A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis

Article abstract of DOI:10.1016/j.ab.2010.12.025

A facile protocol to prepare highly effective and durable in-line enzyme bioreactors inside capillary electrophoresis (CE) columns was developed. To demonstrate the methodology, l-glutamic dehydrogenase (GLDH) was selected as the model enzyme. GLDH was first immobilized onto 38-nm-diameter gold nanoparticles (GNPs), and the functionalized GNPs were then assembled on the inner wall at the inlet end of the CE capillary treated with polyethyleneimine (PEI), producing an in-line GLDH bioreactor. Compared with a GLDH bioreactor prepared by immobilizing GLDH directly on PEI-treated capillary, the GNP-mediated bioreactor showed a higher enzymatic activity and a much better stability. The in-capillary enzyme bioreactor was proven to be very useful for screening of GLDH inhibitors deploying the GLDH-catalyzed α-ketoglutaric acid reaction. The screening assay was preliminarily validated by using a known GLDH inhibitor, namely perphenazine. A Z′ factor value of 0.95 (n = 10) was obtained, indicating that the screening results were highly reliable. Screening of GLDH inhibitors present in medicinal plant extracts by the proposed method was demonstrated. The inhibition percentages were found to be 53% for Radix scutellariae, 45% for Radix codonopsis, 37% for Radix paeoniae alba, and 0% for the other 22 extracts tested at a concentration of 0.6 mg extract/ml.

Full text of DOI:10.1016/j.ab.2010.12.025

Analytical Biochemistry 411 (2011) 88–93
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening
by capillary electrophoresis
a,
⇑
a
b
b,
⇑
Shulin Zhao , Xiaowen Ji , Pingtan Lin , Yi-Ming Liu
a
College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 51004, China
Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 October 2010
Received in revised form 15 December 2010
Accepted 15 December 2010
Available online 22 December 2010
A facile protocol to prepare highly effective and durable in-line enzyme bioreactors inside capillary elec-
trophoresis (CE) columns was developed. To demonstrate the methodology,
L-glutamic dehydrogenase
(GLDH) was selected as the model enzyme. GLDH was first immobilized onto 38-nm-diameter gold nano-
particles (GNPs), and the functionalized GNPs were then assembled on the inner wall at the inlet end of
the CE capillary treated with polyethyleneimine (PEI), producing an in-line GLDH bioreactor. Compared
with a GLDH bioreactor prepared by immobilizing GLDH directly on PEI-treated capillary, the GNP-med-
iated bioreactor showed a higher enzymatic activity and a much better stability. The in-capillary enzyme
bioreactor was proven to be very useful for screening of GLDH inhibitors deploying the GLDH-catalyzed
Keywords:
Enzyme bioreactor
Capillary electrophoresis
Gold nanoparticles
Inhibitor screening
Medicinal plant extracts
a
-ketoglutaric acid reaction. The screening assay was preliminarily validated by using a known GLDH
0
inhibitor, namely perphenazine. A Z factor value of 0.95 (n = 10) was obtained, indicating that the screen-
ing results were highly reliable. Screening of GLDH inhibitors present in medicinal plant extracts by the
proposed method was demonstrated. The inhibition percentages were found to be 53% for Radix
scutellariae, 45% for Radix codonopsis, 37% for Radix paeoniae alba, and 0% for the other 22 extracts tested
at a concentration of 0.6 mg extract/ml.
Ó 2010 Elsevier Inc. All rights reserved.
Enzyme inhibitor screening is an effective approach to identify
drug leads because many enzymes are involved in regulatory cellu-
lar processes and, thus, become therapeutic drug targets [1–3].
