RESEARCH PAPER

Preparation of reversibly immobilized Jack bean ureaseon microchannel surface and application for enzymeinhibition assay

Xiuwen Tang • Sufang Liu • Sifeng Wang •

Qin Zhang • Zhiyi Cheng

Received: 18 July 2013 / Accepted: 2 February 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract We have developed a sensitive enzyme inhi-

bition assay on microfluidic system. The analysis was

carried out by immobilizing enzyme through amide-bond

or disulfide-bond formation with surface. Followed by

detection of reaction product through fluorescent density

were evaluated reusability, stability and sensitivity of

microfluidic enzyme assay. The Michaelis–Menten

parameters for free urease (KM = 1.027 lM) and for

immobilized urease (KM = 1.528 lM of disulfide-bond

immobilization; KM0 = 1.617 lM of amide-bond immo-

bilization) showed reasonable activities maintained after

immobilization with relative standard deviation (RSD) of

4.86 and 6.06 %, respectively. When compared enzyme

activities of five repeated immobilization cycles through

reversible disulfide-bond immobilization, we found that

removal process and reversible immobilization did not

affect efficiency of microreactor with RSD of 4.78 %. The

IC50 value 368 lM of inhibitor acetohydroxamic acid

determined on chip showed good agreement with reported

data 375 lM; Ki of 1.39 lM matched well with Ki of

1.46 lM via the traditional 96-microplate. This microflu-

idic could be extended to screening of enzyme inhibitor

and enzymatic reaction kinetics study, which may be useful

for clinical diagnostics, biotechnological research, drug

discovery and other bioassays.

Keywords Microfluidic chip � Enzyme �Covalent immobilization � Urease inhibition assay

1 Introduction

With rapid development of micrototal analysis system

(l-TAS), revolutionary approaches to detect activity of

biological macromolecule have opened up. Among exten-

sive applications of microfluidic technology in fields of

biochemical studies, clinical diagnostics and environmental

monitoring, the most popular ones include DNA analysis,

enzyme assay, immunoassay and cell capture analysis due

to its high selectivity and sensitivity (Hunter et al. 2011;

Jiang et al. 2011; Chen et al. 2012; Chang et al. 2013).

In 1997, for the first time, Hadd established a micro-

fluidic platform to analyze enzyme kinetics (Hadd et al.

1997). With enzyme anchoring on support, reaction prod-

uct moves into detection part to avoid possible interference

such as primary amines, amino acid groups on enzyme and

other proteins in the system (Gray et al. 2013). Moreover,

immobilized enzymes may exhibit better properties. For

example, multipoint and multisubunit covalent immobili-

zation may improve stability of enzymes (Mateo et al.

2007). For complex enzymes, inactivation is partly the

result of dissociation of enzyme subunits and loss of active

structure. Controlled interaction immobilization may

improve enzyme stability because of increased rigidity

(Fernandez-Lafuente 2009).

Improvement in material science including nanoparticles,

nanofibers, mesoporous has lead to advance in long-term

stabilization by enhancing storage catalytic potency (Hwang

and Gu 2013; Verma et al. 2013). For immobilization via

covalent binding, enzyme is tightly fixed onto support to

prevent leaching from surface (Yakovleva et al. 2002).

X. Tang � S. Wang � Q. Zhang � Z. Cheng (&)

School of Pharmaceutical Sciences,

Sun Yat-Sen University, Guangzhou 510006, China

e-mail: chengzhy@mail.sysu.edu.cn

S. Liu (&)

School of Public Health, Sun Yat-Sen University,

Guangzhou 510080, China

e-mail: liusf@mail.sysu.edu.cn

123

Microfluid Nanofluid

DOI 10.1007/s10404-014-1360-8

However, it can also be a major drawback when enzyme

becomes deactivated, for both enzyme and support are

unusable. Reversible covalent immobilization could solve

enzyme lifetime problem by replacing denatured enzyme

with fresh one. Various groups can be involved in covalent

binding as amino, carboxyl, alcoholic, thiol, phenolic func-

tions (Grazu et al. 2005).

