricate CE-integrated IMERs. The proposed method will greatly extend the application of IMERs in bioassays. The performance and enzymatic kinetics of immobilized AChE and XO IMERs are investigated. The proposed CE-integrated IMER of AChE is successfully applied to detect organophosphorus pesticides (OPs). EXPERIMENTAL SECTION Reagents and materials. AChE, XO, acetylthiocholine (ATCh), xanthine, allopurinol, and tacrine were purchased from Sigma Chemical (St. Louis, MO). Sodium alginate was obtained from Tianjin Gangfu Fine Chemical Research Institute (Tianjin, China). Precipitated CaCO3



particles (~1 µm as reported by the manufacturer) were obtained from Zijin Plastic Products Co., Ltd. (Cangzhou, China). Huperzine-A and MgSO4·7H2O were obtained from Aladdin (Shanghai, China). 2-Pyridinealdoxime methiodide (2-PAM) and paraoxon were purchased from J&K Scientific (Beijing, China). All other reagents were of analytical grade and used without further purification. Alginate solutions (0.5%–1.75%) were prepared by dissolving sodium alginate powder in distilled water, followed by stirring for 2 h. Then, different concentrations of CaCO3 particles (5– 30 mM) were dispersed in sodium alginate solution, followed by stirring for 30 min and ultrasonication for 30 min. No aggregation or settling of the particles was observed over a period of several hours. The alginate and CaCO3 levels were in excess of those required to form strong self-supporting gels. The stock solution of AChE was prepared by dissolving AChE (21 mg/mL) in Tris-HCl buffer (20 mM, pH 7.5). The XO solution (65.8 µg/mL) was directly used as stock solution. ATCh was dissolved in distilled water to a desired concentration before use. MgSO4 (20 mM) was prepared by dissolving MgSO4·7H2O in Tris-HCl buffer (20 mM, pH 7.5). All solvents and solutions were filtered using an inorganic 0.22 µm membrane filter prior to use. Fabrication of CE-integrated IMERs and on-line enzyme/inhibition assays. The principle of the proposed method is illustrated in Scheme 1, in which alginate “egg-box” hydrogel matrix was formed by AChE-mediated hydrogelation for enzyme encapsulation. To fabricate CE-integrated IMERs based on this scenario, we prepared a sol stock suspension (pH ∼7.2) containing enzymes (0.35 mg/mL with/without 3.30 µg/mL XO), alginate (1.25%), CaCO3 (25 mM), ATCh (40 mM), and MgSO4 (20 mM). The suspension was briefly vortexed for 10 s to ensure the homogeneous dispersion of AChE in the mixture solution. The sol mixture solution was


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hydrodynamically injected into the capillary at 20-cm height for 3 min [Scheme 2(1)]. After incubation for 30 min with both ends of the capillary soaked in 10 mM Tris-HCl buffer, hydrogelation and enzyme encapsulation were performed in-capillary [Scheme 2(2)], resulting in a ~2-cm CE-integrated IMER which was ready for on-line enzyme assays of AChE and/or XO [Scheme 2 (3)]. The capillary could be easily regenerated by flushing the IMER with 10 mM EDTA dissolved in 1 M NaOH for 15 min to remove the hydrogel. All CE experiments were performed by a CE instrument equipped with a UV detector under 22 °C cooling air (CL1020 Beijing Cailu Science Apparatus, China). Fused-silica capillaries (50 µm i.d. and 365 µm o.d.) with total length of 40 cm and effective length of 31 cm (Hebei Yongnian Optical Fiber Factory, China) were used to fabricate CE-integrated IMERs and for on-line enzyme assays. Prior to each assay, the CE-integrated IMER was equilibrated electrophoretically at 100 V/cm with 10 mM Tris-HCl buffer at pH 8.0, until a stable current and baseline were achieved. For CE-integrated IMER assays, the enzymatic activity and inhibition kinetic of the AChE-IMER were determined using ATCh as substrates. The ATCh solution with different concentrations prepared in 20 mM borate buffer containing 20 mM MgSO4 (pH 8.0) with or without various concentrations of inhibitors were introduced electrokinetically (2.5 kV, 3 s) into the inlet of the capillary. After incubation for a desired period, high voltage of 12 kV was applied to separate the unreacted substrate and product [Scheme 2(3)]. The enzyme activity or inhibition was determined by measuring the peak area of the product (thiocholine, TCh), which was detected at 230 nm wavelength. For CE-integrated XO-IMER assays, the enzymatic activity and inhibition kinetic were determined under similar CE conditions, except that xanthine was used as substrate,