High-throughput screening (HTS)1 is the most commonly used
method for screening of enzyme inhibitors [4–6]. It is based on col-
orimetric or fluorometric measurements carried out on multiwell
microplates. It works perfectly with large libraries of pure com-
pounds but sometimes is not applicable to assay complicated sam-
ples such as extracts of medicinal plants that may contain many
ultraviolet (UV)-absorbing or fluorescent compounds [7,8]. Over
the past years, the search for enzyme inhibitors present in medicinal
plants has been one of the major interests in drug discovery and
development [9–11]. In fact, many new drugs developed between
sample matrices, screening assays based on liquid chromatography
(LC), mass spectrometry (MS), and capillary electrophoresis (CE)
were developed [13–15]. In most of these methods, an in-line en-
zyme bioreactor prepared by immobilizing the enzyme onto a solid
support that was retained inside the LC or CE column was used. A
test solution containing enzyme substrate was injected and incu-
bated in the enzyme bioreactor for certain time. The product result-
ing from the enzymatic reaction was then separated from other
coexisting components, including unreacted substrate, and was
quantified. If an enzyme inhibitor was present in the test solution,
the amount of product was decreased. Obviously, having an effective
and durable enzyme bioreactor is essential for the success of such a
screening method.
1
981 and 2002, particularly for treating cancer and infectious dis-
To prepare enzyme bioreactors, immobilization techniques
were employed, including physical adsorption, ionic binding, cova-
lent binding, and sol-gel entrapment [16]. Wainer and coworkers
described the immobilization of enzymes on silica particles that
were then packed into LC columns for studying enzyme activity
and screening of inhibitors [17]. Enzyme bioreactors prepared by
entrapping enzymes in sol-gel-derived monolithic capillary col-
umns were reported for protein analysis by CE [18] and for inhib-
itor screening by MS [19]. Using glutaraldehyde as a coupling
reagent, enzymes were covalently immobilized onto amine-
eases, originated from natural sources [12]. To study these complex
⇑
Corresponding authors. Fax: +1 601 979 3674 (Y.-M. Liu).
E-mail addresses: zhaoshulin001@163.com (S. Zhao), yiming.liu@jsums.edu
(
Y.-M. Liu).
1
Abbreviations used: HTS, high-throughput screening; UV, ultraviolet; LC, liquid
chromatography; MS, mass spectrometry; CE, capillary electrophoresis; HPLC–MS/
MS, micro-high-performance liquid chromatography–tandem mass spectrometry;
l
+
GNP, gold nanoparticle; GLDH, L-glutamic dehydrogenase; b-NAD , nicotinamide
adenine dinucleotide; b-NADH, b-nicotinamide adenine dinucleotide (reduced diso-
dium salt); PEI, poly(ethyleneimine); S/N, signal/noise ratio; RSD, relative standard
deviation.
functionalized magnetic nanoparticles to prepare an
a-glucosidase
bioreactor used for inhibitor screening by CE [20]. In addition,
0
003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.12.025

Enzyme bioreactor for inhibitor screening / S. Zhao et al. / Anal. Biochem. 411 (2011) 88–93
89
Zhang and coworkers described a procedure to covalently immobi-
lize trypsin onto a monolithic stationary phase that was activated
by glutaraldehyde [21]. The trypsin bioreactor was used in micro-
high-performance liquid chromatography–tandem mass spec-
trometry (lHPLC–MS/MS) proteomic analysis with a digestion
speed approximately 6600 times faster than that of digestion in
free solutions. Compared with all other immobilization techniques,
ionic binding is certainly the most convenient and fastest tech-
nique to immobilize enzymes. In an immobilization procedure
via ionic binding, the solid support is first coated with a polyelec-
trolyte to obtain a suitably charged carrier surface (either posi-
tively or negatively depending on the predominant charge on the
enzyme). The solid support with a charged surface is then soaked
with an enzyme solution. Due to the electrostatic interactions, en-
zyme molecules bind to the surface of the solid support. The coat-
ing-and-binding process can be repeated several times, forming a
layer-by-layer assembly of enzyme molecules on the carrier
surface. Kang and coworkers described a bioreactor of angioten-
sin-converting enzyme using hexadimethrine bromide as the
polyelectrolyte [22] and a layer-by-layer assembly bioreactor of
acetylcholinesterase using poly(diallyldimethylammonium chlo-
ride) as the polyelectrolyte inside CE columns that were used for
enzyme inhibitor screening [23].