Holden et al. (2004) developed a method for photo-

patterning enzymes inside microfluidic channels via photo-

attachment. The patterning process relied on biotin–strep-

tavidin linkage, and enzyme was more sensitive to envi-

ronment conditions because of flexibility limitation. In

another assay, enzyme was modified with magnetic nano-

particles (MNPs) in microchannel as enzyme microreactor

(Sheng et al. 2012). The enzyme maintained reasonable

activity, yet it had drawback of mass transfer limitation and

enzyme loading was largely determined by characters of

support materials. Kim et al. (2009) immobilized glucose

oxidase covalently on glass microbeads via aminopropyl-

triethoxysilane (APTES); the enzyme-immobilized micro-

beads were retained in reaction chamber by physical

resistance. However, substrate was introduced into the

system at a flow rate of 1.0 lL min-1 because of structural

pressure and diffusion limitation. We have studied on

microfluidic device used for sandwich enzyme-linked

immunoassay. The PDMA surface was immobilized with

target antibodies by protein A covalently via glutaralde-

hyde groups (Hou et al. 2012). All these heterogeneous

assays share distinct advantages such as ease of enzyme

recycling and simple manipulation. The progress and other

immobilization methods have been summarized in several

extraordinary reviews (Wang et al. 2010; Rodrigues et al.

2013; Zhou and Hartmann 2013).

Our group is particularly interested in microfluidic chip

immobilized with Jack bean urease as this kind of enzyme

has been well studied on structure, subunits, molecular

weight and amino acid sequence (Balasubramanian and

Ponnuraj 2010). It was the first enzyme isolated as crystalline

protein and also the first described presence of sulfhydryl

groups (Cvetkovic et al. 2010). Research on urease inhibition

is meaningful in medical, environmental and agronomic

areas (Silva et al. 2000; Kazakova et al. 2011). Nowadays,

urease inhibitors have attracted much attention such as new

anti-ulcer drugs. New results suggest that urease inhibitors

may be useful for treating urinary tract infections caused by

Staph. Saprophyticus (Loes et al. 2014).

Although researches on Jack bean urease cover various

areas, enzyme assay and inhibitor screening on chip remain

elusive. To our knowledge, it is the first time to immobilize

Jack bean urease on chip via disulfide group reacted with

cysteine groups of urease. Herein, we immobilized Jack

bean urease covalently on wells of microfluidic chip for

three specific goals. Firstly, we intended to find a simple

strategy for enzyme immobilization within lab-on-chip

system for use in rapid enzyme activity detection. Sec-

ondly, we wanted to exploit this method for detection

enzyme turnover rates and related kinetic data to screen

inhibitors of urease. Finally, we proposed long-time sta-

bility and repeatability of this method.

2 Experimental sections

2.1 Chemicals

Urease (Jack bean, activity [1.7 EU/mg) was from Qi

Yun Biotechnology Co., Ltd., (Guangzhou, China).

Ammonia chloride, b-mercaptoethanol and urea were

obtained from Guangzhou Chemical Reagent Co., Ltd.

(Guangzhou, China). Aladdin (Shanghai, China) supplied

3-aminopropyltrieth-oxysilane, methyl-triethoxysilane,

succinic anhydride, 1-ethyl-3(3-aminopropyl) carbodiim-

ide hydrochloride (WSCI-HCl), N-hydroxysuccinimide,

cystamine, tributyl phosphine (n-Bu3P), 3-nitro-2-pyr-

idinesulfenyl chloride (Npys-Cl), triethylamine, acetohy-

droxamic acid (AHA) and o-phthalaldehyde. Ultrapure

water (18.4 MX), doubly distilled demineralized water on

Millipore Simplicity (Millipore, Bedford, MA, USA), was

used for preparation of all solutions for all determinations.

