Resurfaced fluorescent protein as a sensing platform for label-free detection of copper(II) ion and acetylcholinesterase activity

Article abstract of DOI:10.1021/ac504390e

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H₃₉GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H₃₉GFP as the signal reporter, a label-free fluorometric sensor for Cu²⁺ sensing was developed based on the unique multivalent metal ion-binding property of H₃₉GFP and fluorescence quenching effect of Cu²⁺ by electron transfer. The high affinity of H₃₉GFP with Cu²⁺ (K_d, 16.2 nM) leads to rapid detection of Cu²⁺ in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H₃₉GFP/Cu²⁺ complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu²⁺ and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL^-1) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H₃₉GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.

Full text of DOI:10.1021/ac504390e

Article
pubs.acs.org/ac
Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free
Detection of Copper(II) Ion and Acetylcholinesterase Activity
†
,‡
†
†
†
†
,†
Chunyang Lei, Zhen Wang, Zhou Nie, Honghua Deng, Huiping Hu, Yan Huang,*
and Shouzhuo Yao†
†State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha 410082, P. R. China
‡
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
*
S Supporting Information
ABSTRACT: Protein engineering by resurfacing is an efficient
approach to provide new molecular toolkits for biotechnology and
bioanalytical chemistry. H GFP is a new variant of green
3
9
fluorescent protein (GFP) containing 39 histidine residues in the
primary sequence that was developed by protein resurfacing.
Herein, taking H GFP as the signal reporter, a label-free
3
9
2
+
fluorometric sensor for Cu sensing was developed based on
the unique multivalent metal ion-binding property of H GFP and
3
9
2
+
fluorescence quenching effect of Cu by electron transfer. The
high affinity of H GFP with Cu (K , 16.2 nM) leads to rapid
detection of Cu in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H GFP/
Cu complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity.
2
+
3
9
d
2+
3
9
2+
2
+
The assay was based on the reaction between Cu and thiocholine, the hydrolysis product of ATCh by AChE. The proposed
−1
sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL ) and is feasible for screening inhibitors of AChE.
Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food
samples. Hence, the successful applications of H GFP in the detection of metal ion and enzyme activity present the prospect of
3
9
resurfaced proteins as versatile biosensing platforms.
rotein resurfacing is an important technique in protein
engineering, which consists of rationally designing a
coordination property, which is expected to be promising in
the development of new biosensing methods.
Recently, the utilization of proteins to develop sensors has
P
protein’s surface while retaining the overall fold, core domain,
1
and boundary regions of the intact protein. Protein resurfacing
attracted much interest, and now several researchers have
2
is especially useful in altering protein−protein interaction,
attempted to exploit fluorescent protein (FP)-based metal ion
3
11−13
modifying antibodies, and endowing a protein with new
functionalities. For example, supercharged green fluorescent
biosensors.
Generally, the metal-binding property is
4
14,15
acquired by introducing natural (e.g., histidine)
or
protein (ScGFP) with high surface net charge has been
developed by resurfacing of GFP. ScGFP shows exceptional
noncanonical amino acid residues (e.g., 3,4-dihydroxy-L-
phenylalanine) with chelating capability into FP via genetic
5
16
solubility and stability against protein aggregation and potent
engineering approaches. The binding site of metal ion is close
to the chromophore of FP; thus, the complexation of metal ion
results in the fluorescence quenching of FP. However, owing to
single metal ion-binding site and correspondingly low binding
5
−7
ability to deliver biomacromolecules into cells,
exhibiting
great promise for application in medicine, biotechnology, and
8
even materials science. Recently, H GFP, a new kind of
3
9
−7
−5
ScGFP containing as many as 39 histidine residues in the
primary sequence, was developed by further resurfacing of
affinity (K , 10 −10 M), the sensitivity of these FP-based
d
sensors is still inefficient and needs to be improved. Multivalent
binding has been proven to be an effective way to improve
9
ScGFP. Owing to their intrinsic fluorescence, robust stability,
17,18
abundant surface functional groups, and cell-penetrating ability,
it will be attractive to adopt the resurfaced GFPs as biosensors.
However, the application of ScGFP in biosensing is scarce so
far. Lately, our group has developed a versatile ScGFP-based
biosensing platform for DNA detection and CpG methylation
binding affinity and capability in antibody and drug design.