Here we describe a new strategy for preparing CE in-column en-
zyme bioreactors based on the ionic binding technique. The inno-
vative aspect of the proposed procedure is that the enzyme is
first immobilized on gold nanoparticles (GNPs) and the functional-
ized GNPs (instead of enzyme molecules) are then assembled onto
the polyelectrolyte-modified carrier surface, forming a GNP-medi-
ated enzyme bioreactor. The rationale for this experimental design
is that enzyme loading in the bioreactor mediated by GNPs is ex-
pected to be much greater than that prepared from a free enzyme
solution because enzyme molecules can be enriched on GNPs with
a vast surface-to-mass ratio. In addition, a much better stability of
the bioreactor can be expected given that enzyme–GNP conjugates
are much more stable than enzyme–polyelectrolyte conjugates. It
is well documented that thiol groups (–SH) present in protein mol-
ecules bind to GNPs strongly, contributing to the high stability of
protein–GNP conjugates [24]. Fig. 1 illustrates an enzyme bioreac-
Fig. 1. Schematic of a GNP-mediated enzyme bioreactor prepared in a CE column.
Preparation of GLDH-functionalized GNPs
GNPs (38 nm diameter) were prepared as described previously
[
4
25]. Briefly, 100 ml of a 0.01% (w/v) HAuCl solution was heated
to boiling, and then 1.0 ml of a 1% trisodium citrate solution was
added with stirring. The solution was refluxed for 30 min. After
cooling to room temperature, the solution was filtered through a
0
.45-
l
m nylon membrane. The pH of the GNP solution was ad-
CO and kept at 4 °C. The size and monodisper-
justed to 9.0 with K
2
3
sity of GNPs prepared were determined by using a JEOL 100CX
transmission electron microscope. To prepare GLDH-functional-
ized GNPs, 95 ll of the GNP solution was mixed with 5 ll of the
above-prepared 10-mg/ml GLDH solution. The mixture was vor-
tor prepared by the proposed approach. In this work, L-glutamic
dehydrogenase (GLDH) was selected as the test enzyme. The pre-
pared CE in-column GLDH bioreactor was evaluated in terms of en-
zyme activity, bioreactor stability, and its usefulness for screening
of GLDH inhibitors present in medicinal plants by CE.
texed for 1 min and then let to stand alone at 4 °C for 1 h.
Preparation of a GLDH bioreactor in a CE column
Materials and methods
A piece of 50
lm i.d. ꢁ 38 cm (29.5 cm to detection window)
Chemicals and solutions
fused-silica capillary was used. The capillary was treated with a
1 M NaOH solution for 1 h and then flushed with water. An approx-
imately 0.5-cm section of the capillary at the inlet end was filled
with the PEI solution as prepared above. After 10 min, the PEI solu-
tion was washed out with water. The GLDH-functionalized GNP
solution was introduced and filled the PEI-modified section of
the capillary. Negatively charged GLDH–GNP conjugates bound to
the positively charged PEI coating on a capillary wall. After 2 h,
the capillary was flushed with water and ready for use in CE
experiments.
+
GLDH, nicotinamide adenine dinucleotide (b-NAD ), b-nicotin-
amide adenine dinucleotide (reduced disodium salt) (b-NADH),
poly(ethyleneimine) (PEI, MW = 750,000, 50% [w/v] solution),
-ketoglutaric acid, and perphenazine were purchased from Sig-
ma–Aldrich Chemicals (St. Louis, MO, USA). All medicinal plants
tested were collected from local markets. The GLDH solution was
prepared by dissolving 0.0020 g of GLDH in 200 ll of 30% glycerol
solution and was kept at ꢀ20 °C before use. The PEI solution was
freshly prepared by diluting the 50% (w/v) PEI solution with water
to obtain a concentration of 4% (w/v) PEI.
a
Preparation of the extracts of medicinal plants
CE system
Air-dried medicinal plants were ground to fine powders. Isopro-
panol–H O (1:1) solution was added to the powder in an Erlen-
2
All CE experiments were performed using an HP3D CE system
(
Hewlett Packard, Waldbronn, Germany). UV detection was at a
meyer flask (40 ml isopropanol solution/10 g powder). The
mixture was sonicated for 2.5 h at a frequency of 100 kHz and
60 °C by using an ultrasonic cleaning bath. The mixture was
fixed wavelength of 254 nm. Data were processed with an HP
ChemStation.