All used chemicals were of analytical grade. Reaction

solutions were supplied to microreactor using a LSP02-1B

syringe pump (Longer Precision Pump Co. Ltd.).

2.2 Fabrication of the microfluidic device

Metal masters were fabricated through a precise embossing

process using a computer numerically controlled (CNC)

machine. PDMS precursor and curing agent (Sylgard 184,

Midland-Michigan, USA) were thoroughly mixed in a

weight ratio of 10:1. After degassing, the mixture was

decanted onto metal master which contained pattern of

designed microchannel, then cured at 65 �C for 1 h. The

cured PDMS film with microchannels was then peeled off

the master. A 16-gauge needle was used to punch holes

through PDMS replica to introduce inlets and outlets.

Finally, two PDMS slabs (layer A and B) with designed

microchannels were oxidized in plasma (Harrick Scientific

Co. Ossining, NY) with a glass slide between them for

3 min. This three-layer chip was heated at 80 �C for 2 h,

resulting in irreversible seal to form enclosed microchannel

(Sia and Whitesides 2003). The three-layer PDMS can

accomplish concentration gradient, enzyme reaction, fluo-

rescence derivation and measurement.

Microfluid Nanofluid

123

2.3 Preparation of immobilized urease reactors

Microchip was treated with piranha solution (7:3 (v/v)

mixture of 98 % H2SO4:30 % H2O2) overnight at a flow

rate of 1.0 lL min-1 at room temperature to get acidic

property surface. After washing with water at a flow rate of

5.0 lL min-1 for 30 min, the chip was treated with 3 %

solution of 3-aminopropyltriethoxysilane and methyltri-

ethoxysilane (60:40 %) in 97 % ethanol in water for 1 h.

After washing with ethanol, the amino-functionalized mi-

crochannel was prepared (Miyazaki et al. 2004); 5 %

polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl

ether (Triton X-100) solution was added into the modifi-

cation system in advance in order to eliminate bubbles in

microchannel.

2.4 Enzyme immobilization through amino-bound

After amino-functionalized, microchip was treated with

1 mM succinic anhydride in N,N-Dimethylmethanamide

(DMF) for 2 h at a flow rate of 5.0 lL min-1 in order to

create carboxyl function. After washing with DMF, car-

boxyl function reacted with 1 M solution of WSCI-HCl

and N-hydroxysuccinimide in DMF for 1 h, then wash with

DMF, water, PBS for 30 min each. The amino modification

microchip was filled with 5 mg mL-1 urease in PBS

solution and react for 12 h at 4 �C.

2.5 Enzyme immobilization through disulfide bond

For a brand new microchip, it was also amino-functional-

ized prepared and treated with succinic anhydride, WSCI-

HCl and N-hydroxysuccinimide. To create disulfide bond,

the microchip was treated with 1 mM cystamine in PBS for

2 h and 2.4 mM n-Bu3P in 50 % methyl alcohol for 1 h in

turn. To incorporate active SH group on the surface, the

chip was treated with 1 mM solution of Npys-Cl and tri-

ethylamine in DMF for 2 h. After wash with DMF, water,

PBS, enzyme was loaded on chip for overnight at 4 �C.

Immobilized enzyme could be completely unloaded by

injecting n-Bu3P on chip with regenerated sulfhydryl

groups of enzyme.

2.6 Evaluation of performance of urease

The prepared microchip device was placed in a controlled-

temperature bath and maintained at 35 �C. Urea was used

as substrate, AHA as inhibitor and phthalaldehyde-mer-

captoethanol (OPA) as fluorescence derivation reagent. All

solutions were freshly prepared and filtered before injected

into microfluidic system controlled by a syringe pump.

OPA reagent was freshly prepared by mixing a 750 mM

solution of phthalaldehyde (100 mg mL-1) in absolute

ethanol with a 72 mM solution of mercaptoethanol

(5 lL mL-1) in ethanol and 0.2 M phosphate buffer in

relative proportions of 1:1:18 (v/v/v). All solutions were

kept in freezer at 4 �C. Fluorescence detection via window

on chip was performed using a microplate reader (flex

station 3). All data were normalized by subtracting the

intensity obtained in negative control to obtain relative

fluorescence.

