Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay
Siming Wang,Ping Su,Yi Yang ⇑
College of Science,Beijing University of Chemical Technology,Beijing 100029,People’s Republic of China
a r t i c l e i n f o Article history:
Received 27February 2012
Received in revised form 21April 2012Accepted 15May 2012
Available online 23May 2012Keywords:
Glucose oxidase
Online immobilized capillary enzyme microreactor
Capillary electrophoresis Enzyme assay
Inhibitor screening
a b s t r a c t
An online immobilized glucose oxidase (GOx)capillary microreactor was developed based on an enzy-matic redox reaction with 1,4-benzoquinone as an acceptor of electrons,replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation.A high effi-ciency of enzymolysis was obtained at 1mg ml À11,4-benzoquinone for 5min of incubation at 25°C,
and baseline separation of the substrate and product could be achieved with a resolution of 3.85by employing 20mM phosphate buffer (pH 8.0)containing 40mg ml À1sulfated b -cyclodextrin as an addi-tive,a constant voltage of 15kV,and a detection wavelength of 220nm.In addition,an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag +and Cu 2+used as model inhibitors.The results indicate that Ag +(IC 50=69.16l M)has a markedly higher inhibitory effect than Cu 2+(IC 50=1.33mM).The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors.
Ó2012Elsevier Inc.All rights reserved.
Online immobilized capillary enzyme microreactor is a very use-ful tool for the study of enzyme reactions,inhibitor screening,pro-teomic peptide mapping,and diagnostics [1–5].In this approach,the enzyme is immobilized on either the inner wall of the capillary or a suitable carrier such as a monolith fixed at the capillary inlet to form a microreactor.After introduction of the substrates,the reac-tion occurs in the microreactor area of the capillary that contains the immobilized enzyme,and the products are subsequently sepa-rated electrophoretically [6,7].The online immobilized enzyme microreactor possesses several remarkable advantages in that auto-mation of all steps is straightforward,there is no requirement for sample transfer,it is compatible with multiple detectors,and the enzyme reaction and a
nalyte separation are performed in the same capillary [8].Hence,the use of online immobilized enzyme microre-actor has attracted widespread attention during recent years,espe-cially in the field of enzyme inhibitory assay.Some immobilized enzyme microreactors were prepared through ionic binding and used for the characterization and inhibition studies of angioten-sin-converting enzyme [1],acetylcholinesterase [9],adenosine deaminase [10],and alkaline phosphatases [11].Furthermore,online capillary enzyme microreactors have been extended by cou-pling with chromatography and mass spectrometry [12,13].
Glucose oxidase (GOx)1is often used as a model enzyme in ana-lytical chemistry,biochemistry,and clinical chemistry as well as in the development of microreaction systems [14–16].It has been ap-plied in various biosensors for the detection and estimation of glu-cose in body fluids such as blood and urine and has played an important role in clinical diagnosis for diabetes mellitus,hyperglyce-mia,hypoglycemia,and hepatocirrhosis [17,18].The inhibition study of GOx activity and screening of its inhibitors are required in the clinical diagnosis to predict some potential disruptors and,thus,may avoid false negative results.In addition,the inhibitory assay for GOx was also successfully applied to the determination of several heavy metals that would cause serious environmental and healthy consequences,providing the possibility for development and appli-cation of GOx inhibition assays in en
vironmental monitoring [19,20].However,many spectrophotometric and fluorometric assays for GOx may be difficult to realize in the absence of a second enzyme,peroxidase,to generate light-absorbing or fluorescent sub-stances [21,22].Therefore,easy and efficient methods are needed for the GOx enzymatic and inhibitory assay.
Our previous work resulted in a new type of offline immobilized GOx capillary microreactor that proved to be highly stable and effi-cient [23].In this study,GOx was immobilized at the inlet end of the capillary as a plug using a crosslinking method and an online immobilized GOx capillary microreactor was prepared.To achieve direct ultraviolet detection without derivation,an enzymatic redox reaction was developed for the microreactor based on 1,4-benzo-quinone as an acceptor of electrons,replacing the molecular oxygen in the typical GOx reaction.The corresponding reduction product was hydroquinone [24,25].Due to strong ultraviolet absorbance,both the substrate (1,4-benzoquinone)and product (hydroquinone)can be monitored spectrophotometrically and do
0003-2697/$-see front matter Ó2012Elsevier Inc.All rights reserved./10.1016/j.ab.2012.05.014
⇑Corresponding author.Fax:+861064434898.
E-mail address:yangyi@mail.buct.edu (Y.Yang).
1Abbreviations used:GOx,glucose oxidase;APES,c -aminopropyltriethoxysilane;CE,capillary electrophoresis;RSD,relative standard deviation.
not require peroxidase as a coupling enzyme.The optimal separa-tion and enzymolysis conditions for the online microreactor capil-lary were evaluated.Metal ions Ag+and Cu2+were used as model inhibitors,and an online enzyme inhibition study was performed on the microreactor.