9
0
Enzyme bioreactor for inhibitor screening / S. Zhao et al. / Anal. Biochem. 411 (2011) 88–93
filtered. The filtrate collected was evaporated to nearly complete
dryness. The residue was dried in a vacuum dryer at 30 °C and
0
.07 MPa, obtaining the extract. Solutions of extracts were pre-
pared in 50% isopropanol at 6 mg /ml. The solution was appropri-
ately diluted with the CE running buffer before assay.
Screening of GLDH inhibitors by CE
The screening assay was based on the following enzymatic
reaction:
GLDH
a
ꢀ ketoglutaric acid þ CH
3
CO
2
NH
4
þ b-NADH
þ
!
glutamic acid þ b-NAD þ CH
3
CO
2
H þ H
2
O
For enzyme activity studies, a substrate solution was injected
into the CE capillary with an enzyme bioreactor at the inlet end
by pressure at 40 mbar for 5 s. A voltage of ꢀ28 kV was applied
+
to separate b-NAD from b-NADH and potentially any other exist-
ing compounds in the sample solution. After the separation, b-
Fig. 2. CE separation of b-NAD and b-NADH. Experimental conditions: CE column,
+
+
NAD was quantified by UV absorbance at 254 nm. Enzyme activity
50 mm i.d. ꢁ 29.5 cm effective length fused-silica capillary; injection, 40 mbar for
+
5 s; running buffer, 25 mM borate buffer (pH 9.0); voltage applied, ꢀ28 kV; column
was assessed based on the amount of b-NAD produced from the
+
temperature, 25 °C; and UV detection at 254 nm. Peak identifications: (1) b-NAD
enzymatic reaction.
and (2) b-NADH (0.10 mM each).
For inhibition studies, the CE running buffer containing a known
inhibitor (or extract of a medicinal plant) was injected into the
capillary and left in place for 1 min. An inhibitor (or extract)-
containing substrate solution was then injected. The CE separation
was started, and b-NAD+ was quantified as described above. The
inhibition percentage was calculated according to the peak area of
Performance of in-line GLDH bioreactor
The GLDH bioreactor was first evaluated in terms of GLDH
activity of the bioreactor. In these studies, a substrate solution con-
taining 16 mM a-ketoglutaric acid, 0.050 mM b-NADH, and 80 mM
ammonium acetate was injected into the CE capillary with an in-
line GLDH bioreactor at the inlet end, and the solution was left in
the bioreactor for some time (0–3 min) before a CE voltage was ap-
+
b-NAD compared with the reference electropherogram obtained
in the absence of the inhibitor (or extract).
Results and discussion
+
plied to separate and quantify b-NAD produced from the enzy-
matic reaction. The results indicated that the enzymatic reaction
was more than 75% complete with zero incubation time (i.e., the
+
CE quantification of b-NAD
CE separation was started immediately after injecting the substrate
b-NAD+ is a coenzyme found in all living cells. b-NAD+ and its
reduced form, b-NADH, are involved as a reactant/product pair in
many enzymatic reactions. For example, in the GLDH-catalyzed
solution). Fig. 3 shows the relationship between the b-NAD+
amount produced and the incubation time. Within 1.0 min, the
enzymatic reaction was more than 97% complete. The reaction
velocity was unexpectedly high considering that the enzyme quan-
tity loaded in the bioreactor was extremely small. The high reac-
tion velocity was likely due to the vast GNP surface area for
reaction of
a-ketoglutaric acid selected in this work for screening
+
of GLDH inhibitors, b-NADH is converted to b-NAD . Therefore,
quantification of b-NAD+ in the reaction mixture serves well the
+
purpose of monitoring these enzymatic reactions if b-NAD does
not occur in the sample matrix (i.e., the herbal extracts in this
work). CE was proven to be a very effective technique to separate
these two compounds. Thus,
29.5 cm effective length) was used to achieve a fast quantification.