3 Results and discussion

3.1 Microfluidic chip design

As illustrated in Fig. 1, the microfluidic chip has dimension

of 6.35 9 6.35 cm, composed of four functional parts:

concentration gradient generator (250 lm width, 100 lm

depth, part 2); reaction wells (3.0 mm in diameter and

1.5 mm in height with a volume of 10 lL, part 1); fluidic

distribution of OPA (part 3); fluorescence derivation

channels and detection windows (3 mm width, 5 mm

length, 1 mm depth, part 4). Part 3 and part 4 are patterned

on the top layer (layer A), and part 2 is on the bottom layer

(layer B). The middle one (layer C) has an array of six

parallel holes to connect liquid flow and two holes at the

same place of C1 and C2. The top and bottom layers are

fabricated of PDMS, and the middle one is made of glass.

In surface modification process, C5 was blocked;

reagent was injected into the microfluidic system from C1

and C2, then flowed through concentration gradient gen-

erator and reaction wells and finally removed from C3

and C4. After surface was modified with active groups,

enzyme was injected into the system from C3 and C4. As

volume of the wells could be calculated (10 lL) and the

volume of injected solution was controlled by pump, C1

and C2 were opened until the wells were filled with

enzyme solution (about 60 lL) and then C1 and C2 were

blocked and C5 was open, liquid was injected from C3

and C4 to C5. In this case, enzyme was immobilized on

limited area on chip. In enzyme activity detection process,

substrate urea (or urea mixed with AHA in inhibition

assay) was injected into the system from C1 and C2 (high

and low concentrations) to generate concentration gradi-

ent and catalyzed by immobilized enzyme in reaction

wells. OPA was injected from C3 and C4, and then mixed

with reaction product ammonia through long sinuous

microchannel by diffusion effect. Detection windows

were adapt to well architecture in the 96-microplate so

that fluorescence strength could be detected by a micro-

plate reader.

Microfluid Nanofluid

123

3.2 Determination of urease activity

A neutral condition was necessary for preparation of Jack

bean urease in order to avoid protein denaturation. For

analysis of enzyme reaction, a combination of reaction step

(enzyme with substrate and inhibitor) and detection step

(enzymatic reaction products and fluorescence derivation

reagent) was required. Urease catalyzed hydrolysis of urea

to produce ammonia. The enzymatic reaction product

ammonia mixed with OPA reagent and generated fluores-

cence product NH4?-OPA, which excited at 420 nm and

detected at 475 nm wavelengths (Roth 1971). The fluo-

rescence could be measured by a microplate reader in

detection chamber with a volume of 15 lL. It is mean-

ingful to correlate enzyme relation rate to relative fluo-

rescence. Accordingly, this assay can be used for high-

throughput screening of enzymatic activity. In order to

achieve more sensitive and precision results, we optimized

reaction conditions such as reaction time and pH.

The effect of pH on fluorescence measurement is shown

in Fig. 2, and it can be concluded that the highest fluo-

rescence can be achieved at pH 7.2. From changes in

fluorescence intensity, it is demonstrated that enzyme

activity increased with growth of pH in the range from 6.8

to 7.2. At pH value above 7.2, fluorescence intensity

decrease with enhance of pH value. In order to obtain

optimum mixture time to ensure that ammonia react

completely with OPA, an experiment was conducted by

plotting relative fluorescence against reaction time (in the

range of 5–30 min). The results showed that fluorescence

signal reach peak value and maintain stable after ammonia

react with OPA for 15 min, and fluorescence intensity

would not quench for as long as 6 h. As for the same

reaction on 96-well microplate, 30 min was required for

completely reaction; this phenomenon was mainly due to

rapid diffusing and mixing on chip. From Fig. 3, it can be

concluded that relationship between enzyme reaction rate

and relative fluorescence could be measured in the range

from 0 to 1.0 mM and exhibited good linearity with r2 of

0.992 in the condition optimized above.