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and the product (uric acid, UA) was detected at 290 nm wavelength. The inhibition rate (I%) was used to evaluate the degree of the enzyme inhibition and calculated as follows: ‫ܫ‬% =

஺బ ି஺೔ ஺బ

× 100

(1)

where A0 and Ai are the peak areas of the product without and with inhibitor in the samples, respectively. Detection of paraoxon in spiked apple juice using the CE-integrated IMERs. Edible parts (2.5 g) of an apple, including the peel, were chopped and crushed into a homogenate with the addition of 10 mL of 20 mM PBS (pH 7.4). After ultrasonic treatment and stirring of the homogenate for 60 min, the homogenate was centrifuged for 30 min at 1509.3 g. The upper solution was filtered through a 0.22 µm filter membrane, and the apple juice was collected for analysis. Different concentrations of standard paraoxon solutions were then added to the apple juice to obtain spiked apple juice with final paraoxon concentrations of 0.1, 0.5, and 1.0 ppm. The concentration of paraoxon in the spiked apple juice was determined by the CE-integrated AChE-IMER. Off-line enzyme/inhibition assay. For off-line analysis of the AChE-catalyzed reaction, the reaction mixture (50 µL), which contained 20 mM MgSO4, and the substrates (ATCh) with different concentrations were mixed in 20 mM borate buffer (pH 8.0). For the inhibition study, the reaction mixture also contained different concentrations of inhibitor. The reaction was started by quickly adding 1 µL of enzyme (2.5 mg/mL) into the mixture. After incubation at 37 °C for 2 min, the enzyme reaction was stopped by boiling for 5 min in boiling water. The resulting sample was electrokinetically (2.5 kV, 5 s) injected into a capillary (75 µm i.d., 365 um o.d.) from the inlet of the capillary. The unreacted substrate ATCh and product TCh were separated by CE and detected


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by UV absorption at 230 nm. The CE running buffer was 10 mM Tris-HCl buffer (pH 8.0). The off-line analysis of XO catalyzed reaction was similar to that of the AChE-catalyzed reaction as above. The reaction mixture (50 µL) contained the substrates (xanthine) of different concentrations with or without the inhibitor in 20 mM borate buffer (pH 8.0). The reaction was started by quickly adding 1 µL of enzyme (65.8 µg/mL) into the mixture. After incubation at 37 °C for 2 min, the enzyme reaction was stopped by boiling for 5 min in boiling water. The resulting sample was injected electrokinetically (5.0 kV, 5 s) into a capillary (75 µm i.d., 365 um o.d.) from the inlet of the capillary. The unreacted substrate xanthine and product UA were separated by CE and detected by UV absorption at 290 nm. The CE running buffer was 10 mM Tris-HCl buffer (pH 8.0). RESULTS AND DISCUSSION Investigation on AChE-mediated alginate hydrogelation for CE-integrated IMERs. The key issue for the fabrication of CE-integrated IMERs using the present method is the in situ alginate hydrogelation induced by AChE enzymes. The following reactions are involved in the process: 1) The AChE enzyme catalyzes the hydrolysis reaction of ATCh to produce acetic acid. This process gradually changes the pH value of the reaction sol solution to acidic; 2) The reaction of CaCO3 and acetic acid occurs to release Ca2+; and 3) The Ca2+–alginate hydrogel is formed for enzyme encapsulation. Different concentrations of the reactants that may affect the alginate hydrogelation were investigated. The dependence of hydrogelation time on concentrations of the substrate ATCh and enzyme AChE of the hydrolysis reaction are shown in Fig. 1(A) and (B), respectively. The concentration of AChE was kept at 0.25 mg/mL in Fig. 1(A), and the concentration of ATCh was kept at 40 mM in Fig. 1 (B). Hydrogelation time refers to the time