Materials and methods
Chemicals and materials
GOx(EC 1.1.3.4from Aspergillus niger,109U/mg)was pur-chased from Amresco(Solon,OH,USA).b-D-Glucose was obtained from TCI(Tokyo,Japan).c-Aminopropyltriethoxysilane(APES),so-dium cyanoborohydride,and1,4-benzoquinone were obtained from Acros Organics(Geel,Belgium).Sulfated b-cyclodextrin was purchased from Sigma–Aldrich(St.Louis,MO,USA).Silver sulfate was obtained from Alfa Aesar(Ward Hill,MA,USA).Hydroquinone and Copper(II)sulfate pentahydrate were supplied by Beijing Yili Chemical(Beijing,China).All other chemical reagents used in the Preparation of immobilized GOx capillary microreactor
The60-cm fused-silica capillary was rinsed sequentially with 1M NaOH for2h,deionized water for15min,0.1M HCl for 30min,deionized water,and then methanol for10min.After the pretreatment,a1-cm-long plug of30%(v/v)APES solution was in-jected into the capillary at0.5psi for6s to introduce amino groups within the inlet end of the capillary.The APES solution was kept for 2h,and then the capillary was washed with methanol by applying pressure to the outlet end of the capillary.Subsequently,with nearly the same length,5%glutaraldehyde solution was injected into the capillary and reacted for3h.The capillary was then washed with phosphate buffer for5min.Then,2.5mg mlÀ1GOx and sodium cyanoborohydride phosphate solution(pH8.0)was circulated through the prepared capillary in a similar way and incubated overnight.Free(unbound)enzyme was washed out of the capillary with phosphate buffer by applying pressure to the outlet end of the capillary.Finally,the immobilized capillary en-zyme microreactor,filled with phosphate buffer,was stored at 4°C before use.In this way,GOx was covalently attached at the in-
of the process for online enzyme assay by the immobilized GOx microreactor.Insets:Typical electropherograms for the online enzyme and sample containing10l g mlÀ1Ag2SO4(b).Peaks:(1)1,4-benzoquinone;(2)hydroquinone.Conditions:capillary,75l m inner diameter,
cm total length,effective length50cm;capillary thermostat temperature,25°C;applied voltage,15kV;runni
ng buffer,20mM phosphate mlÀ1sulfated b-cyclodextrin as an additive;detection wavelength,220nm;incubation time,5min;sample solution,1mg mlÀ11,4-benzoquinone -glucose solution;sample injection,0.5psi for3s.
140Online immobilized enzyme microreactor/S.Wang et al./Anal.Biochem.427(2012)139–143
D-glucose was injected into the enzyme microreactor at a pressure of0.5psi for3s.After incubation,the product was separated from the unreacted substrate by capillary zone electrophoresis.The optimal enzymolysis conditions were obtained according to the peak area of the enzymolysis product,hydroquinone.For the inhi-bition study,a mixture of substrate and inhibitor was injected and incubated.The inhibition percentage was calculated according to the reduction of the peak area of product.Before each analysis, the capillary was conditioned by passing phosphate buffer at 25psi for2min.1,4-Benzoquinone solutions were always stored in the dark and used soon after preparation.
Results and discussion
Optimization of CE conditions
Control experiments were performed on a bare capillary and the microreactor capillary to validate the e
ffect of the immobilized enzyme.In the absence of immobilized GOx,only the1,4-benzo-quinone(substrate)peak was observed.In contrast,for the mic-roreactor capillary,another peak could be detected and identified as the enzymolysis product,hydroquinone,according to the reten-tion time.The results demonstrate the feasibility of the protocol for the online immobilized GOx microreactor.Characterization and detection of substrate and product by ultraviolet spectrophotome-try was straightforward because they exhibit a strong absorption at 220nm.It was found in our previous work that the immobilized GOx microreactor displays maximum activity in20mM phosphate running buffer at pH8.0[23].Therefore,these conditions were adopted in our experiments.
The sulfated b-cyclodextrin concentration is such an important factor in electrophoretic separation of the1,4-benzoquinone and hydroquinone that their separation could not be realized without it as an additive in the running buffer.In the presence of sulfated b-cyclodextrin,hydroquinone had a longer migration time than 1,4-benzoquinone as a result of the stronger binding to the nega-centration.Consequently,a high current could induce excessive Joule heat that ruined the resolution and repeatability.This may be the reason for the slower growth of resolution as the concentra-tion of sulfated b-cyclodextrin continued to increase.The optimal sulfated b-cyclodextrin concentration was40mg mlÀ1.
The effect of voltage on separation efficiency was also consid-ered.Voltage is a major parameter in electrophoretic separation because a higher voltage allows the migration time of the analytes to decrease and the peak shapes to sharpen.However,a higher voltage results in larger Joule heat,limiting the resolution.An opti-mal voltage of15kV was selected for the subsequent experiments. Baseline separation of the substrate and product with a resolution of3.85could be achieved at the optimal separation conditions.