3.3 Performance of concentration distribution

In our study, concentration distribution on chip is signifi-

cant; a linear concentration gradient should be generated

based on principles of laminar flow in microchannels. Five

flow rates (2.5, 5, 10, 15, 20 lL min-1) were chosen to

optimize flow rate. We just injected a low (0 mM) and a

high (1.0 mM) concentration of urea solution into the

system; solutions mixed gradually when moving through

microchannels and generated six different concentrations.

The corresponding theoretical concentrations value were

calculated based on equation below and compared with

experimental data (Yang et al. 2011).

Cði;NÞ ¼ ðN � iÞC1

Nþ iC2

Nð1Þ

Fig. 1 Schematic of

microfluidic chip for urease

inhibition assay (I). Inlet ports

C1, C2, C3 and C4 are

connected to syringe pumps for

reagent delivery, and outlet port

C5 is for fluid removal. The

main functional regions of

microchip include:

concentration gradient generator

(part 2); reaction wells (part 1);

fluidic distribution of OPA (part

3): fluorescence derivation and

detection windows (part 4). II A

real picture of the device; IIIchannels of dilution device on

layer A; IV channels and

detection windows on layer B

Microfluid Nanofluid

123

In this equation, C(i, N) represents different concentra-

tion at the end of concentration distribution microchannels,

N stands for the number of sequence of channel, i means

the number of sequence of each branch point. When the

concentrations (C1, C2) inject into the system changed, a

wide range of solution concentrations can be generated and

access to immobilized enzyme.

Data from Fig. 4 indicate that when flow rate was below

5 lL min-1, experimental outcomes matched up well with

theoretical data. Greater deviation occurred when flow rate

was higher than 10 lL min-1; the variance was largely

attributed to the roughness of microchannel surface after

chemical modification, which may lead to incompletely

reaction between substrate and enzyme as well as product

and OPA reagent.

3.4 Comparison between performances

of immobilized enzymes

In this study, we used PDMS-glass microchip. To adhere

PDMS to glass surface, PDMS layers were oxidized in

oxygen plasma. Contact of oxidized PDMS in pH 7–9 is

104.5�; it is more hydrophobic than other materials, which

may lead to an increased adsorption of proteins. The extent

and stability of PDMS absorption were showed by treating

microchip with no modification but filled with enzyme

solution and kept in 4 �C for 12 h and compared with

enzyme immobilized through chemical modification.

The catalyzed reaction of urease was evaluated in the

concentration range 0.2–3 lM by automatically concen-

Fig. 2 a Fluorescence intensity plotted against time at a single

concentration (0.1 mM); b effect of OPA reagent pH on the

fluorescence of NH4?-OPA product. Error bars indicate the standard

deviation of the measurements (n = 3)

Fig. 3 Relative fluorescence intensity in related to the concentration

of urea. The line in the inset is a linear fit; the correlation coefficient is

0.992. Error bars indicate the standard deviations of the measure-

ments (n = 3)

Fig. 4 Performance of the integrated concentration gradient gener-

ator. Comparison between prediction and experimental data on

fluorescence intensity in six detection chambers at five different flow

velocities. Error bars indicate the standard deviations of the

measurements (n = 3)