required to form a self-supporting hydrogel, which was tested in vials as displayed in Fig. 1(C) as an example for the results of different AChE concentrations. As the ATCh concentration increased from 20 mM to 40 mM, the hydrogelation time decreased rapidly from 240 min to 20 min, and then remained almost constant at 20 min as the concentration was further increased from 40 mM to 100 mM [Fig. 1(A)]. Similarly, as the AChE concentration was increased, the reaction rate of hydrolysis of ATCh increased, resulting in decreasing hydrogelation time [Fig.1 (B)]. The shortest hydrogelation time was observed at 0.25 mg/mL AChE and remained almost constant at concentrations in the range of 0.25–0.45 mg/mL. This phenomenon indicated that the acid released from the enzymatic reaction became stable at higher AChE concentrations. Figures 1 (D) and (E) present the hydrogelation time at different concentrations of CaCO3 particles (5–30 mM) and alginate (0.5%–1.75%), respectively. The ATCh and AChE concentrations were maintained at 40 mM and 0.25 mg/mL respectively. The hydrogelation time decreased gradually when either the amount of CaCO3 or alginate concentration was increased. The formed hydrogel was weak and the hydrogelation time became longer than 40 min if less than 20 mM CaCO3 was added [Fig. 1 (D)]. As shown in Fig. 1(E), solutions prepared with >1.25% alginate formed hydrogels within 30 min, whereas solutions prepared with 0.75% alginate formed weak hydrogels only after 60 min. As the alginate concentration was decreased to less than 0.5%, no hydrogelation was formed even after overnight incubation. Performance of CE-integrated IMERs. According to the results in the previous section, homogenous hydrogelation of alginate could be achieved in 30 min for a sol solution containing 40 mM ATCh, 0.25 mg/mL AChE, 30 mM CaCO3 and 1.25% alginate. When the sol solution was injected into the capillary and incubated to grow the alginate network, AChE and XO initially


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contained in the solution will be encapsulated to form CE-integrated IMERs. This process can be used for the assay of either AChE- or XO-catalyzed reaction depending on the substrate injected for the CE analysis. In this section, we analyzed the AChE-catalyzed reactions as an example to show the performance of the fabricated CE-integrated IMERs by measuring the consumption of the substrate ATCh and the formation of the product TCh. The enzyme loading capacity was quantitatively determined by encapsulation of AChE in a long capillary column (50-cm long and 75-µm i.d.) under the same hydrogelation conditions as those for the CE-integrated AChE-IMER. After incubation for hydrogelation for 30 min followed by electrophoresis for 60 min under 300 V/cm electric potential, encapsulation of AChE into the hydrogel matrix was evaluated by measuring the protein content in the hydrogel using the Bradford method. The results show that approximately 68% of the original concentration of AChE in the sol mixture was incorporated into the hydrogel matrix. Thus, the average amount of AChE encapsulated in the CE-integrated IMER was evaluated to be ~0.01 µg/(cm hydrogel) for an initial AChE concentration of 0.25 mg/mL in the sol solution. The run-to-run reproducibility of the CE-integrated IMER was investigated by analyzing 40 consecutive runs over a day. The results are presented in Figure 2 A. The CE-integrated IMERs exhibit excellent run-to-run reproducibility, with RSDs of 3.8% and 3.3% for the peak heights of ATCh and TCh, respectively. The inter-day run-to-run repeatability was evaluated by testing the same IMER in five days with five runs per day. After tests each day, the capillary was kept in the running buffer at 4 °C for use the following day. The results show that the enzyme maintained 85.5% of the initial activity after five-day usage with RSDs of 3.3% and 2.8% for the peak height and migration time of TCh, respectively. For the batch-to-batch reproducibility, six freshly