Because phosphate ions affect GLDH activity and borate ions do not
26], a borate buffer solution (25 mM at pH 9.0) was chosen as the
a
relatively short CE column
6.8
(
[
6.4
6.0
5.6
5.2
CE running buffer. Under the experimental conditions selected,
+
b-NAD and b-NADH were separated within 3 min (Fig. 2). For
quantification, seven-point calibration curves were prepared by
analyzing b-NAD+ standard solutions at varying concentrations
+
from 0.0100 to 0.400 mM. b-NAD was quantified using the CE
peak area. Linear regression resulted in the following regression
equation:
2
y ¼ 136x ꢀ 0:108; r ¼ 0:9993;
+
where y is the peak area, x is the b-NAD concentration (in mM), and
2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
r is the correlation coefficient. The detection limit was calculated to
+
be 0.002 mM for b-NAD (signal/noise ratio [S/N] = 3). To determine
Incubation time (min)
+
the assay reproducibility, a 0.050-mM b-NAD standard solution
Fig. 3. Enzyme activity tests: incubation time versus amount of b-NAD+ produced
from the enzymatic reaction. The substrate solution injected contained 80 mM
ammonium acetate, 16 mM
running buffer. CE experimental conditions were as in Fig. 2
was analyzed 10 times. The assay reproducibility was found to be
1
.4% (relative standard deviation [RSD], n = 10). The reproducibility
a-ketoglutaric acid, and 0.050 mM b-NADH in CE
+
of b-NAD migration time (RSD, n = 10) was 0.65%.

Enzyme bioreactor for inhibitor screening / S. Zhao et al. / Anal. Biochem. 411 (2011) 88–93
91
enzyme immobilization and, thus, a high level of contact between
the immobilized enzyme and the substrate.
The value of the Michaelis–Menten constant (K ), an important
m
parameter of an enzymatic reaction, was also determined for the
GLDH bioreactor by using the Lineweaver–Burk plotting method:
and quick monitoring of the GLDH reaction. These results com-
bined suggested that the CE-based method was well suited for
screening of GLDH inhibitors. To evaluate its applicability for GLDH
inhibitor screening, inhibition studies using a known GLDH inhib-
itor, perphenazine, were performed. In these tests, a perphenazine
solution prepared in the CE running buffer was injected and left in
place for 1 min. The substrate solution containing perphenazine at
0.125 mM was then injected. It was noted that without the inhib-
itor in the bioreactor, a significant amount of the substrate was
converted to the product before the enzyme was inhibited due to
a very fast enzymatic reaction rate. The inhibition percentage
was calculated using the following equation:
1
v
K
max½Sꢂ
m
1
¼ V
þ V
;
ð1Þ
max
where
v and Vmax are the initial and maximal velocities of the reac-
tion, respectively, and [S] is the substrate concentration. Six
b-NADH solutions at different concentrations ranging from 0.020
to 0.100 mM were analyzed. Each of the solutions was analyzed
ꢀ
ꢁ
+
x
three times. The peak area of b-NAD was used to represent the ini-
Inhibition % ¼ 100 ꢀ
ꢁ 100
ð2Þ
blank
tial reaction rate. A double-reciprocal plot defined by Eq. (1) was
constructed. Linear regression analysis of the data produced the fol-
+
where x and blank are the peak areas of b-NAD measured with and
without the inhibitor, respectively. The quality of screening assays
can be assessed by using the Z’ factor, calculated as follows [28]:
2
lowing equation: 1/v = 0.373 (1/[S]) + 24.85 (r = 0.9961). From this
equation, K
that obtained from the GLDH reaction in free solutions (0.02 mM)
27]. The results suggested that no significant change in the sub-
m
was calculated to be 0.015 mM, which was similar to
Z0 ¼ 1 ꢀ 3
r
ls
s
þ 3
r
c
ð3Þ
[
j
ꢀ
l
j
c
strate–GLDH binding property was caused by enzyme
immobilization.