Microfluid Nanofluid

123

tration generation via distribution part. For most ureases,

the Michaelis constants KM keep in the range of 1–4 lM

(Fidaleo and Lavecchia 2003). For immobilized enzyme

through disulfide bond, a KM of 1.528 lM was derived

from an on-chip assay by plotting the fluorescence signal

taken from Fig. 5 versus substrate concentration to the

Michaelis–Menten equation. This value shows vital dif-

ference with KM of enzyme immobilized through amino

group of 1.617 lM (Table 1). As for enzyme immobilized

by PDMS adsorption, affinity of enzyme and substrate is

higher with a KM of 1.071 lM. The expressed activity

value for urease immobilized by PDMS adsorption is

almost the half of urease immobilized via chemical meth-

ods, and repeatability of immobilized enzyme activity

assay is poor with relative standard deviation (RSD) of

0.647 within 7 days, compared with 0.0278 and 0.0168 for

disulfide and amino-bound immobilization enzyme,

respectively. The loss of enzyme activity may be partly

because of weak physical absorption and partly because of

the lack of particular combination site and multi-interac-

tions between enzyme and support may lead to enzyme

distortion and active center may be blocked through the

reaction (Garcia-Galan et al. 2011). From known infor-

mation, most cysteine groups are not essential in catalysis

process, yet one particular cysteine (Cys592 in Jack bean

urease) (Krajewska and Zaborska 2007) is involved in the

catalysis becase this cysteine is positioning other key res-

idues in the active site. In this assay, immobilization

through cysteine group would not influence enzyme

activity to a large extent.

Among three kinds of immobilization methods, we

preferred the disulfide one mainly because disulfide

immobilized enzyme was reversible, while stability of

immobilized enzyme was also reasonable. Fresh enzyme

with high activity could take place of denatured one so that

lifetime of the microchip system was largely extended.

3.5 Long-term stability

The precision value through five successive experiments

rendered the RSD of 2.87 %; RSD of measurements on

seven successive days on the same microchip is 4.59 %.

The long-term stability of immobilized enzyme assay is

intimately related to properties of support material and

immobilized chemical method, as well as other factors

such as number of consecutive assays and regeneration

time. We immobilized urease through disulfide bond, then

detected activity of enzyme on continuous days and com-

pared outcomes with the first day to observe activities

variance within days. After 15 days of successive opera-

tion, RSD of relative fluorescence data on different days is

5.28 % (Table 2), 20 days with RSD of 5.46 % and

25 days with RSD of 8.46 %, the relative fluorescence

signal changed 16.3 % compared to day 1. The results

compared well with enzyme immobilized via ionic inter-

actions on mesoporous silicates which could be reused 20

times with retention of activity (Hudson et al. 2007).

3.6 Repeatability of different immobilization cycles

As mentioned above, immobilization process through

disulfide bond is reversible; we identified the removal of

enzyme by comparison with baseline value (the same assay

system without enzyme). The results showed that urease

could be completely removed from the surface of micro-

channel. Next, we repeated immobilization and removal

Fig. 5 Measurement of KM of immobilized enzyme by disulfide

bond, amino bond, PDMS absorption and free enzyme

Table 1 Kinetic characteristics and repeatability of urease immobi-

lized through disulfide immobilization, amino immobilization and

PDMS absorption

Disulfide-bond

immobilization

Amino-bond

immobilization

PDMS

absorption

Relative

fluorescence

1,731.323 1,764.7 852.35

KM 1.528 1.617 1.071

RSD (n = 7) 0.0486 0.0606 0.340

Change in relative

fluorescence

(n = 7)

0.0278 0.168 0.647

Table 2 Effect of reversible immobilization through disulfide bond

for long-time stability

Day 5 10 15 20 25

RSD 0.0139 0.0548 0.0528 0.0546 0.0846

Change in relative

fluorescence

0.0245 0.1310 0.0596 0.0617 0.1630

Microfluid Nanofluid

123

process for five times and compared the activities of

immobilized enzyme. Repetition of immobilization process

did not alter activity of immobilized enzyme with the RSD

of 4.78 % (Table 3). As stability of relative fluorescence

only could not overall represent enzymatic assay stability,

monitoring other parameter such as IC50 of inhibitor could

provide more information; detection of IC50 data showed

reasonable repeatability with RSD of 2.98 %. The results

indicated that the process of re-immobilization would not

affect efficiency of enzyme assay on chip.