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prepared CE-integrated IMERs were tested under the same experimental conditions, and an average of five runs for each IMER was provided. Good batch-to-batch reproducibility is demonstrated with RSDs of 1.6% and 2.4% for peak height and migration time of the product, respectively. The excellent intraday and interday stability and batch-to-batch reproducibility imply that the present strategy using enzyme-mediated method to induce in situ hydrogelation to fabricate CE-integrated IMERs is reliable and applicable for accurate on-line enzyme assay. The storage stabilities for the free and immobilized AChE were compared, and the results are presented in Figure 2B. The free AChE solution and AChE-IMER were stored at 4 °C, and their activities were measured in five days. The immobilized enzymes retained 85.4% of its activity after five days of usage, whereas the activity of free AChE decreased to 38.5%. These results indicate greater storage stability of immobilized enzymes than that of free enzymes. Enzyme and inhibition assays using the CE-integrated IMERs. On-line assays for enzyme kinetics and inhibition of AChE and XO were performed using the fabricated dual CE-integrated IMERs by measuring the peak area of the product TCh (for AChE reaction) or uric acid (for XO reaction). The resulting Michaelis–Menten diagrams are presented in Figs. 3A and B for AChE and XO, respectively. Based on the nonlinear regression of the Michaelis–Menten diagrams, the Michaelis constant (Km) of an enzyme can be determined using the following equation: ଵ ௏

=௏

௄೘

ଵ

೘ೌೣ [ௌ] ௏೘ೌೣ

(2)

where V and Vmax are the initial and maximum reaction velocities, and [S] is the substrate concentration. Using the proposed CE-integrated IMERs, the Km values were determined to be


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1.01±0.04 mM for AChE and 0.089±0.005 mM for XO, which are consistent with those measured using free enzymes (0.89±0.08 mM for AChE and 0.065±0.004 mM for XO). The results indicates the absence of significant structural change in the enzymes or reduction in the accessibility of the substrate to the active sites of the immobilized enzymes in the proposed dual CE-integrated IMERs. The typical electropherograms shown in the inserted figures in Figs. 3A and B indicate that the substrates and products are well resolved, with separation resolutions of 6.8 for ATCh and TCh and 4.9 for xanthine and UA. Rapid analysis for enzyme assay of either AChE or XO can be achieved in less than 3.5 min using the proposed CE-integrated IMERs. To demonstrate the feasibility of on-line inhibition assays using the proposed IMERs, inhibitors tacrine (TAC)35-36 and huperzine-A (Hup-A)37 for AChE and allopurinol (All)38 for XO were investigated. The resulting Lineweaver-Burk plots are presented in Figs. 4A, B, and C for inhibitors TAC, Hup-A, and allopurinol, respectively, with different inhibitor concentrations as labeled in each figure along with that measured without the inhibitor. The Ki values of TAC and Hup-A on the immobilized AChE enzyme were determined to be 0.31±0.08 µM and 1.47±0.14 µM. respectively, while that of allopurinol on immobilized XO enzyme was determined to be 3.24±0.12 µM. The corresponding Ki values for the free enzymes were also measured, which were 0.21±0.05, 1.12 ±0.08, and 2.29± 0.10 µM for TAC, Hup-A, and allopurinol, respectively, in good agreement with those of the CE-integrated IMERs. These results demonstrated that the proposed hydrogelation matrix prevents the denaturation of enzymes and does not affect the enzymatic activity and kinetics. In-depth analysis of the Lineweaver-Burk plots for TAC and Hup-A inhibition show increasing y-intercept (decrease Vmax at increasing inhibitor concentrations) and increasing