+
where
s c
l and l are the means of the signal (i.e., b-NAD peak area
Stability of the in-line GLDH bioreactor was assessed. A sub-
strate mixture solution was repetitively injected for analysis up
to 110 times on the same CE capillary with an in-line GLDH biore-
in this work) obtained from the standard assay (in the absence of
inhibition) and the control assay (100% inhibition by a known inhib-
itor), respectively, and
data. A Z factor value between 0.5 and 1 indicates that the quality
s c
r and r are the standard deviations of the
+
actor. Fig. 4 shows the results of the b-NAD peak area versus the
0
number of assays. As can bee seen, the efficacy of the GLDH biore-
of the screening data is excellent (i.e., the inhibitor screening assay
+
actor (measured by b-NAD peak area) tended to decrease as the
is accurate and reliable). In this work, perphenazine solutions at
number of assays increased. However, the decrease was relatively
insignificant (<15% after an extensive use of 110 times). In addi-
tion, the bioreactor could be regenerated easily by dipping the inlet
end of the capillary into the GLDH-functionalized GNP solution for
0
0
.06 and 0.12 mM were assayed. The Z factor value was calculated
as 0.95 (n = 10). The result indicated that the accuracy of the current
CE-based screening method was excellent for the screening of GLDH
inhibitors.
1
0 s and flushing the capillary with water 2 h afterward. For com-
parison, a GLDH bioreactor was prepared by immobilizing GLDH
instead of GLDH-functionalized GNPs) onto the inner wall of cap-
Screening of GLDH inhibitors in Chinese medicinal plants
(
illary treated with PEI. It was found that the enzymatic activity de-
creased very quickly. The bioreactor lost approximately 40% of its
initial activity after only five assays. The improved stability of
the GNP-mediated bioreactor likely resulted from the fact that
the GLDH–GNP conjugate was much more stable than the GLDH–
PEI conjugate.
The above-described studies showed that the in-line GLDH bio-
reactor had not only a high enzymatic activity but also a good sta-
bility. In addition, the CE quantification allowed accurate, reliable,
Isopropanol extracts were prepared from 25 Chinese medicinal
plants. In addition, perphenazine (a known GLDH inhibitor) was
also included in the chemical library for the purpose of assay val-
idation (as a positive control). Triplicate assays were made for each
isopropanol extract. The averaged peak areas from the three mea-
surements were used to calculate the inhibition percentage by Eq.
(
2). Fig. 5 shows several electropherograms from the screening as-
says. Fig. 5A was from a blank solution (i.e., in the absence of inhi-
bition). The injected substrate solution contained -ketoglutaric
a
acid, ammonium acetate, and b-NADH. Fig. 5B and C were from
assaying Radix scutellariae and Radix codonopsis extracts (both at
1
0
8
6
4
2
0
0
.6 mg/ml), respectively. The injected sample solutions were the
CE running buffer containing the extract at 0.6 mg/ml for preincu-
bation and then the substrate solution containing the extract. As
+
can be seen, the peak areas of b-NAD , the product of the
enzymatic reaction, were clearly reduced compared with that in
Fig. 5A from a blank solution, clearly indicating GLDH inhibition
(
53% inhibition from R. scutellariae extract and 45% from
R. codonopsis extract). It is worth noting that several endogenous
compounds present in the extract were separated from b-NAD+
and b-NADH, as shown in Fig. 5B. This might eliminate potential
interference with the quantification and, therefore, false screening
results. As can be seen in Table 1, perphenazine (at 0.125 mM) and
three extracts at 0.6 mg/ml were found to be positive for GLDH
inhibition. The other extracts did not show any inhibitory effects.
They served well as negative controls for the screening assay,
whereas perphenazine served as a positive control. The extract of
0
20
40
60
80
100
120
R. scutellariae showed
a 53% inhibition on GLDH activity.