Hence, it is reasonable to expect that highly sensitive detection
of metal ions would be achieved if H39GFP was used as a
biosensor probe, because so many histidine residues on the
1
0
analysis. Because of its multiple histidine residues, the
newcomer of ScGFPs, H GFP, possesses several advantages,
Received: November 24, 2014
Accepted: January 6, 2015
3
9
such as pH-mediated surface multicharge and metal ion
©
XXXX American Chemical Society
A
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Article
surface of H GFP provide multivalent binding sites for metal
ultrapure water (18.2 MΩ·cm) from Millipore (Milli-Q)
system.
39
ions.
Copper is an essential trace element in the human body, and
Cloning, Protein Expression, and Purification. The
19
it plays an important role as an enzymatic cofactor. Imbalance
of copper homeostasis leads to many pathological sequences,
including Alzheimer’s, Menkes, and Wilson’s diseases as well as
amino acid sequence of H GFP was referred to the reported
3
9
9
literature. The gene sequence encoding H GFP (h gfp) was
39
39
reversely translated from the amino acid sequence of H GFP
3
9
20
tumor development and progression. Copper is also widely
used in many industrial processes and has become one of the
major components of environmental heavy metal ions
and optimized for E. coli codon usage. The full-length gene was
synthesized and inserted in plasmid pUC19 by Takara (Dalian,
China). Then h gfp was transferred from pUC19-h gfp to
39
39
2
1
pollution. Currently available methods, such as atomic
pET28a. The reconstructed plasmid pET28a-h gfp was
39
22
absorption spectroscopy and inductively coupled plasma
transformed into E. coli BL21 DE3 with electroporation.
Cells were cultured inLuria broth (LB) medium at 37 °C
until OD600 (optical density at 600 nm) reached ∼0.6 and then
were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) at 30 °C for 3 h for protein expression. After being
harvested by centrifugation and resuspended in buffer (10 mM
Tris pH 7.4 and 2 M NaCl), cells were lysed by sonication.
Protein was purified by Ni-NTA (NTA = nitrilotriacetic acid)
2
3
mass spectroscopy, are complicated and sample-destructive
for determination of copper levels in both biological and
environmental samples. Therefore, several FP-based sensors
12−14,16
have been proposed for measuring the level of copper.
However, most of these biosensors only give satisfactory
2
+
responses at high concentrations of Cu (>20 μM), which is
not sufficient in practice.
Acetylcholinesterase (AChE) is a critical enzyme in the
central and peripheral nervous system, which has pivotal
agarose chromatography (A
exchanged into 10 mM Tris, 100 mM NaCl, and pH 7.4 by
desalination chromatography (AKTA, GE). The purified
H GFP was quantified by using the improved Bradford
̈
KTA, GE), and then the buffer was
24
functions in Alzheimer’s disease and nerve agent poisoning.
AChE inhibitors that penetrate the blood−brain barrier are
̈
39
25
useful in the symptomatic treatment of Alzheimer’s disease.
protein assay dye reagent kit with bovine serum albumin
In addition, pesticides including carbamates and organo-
phosphorus compounds cause the irreversible inhibition of
AChE activity, leading to acute toxicity on human health.
(BSA) as the standard and was stored at −20 °C.
2
+
Typical Experimental Process for Cu Detection. A 10
μL solution of Cu2+ with different concentrations was mixed
with 20 μL of 5× buffer (1× buffer: 10 mM 2-(N-
morpholino)ethanesulfonic acid (MES), pH 7.0) and replen-
ished to 90 μL by H O. Then 10 μL of H GFP (1 μM) was
26
Therefore, many efforts have been made to probe the activity of
AChE and to screen its potential inhibitors. Ellman’s
colorimetry is the technique mostly applied in the detection
2
39
27
of AChE activity but is limited by its low sensitivity. In recent
added to a final volume of 100 μL and incubated at room
temperature for 5 min. Fluorescence spectra of each sample
were recorded on an F-4500 fluorescence spectrophotometer
28
years, nanomaterials, such as quantum dots, gold nano-
2
9
30
particles, and fluorescent conjugated polymers, have also
been employed for the detection of AChE activity. However,
certain drawbacks still exist, such as multistep operations,
sophisticated probe preparation, and long measuring time.
Therefore, a novel label-free, simple, sensitive, and selective
method for AChE activity and inhibitor monitoring is highly
desirable.
(
Hitachi, Japan). The excitation wavelength was 480 nm, and
the emission wavelengths were in the range from 500 to 600
nm with both excitation and emission slits of 5 nm.