Optimization of enzymolysis conditions
The effect of incubation time on the yield of the reaction prod-uct hydroquinone is shown in Fig.3A.A longer incubation time al-lows the sample1,4-benzoquinone to react more completely with the immobilized enzyme,and therefore a higher product peak area will be detected.On the other hand,longer incubation times re-duce the speed of analysis.The maximum yield of the enzymatic reaction was achieved when the incubation time was more than 5min.The effect of capillary temperature on immobilized enzyme activity and separation efficiency was also investigated.The yield of product initially increased with rising temperatures,whereas higher temperatures led to a decline in the resolution and enzyme denaturation,which severely lowered enzymolysis efficiency.An optimal capillary temperature of25°C was chosen to give accept-able peak area and resolution.
Based on the results obtained above,the optimal substrate con-centrations and the kinetic behavior for the online immobilized capillary enzyme microreactor were investigated.The results for injected samples with different1,4-benzoquinone concentrations are shown in Fig.3B.The enzymolysis efficiency increased with increasing substrate concentrations up to1mg mlÀ1.However,fur-ther increases in concentration did not increase enzymolysis effi-ciency significantly because of saturation of thefixed length of immobilized enzyme.The most important kinetic constant of the enzyme reaction,the Michaelis constant,can be determined by the Lineweaver–Burk method[26].The Lineweaver–Burk plot was linear and gave a Michaelis constant of7.94mM,similar to our previous result(7.46mM)[23]and the values reported in the literature(7.8mM)[27,28].The result demonstrates that no signif-icant change in enzyme properties was observed for the proposed method.
The repeatability and stability of the online immobilized GOx microreactor were evaluated.The results suggest a satisfactory repeatability in the peak area of the product and its migration time with a relative standard deviation(RSD)of3.59%.The stability of the microreactor was tested both during continuous use and after storage in buffer at4°C.The operational stability was examined by the injection of substrate solution six times a day for7days,and the activity remained at90.19%with an RSD of3.76%.The storage stability was assessed by measuring activity after storage in buffer at4°C for30days,and the activity remained at82.91%.
Online immobilized GOx capillary microreactor for metal ion inhibition assay
Abnormal activity or expression of enzymes occurs with the emergence and metastasis of diseases.Hence,enzymes are a useful class of indicators for disease diagnosis and play an important role during drug discovery processes.One of the most useful applica-tions of the online immobilized enzyme microreactor is to screen enzyme inhibitors as potential new drugs.In this study,Ag+and
Fig.2.Effect of sulfated b-cyclodextrin concentration on electrophoretic separation
and current.Conditions:sulfated b-cyclodextrin concentration range,20to
60mg mlÀ1;other conditions as in Fig.1.
Online immobilized enzyme microreactor/S.Wang et al./Anal.Biochem.427(2012)139–143141
Cu2+were used as model metal inhibitors to evaluate the utility of the prepared microreactor.In contrast to the absence of inhibitor (inset a of Fig.1),inset b of Fig.1represents the assay for a sample containing10l g mlÀ1Ag2SO4,showing a dramatic reduction in product peak area.
The enzyme kinetics in the presence of inhibitors was investi-gated in the microreactor using the optim
al conditions described above.The Lineweaver–Burk plots for the immobilized GOx in the presence of10l g mlÀ1Ag2SO4(or CuSO4Á5H2O)are shown in Fig.4.The inhibition constant K i for the two model metal inhibitors were0.64mM(Ag+)and1.87mM(Cu2+).The smaller K i value for Ag+shows a stronger inhibition and interaction with the enzyme.
values reported here are higher than those obtained with GOx in solution,reflecting improved stability a
nd resistance inhibition by the immobilized GOx.
inhibition efficiency curve for each inhibitor was estab-
by varying the inhibitor concentration(maintaining a con-substrate concentration).These results indicate that the value for Ag+(69.16l M)was obviously smaller than that for
(1.33mM);this suggests that,compared with Cu2+,Ag+dis-plays a markedly stronger inhibitive effect,in agreement with the literature results[29,30].The mechanism of GOx inhibition by some metal ions,such as silver,copper,and mercury,involves the coordination of the metal ion with the reduced form of thefla-vin cofactor(FADH2)in the active site of GOx.This prevents FADH2 from reducing O2,1,4-benzoquinone,and other electron acceptors, so that the GOx activity is partly inhibited[30].
Conclusion
A new type of online immobilized GOx capillary enzyme mic-roreactor has been developed,based on an enzymatic redox reac-tion with1,4-benzoquinone as an acceptor of electrons replacing molecular oxygen in the typical GOx reaction.Direct detection could be achieved using this approach without derivation.The enzymatic hydrolysis and enzyme inhibition assay were performed using the online imm
obilized GOx capillary.The results demon-strate that the new microreactor possesses high efficiency,good accuracy,and satisfactory repeatability.It combines the advanta-ges of both enzyme microreactors and CE so that enzymatic hydro-lysis,electrophoretic separation,and detection all could be accomplished simultaneously in the prepared capillary.The meth-od not only has low cost because of the nanoliter-level reagent consumption and reusable enzyme but also provides a consistent level of enzyme for all assays.The current online immobilized en-zyme microreactor approach can be easily adapted to capillary ar-ray electrophoresis,which can potentially realize high-throughput screening of enzyme reactions and enzyme inhibitors. Acknowledgment
This work was supported by the National Natural Science Foun-dation of China(20375004,90713013,and21075008). References
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