Number of assays
R. scutellariae is widely used for the treatment of inflammation,
fever, hepatitis, allergic diseases, hypertension, and the like in
Chinese traditional medicine [29]. Studies have shown that
Fig. 4. Stability of the in-line GLDH bioreactor: enzyme efficacy versus number of
assays. Experimental conditions were as in Fig. 3.

9
2
Enzyme bioreactor for inhibitor screening / S. Zhao et al. / Anal. Biochem. 411 (2011) 88–93
Fig. 5. Electropherograms obtained from the screening assays: (A) blank solution (i.e., the substrate solution); (B) Radix scutellariae extract at 0.6 mg/ml; (C) Radix codonopsis
+
extract at 0.6 mg/ml; (D) perphenazine (a known GLDH inhibitor) at 0.125 mM. Assay conditions were as in Fig. 3. Peak identifications: (1) b-NAD and (2) b-NADH.
Table 1
Results of GLDH inhibitor screening in medicinal plant extracts.
Medicinal plant
Inhibition %a
Medicinal plant
Inhibition %a
Perphenazine (a known GLDH inhibitor)
Radix astagali
Fructus crataegi
65
0
0
Exocarpium citri rubrum
Wrinkled gianthyssop
Herba verbenae
0
0
0
Poria
0
0
0
37
0
0
0
0
Radix scutellariae
53
0
0
0
0
0
0
0
Radix achyranthis bidentatae
Fructus hordei germinatus
Radix paeoniae alba
Fructus piperis
Rhizoma chuanxiong
Rhizoma anemarrhenae
Radix ophiopogonis
Radix scrophulariae
Semen lablab album
Pericarpium citri reticulatae
Leaf of Henon bamboo
Fructus amomi rotundus
Radix et rhizoma rhei
Semen arecae
Rhizoma pinelliae
Rhizoma atractylodis macrocephalae
0
0
Radix codonopsis
Bulbus fritillariae cirrhosae
45
0
Note. The concentration of extract was 0.6 mg/ml, and the concentration of perphenazine was 0.125 mM.
a
Means of triplicate assays.
R. scutellariae lowers blood pressure and has sedative effects on the
central nervous system [30,31].
bilization. Use of the highly effective and durable in-line GLDH
bioreactor in combination with the proposed CE quantification of
b-NAD allowed accurate and reliable GLDH inhibitor screening.
+
The proposed screening method was applied to analyzing extracts
of 25 medicinal plants. The inhibition percentages were found to
be 53% for R. scutellariae, 45% for R. codonopsis, 37% for Radix paeo-
niae alba, and 0% for the other 22 extracts tested. The screening
method was proved to be accurate, easy to carry out, and well sui-
ted for assaying complex samples such as medicinal plant extracts.
Conclusion
GLDH could be easily immobilized on 38-nm-diameter GNPs.
The functionalized GNPs were assembled on the inner wall at the
inlet end of a CE capillary modified by PEI, producing an in-line
GLDH bioreactor. The GNP-mediated GLDH bioreactor showed a
high enzymatic activity and a very good stability. The value of
the Michaelis–Menten constant (K
m
) was 0.015 mM, which was
Acknowledgments
very similar to that obtained from the GLDH reaction in free
solutions. The result suggested that no significant changes in the
substrate–GLDH binding property were caused by enzyme immo-
Financial support from the National Natural Science Foundation
of China (20875019 to S.Z.), the Guangxi Science Foundation of Chi-

Enzyme bioreactor for inhibitor screening / S. Zhao et al. / Anal. Biochem. 411 (2011) 88–93
93
na (0832004 to S.Z.), and the US National Institutes of Health
SC1GM089557 to Y-M.L.) is gratefully acknowledged.
[17] M. Bartolini, V. Andrisano, I.W. Wainer, Development and characterization of
an immobilized enzyme reactor based on glyceraldehyde-3-phosphate
dehydrogenase for on-line enzymatic studies, J. Chromatogr. A 14 (2003)
(
331–340.
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