3.7 Application on inhibitor screening

To identify application of microchip on inhibition screen-

ing, solution of a known inhibitor AHA was introduced

into microfluidic system and mixed with enzyme in indi-

vidual channel. All residual activities of inhibitor-substrate

complexes were compared to activity of naught concen-

tration inhibitor-substrate, account as a control activity of

100 %. For further experiment, 10 min was required for

sufficient equilibration of the inhibitor and enzyme. In

order to determine IC50 of AHA, the inhibitor solution flow

through the concentration distribution part and generated

six difference concentrations solutions and incubated with

immobilized enzyme in flow stop condition. Reduced

fluorescence signal and the inhibition ratio of AHA were

calculated according to Eq. 2. Here, X is for the relative

fluorescence determined in the presence of the inhibitor,

and Blank is the relative fluorescence determined without

inhibitor.

Inhibitionð100 %Þ ¼ ðBlank� XÞ=Blank� 100 % ð2Þ

The IC50 of inhibitor AHA determined via experimental

data is 368 lM (Fig. 6), which compared reasonably well

with reported IC50 375 lM in reported assays (Tanaka

et al. 2004), which could prove the fact that this enzyme

inhibition assay is reliable and objective. For competitive

inhibitors, equilibrium between inhibitor and enzyme

reduces the concentration of enzyme available for catalysis

of substrate. From the formula below, it is clear that the

constant Ki, represents inhibitor equilibrium dissociation, is

related to initial inhibitor concentration [I] and the enzyme

reaction rate, Vinitial (Hadd et al. 1999)

Vinitial ¼Vmax½S�

1þ ½I�Ki

� �KM þ ½S�

ð3Þ

Here, Vmax stands for maximum reaction rate, [S] means

substrate concentration and KM represents the Michaelis

constants. When inhibitor concentration is zero, this

equation reduces to Michaelis–Menten expression for

enzyme kinetics. As KM is already known and substrate

concentration is fixed, Ki can be determined by enzyme

catalysis reaction rate under influence of the inhibitor.

V � Vi ¼V ½I�

Kið1þ ½S�KMÞ þ ½I�

ð4Þ

In Eq. 4, V means the reaction rate without effect of

inhibitor; Vi is the reaction rate in the presence of inhibitor.

Thus, the Ki constant of AHA can be derived from a

nonlinear least-square of Eq. 4 to a plot of V - Vi versus

inhibitor concentration. As mentioned above, the reaction

rate was proportional to fluorescence of corresponding

situation. From a nonlinear regression fit of change in

relative fluorescence versus urea concentration, a Ki of 1.39

for the immobilized urease through microchip assay and a

Ki of 1.46 for free urease via the 96-microplate were

derived. In conventional microplate assay (Firdous et al.

2012), more reagent volume and reaction time were

required to get equilibrium.

Table 3 Effect of reversible immobilization through disulfide bond for repeatability of different immobilization cycles

Times

1 2 3 4 5

Relative fluorescence 2,217.7 2,051.6 1,974.8 1,979.1 2,065.8

Change in relative fluorescence 0 0.0748 0.109 0.107 0.0685

IC50 data 0.368 0.341 0.362 0.362 0.352

Fig. 6 Measurement of inhibitor IC50 curves on microfluidic device.

Error bars indicate the standard deviations of the measurements

(n = 3)

Microfluid Nanofluid

123

4 Conclusions

The present study developed a chemical modification

method to prepare active groups on microchannel surface.

We demonstrated its application in preparing an enzyme

microreactor. The resulting immobilized urease maintained

reasonable activity and showed high performance in inhi-

bition assay. As immobilization through disulfide bond is

reversible, we tested relative fluorescence from different

immobilization cycles; results showed that inhibition assay

on chip is reliable and stable. The enzyme-immobilized

microchip platform might be useful in enzyme inhibition

assay.

Acknowledgments This work is financially supported by the

National Natural Science Foundation of China (No. 20875105).

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