x-intercept (increase Km) with higher inhibitor concentration (Figs. 4A and B), indicating a mixed-type inhibition on AChE reaction. For allopurinol inhibition on XO, Figure 4 C illustrates that the Lineweaver-Burk plots with and without the inhibitor have the same y-intercept. This phenomenon indicates that the Vmax value is not affected by the addition of the inhibitor or its concentration. This result is a typical characteristic of a reversible competitive inhibition reaction. The above studies show that the proposed CE-integrated IMERs can be used for efficient on-line enzyme and inhibition assay, which can be of valuable potential application for real sample analysis. As an example, we analyzed paraoxon in apple juice samples using the proposed IMERs. Paraoxon is a representative OP that is the irreversible inhibitor of AChE because of the covalent link of OPs to AChE39-42. We determined the percentage inhibition of the enzymatic activity against paraoxon concentration (Fig. 5). The results show a good linear response of inhibition over the concentration range of 0.02–1.2 µg/mL (R2 = 0.9922), with a limit of detection (LOD) of 53 ng/mL of paraoxon (S/N=3). The obtained LOD of paraoxon with the proposed CE-integrated IMER is well below the maximum allowable residual concentration (MRL) as reported by the European Union pesticide database (0.02–0.05 mg/kg meat or fruit). A crucial problem for practical applications is the irreversible inhibition mechanism forming a covalent link between AChE and pesticide. 2-PAM has been generally employed as an efficient chemical reactivator of AChE to recover the activity of inhibited AChE43-44. The proposed IMER can be reactivated by introducing electrokinetically (2.5 kV, 10 s) 5 mM PAM from the inlet of the capillary and incubation for 10 min, and the original enzymatic activity of AChE could resume to 94.3%. Thus, 10 min of incubation time in 5 mM 2-PAM solution was chosen after each inhibition assay for reactivation of the constructed IMER system. This reactivation allowed the repeated use


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of the CE-integrated IMER for the detection of pesticides. Paraoxon was determined in apple juice samples by employing a standard addition method using the proposed IMERs. The recoveries of paraoxon in the apple juice sample were determined to range from 92.9% to 108.2%, with RSD between 1.8% and 2.3% (Table 1). The fact that the measured values can be either less or larger than the ideal recovery of 100% indicates there does not exhibit apparent systematic errors of the proposed method. The results indicate satisfactory levels of accuracy and reproducibility of the proposed CE-integrated IMERs for real sample analysis. Discussion.

Ca-alginate

hydrogel

matrix

with

unique

“egg-box”

structure

is

environment-friendly for efficient protein encapsulation. The present study is performed to develop a simple and robust method for fabricating CE-integrated IMERs for on-line enzyme/inhibition CE assays based on the Ca–alginate hydrogel matrix. In our method, the alginate hydrogel formed inside a capillary is initiated with the release of Ca2+ by changing the pH of the solution. This process is accomplished by AChE-catalyzed hydrolysis reaction of ATCh to produce acetic acid. Such AChE enzyme-mediated hydrogelation, which can be viewed as an internal setting hydrogelation approach, can provide a homogeneous and mild matrix for enzyme encapsulation. As presented in the experimental section, the hydrogelation time was adjustable by simply changing the concentrations of the reactants in the mixed solution before injection to the capillary (Fig. 1). In our experimental conditions, only 30 min elapsed to the form self-supporting hydrogel in the capillary. The proposed method is a single-step in-situ strategy for the hydrogelation of alginate and encapsulation of enzymes. This feature offers several essential benefits for fabricating



CE-integrated IMERs and on-line enzyme assays. First, the fabrication procedure was greatly simplified, and the aggregation or denaturation of enzymes could be avoided, because the enzymes were easily encapsulated in situ in the “egg-boxed” alginate-hydrogel network without additional heating or light radiation. Second, the single-step in-situ encapsulation enhanced the loading capacity of enzymes. Using AChE as an example, we estimated that 68% of the original enzyme amount in the sol mixture could be incorporated in the hydrogel matrix. Such high enzyme loading capacity ensures the efficient on-line assays using the IMERs, as determined by analyzing activity and inhibition of the AChE and XO enzymes (Figs. 3 and 4). Moreover, the proposed method could serve as a universal strategy for CE-integrated IMERs. Theoretically, any enzyme can be encapsulated in the hydrogel matrix, because the size of an enzyme is usually larger than the porous structure of hydrogel. The proposed method employs AChE enzyme-mediated hydrogelation, which occurs under mild conditions. Thus, the activity of the target enzyme will not be affected. The enzyme leakage of the IMERs is negligible, as proved by the excellent inter-day run-to-run repeatability in Fig. 2. Therefore, we expect that the proposed CE-integrated IMERs can be broadly applied for on-line enzyme assays, such as enzyme-catalyzed cascade reactions which are important in metabolomics, biomass conversion, and other biochemical analysis. CONCLUSIONS In summary, we report a novel method for the fabrication of CE-integrated IMERs. This process was achieved by single-step in-situ AChE-mediated alginate hydrogelation and enzyme encapsulation. The performance of the proposed CE-integrated IMERs was evaluated, and the enzymatic kinetics and inhibitions of immobilized AChE and XO enzymes were investigated. Our


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study provides a simple, efficient, and universal approach for on-line assays with immobilized enzymes, and this approach can be extensively applied in bioanalysis.

ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant No. 21475019 and 21775017) and the Natural Science Foundation of Jilin Province, China (Grant No. 20180101174JC). L. Yang would like to thank the support from Jilin Provincial Department of Education and Jilin Provincial Key Laboratory of Micro-nano Functional Materials (Northeast Normal University).

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Table 1. Recovery studies of paraoxon in apple juice. Added (ppm)

Found (ppm)

Recovery (%)

RSD (%) (n=3)

0.10

0.097 + 0.004

97.0

2.1

0.50

0.464 + 0.007

92.9

1.8

1.00

1.082 + 0.003

108.2

2.3



Figure captions Scheme 1. Schematic illustration of acetylcholinesterase (AChE)-mediated hydrogelation of alginate. AChE-catalyzed oxidation of acetylthiocholine generates protons, which solubilize the Ca2+ ions that trigger alginate’s hydrogel formation. Scheme 2. Fabrication of CE-integrated IMERs and the application for on-line enzyme assays. Figure 1. The effects of the experimental parameters involved in AChE-mediated hydrogelation process of alginate-Ca hydrogel on the hydrogelation time. (A) concentration of ATCh; (B) concentration of AChE; (C) vial inversion tests providing visual evidence for the effect of AChE concentration on hydrogelation time and strength of formed gel (self-supporting); (D) concentration of CaCO3; (E) concentration of sodium alginate. Figure. 2. (A) Peak areas of the substrate ATCh and the product TCh as a function of run number in 40 consecutive runs using the proposed CE-integrated IMER. (B) Storage stability of the free and immobilized AChE. Each data point in the figures is an averaged result of three repeatable assays. 5 mM ATCh solution was injected into the AChE-IMER at 100 V/cm for 3 s, incubation 2 min. Other conditions are the same as those in Figure 1. Figure. 3. Michaelis–Menten diagram of ATCh (A) and xanthine (B) on the proposed CE-integrated IMER. Typical electropherograms are shown in the inserted figures in Figure 3 A and B. Figure 4. Lineweaver–Burk plot of CE-integrated IMERs in the presence of different inhibitor concentrations of Tacrine (A) and Huperzine-A (B) for AChE inhibition, and allopurinol (C) for XO inhibition, respectively. Other conditions are the same as those in Figure 3. Figure 5. Sensitivity and linear range of the CE-integrated IMER for paraoxon determination


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under the optimum conditions. Each point is an averaged result of three replicated runs.



IMER

Na

Separation

Na

Na Na Na Na Na

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Detection

Ca In situ AChE-mediated hydrogelation Enzyme encapsulation

Ca

Ca Ca Ca

Na

For TOC only


Ca

Ca Ca

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+ H2O

Na

Na

Na Na Na

Na

Ca2+

H+

Ca

Ca

CaCO3

Ca

Ca

Ca

Ca

Na

Na

Sol solution Na Na

Na

Hydrogel

Sodium alginate

AChE

Na


XO

Ca Ca


(1) Injection of AChE-alginate sol

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(2) Hydrogelation of sol

Capillary Buffer

Buffer

(3) Application of AChE-IMER Sample H = 20 cm t = 3 min Product Unreacted substrate Alginate sol Alginate hydrogel AChE / XO Sample Buffer

Product

Scheme 2


Detection

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--20 min

(C)

--30 min

--60 min

--75 min

--100 min

--190 min

Low

Figure 1


AChE concentration

High


Figure 2


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(A)

(B)

Figure 3


Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

(A)

(B)

Figure 4


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(C)

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Figure 5


A single-step in-situ acetylcholinesterase-mediated alginate

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