H GFP-dropped agarose hydrogels were prepared as
3
9
follows: 100 mg of low-melting-point agarose was dissolved
in 10 mL of water under microwave heating to produce 1%
agarose, and then the solution was cooled to 50 °C by water
bath. H GFP (133 μL, 15 μM) was mixed with 1% agarose to
Herein, we present a simple and novel method for metal ion
assay using H GFP as a fluorescent probe. Benefiting from the
39
39
multivalent metal ion-binding sites of H GFP and the
3
9
a final volume of 1 mL at 50 °C. Subsequently, an aliquot of 40
μL of the mixture was added into the mold (cap of 0.5 mL
Eppendorf tube) and then was kept at 4 °C for 15 min to form
the hydrogels. The H GFP-dropped agarose hydrogels were
2
+
fluorescence quenching effect of Cu , this method provides a
label-free, rapid, and highly sensitive detection of Cu .
Moreover, this H GFP-based Cu assay is further applied to
2
+
2
+
39
39
analyze AChE activity and AChE inhibitors by using
acetylthiocholine (ATCh) as the substrate. In this assay,
thiocholine (TCh), generated from the hydrolysis of ATCh by
immersed in 1 mL of tap water samples containing varying
concentrations of Cu2+ at room temperature for 30 min. The
fluorescence was visually observed under UV light, and the
images were taken by a Canon digital camera.
2
+
AChE, reacts with Cu to form a Cu(I)−TCh complex. As a
result, fluorescence of H GFP is restored and provides a facile
3
9
AChE Activity Assay. Four μL of 5× buffer, 1 μL of ATCh
10 mM), and 2 μL of AChE with various concentrations were
switch-on assay for AChE activity detection. This work
demonstrates that H GFP, a resurfaced GFP, can be used as
(
3
9
mixed in a final volume of 20 μL and incubated at 37 °C. Thirty
a novel and versatile biosensing platform for the label-free
detection of metal ion and enzyme activity, which will inspire
the exploration of more widespread applications of engineered
proteins in analytical chemistry.
2+
min later, 70 μL of 1× buffer containing 1 nmol Cu and 10
μL of H GFP (1 μM) were added to a final volume of 100 μL
39
and incubated at room temperature for 5 min. Fluorescence
spectra of each sample were recorded on an F-4500
fluorescence spectrophotometer.
Inhibitor Screenings. For tacrine, AChE (0.5 mU) was
incubated with different concentrations of tacrine in a final
volume of 19 μL (1× buffer) at 37 °C for 5 min. Then 1 μL of
ATCh (10 mM) was added and incubated at 37 °C for 30 min.
EXPERIMENTAL SECTION
■
Materials. Acetylcholinesterase (AChE, from Electrophorus
electricus), acetylthiocholine (ATCh), and tacrine were
purchased from Sigma-Aldrich (Shanghai, China). Carbaryl
was obtained from Agro-Environmental Protection Institude,
Ministry of Agriculture (Tianjin, China). All other chemicals
were of analytical grade. All solutions were prepared using
2
+
After that, 70 μL of 1× buffer containing 1 nmol Cu and 10
μL of H GFP (1 μM) were added to a final volume of 100 μL
39
and incubated at room temperature for 5 min. Fluorescence
B
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Article
spectra of each sample were recorded on an F-4500
fluorescence spectrophotometer.
For carbaryl, AChE (0.2 mU) was incubated with different
concentrations of carbaryl in a final volume of 19 μL (1×
buffer) at 37 °C for 10 min. The following process was the
same as that of tacrine.
the fluorescence emission of H GFP by electron transfer.
39
Figure 1B displays the fluorescence emission spectra of H GFP
3
9
2+
2+
with or without the presence of Cu . The addition of Cu (10
μM) induced a significant decline in the fluorescence emission
of H GFP (decreased ∼98%), which indicated that H GFP
3
9
39
2+
can be used as a sensor for Cu detection. Furthermore, the
addition of ethylenediaminetetraacetic acid (EDTA) (a
stronger chelator for Cu ) could completely restore the
Analysis of Carbaryl Residues in Food Samples. Apple,
tomato, and cucumber were chosen as the sample matrixes to
evaluate the carbaryl residue levels in the real application tests
of this pesticide assay. A standard carbaryl solution (100 mg
2+
fluorescence of H GFP, implying that the interaction between
3
9
2+
Cu and H GFP is reversible.
39
−1
2+
L ) was sprayed onto skins of the food samples. Twenty-five g
of each sample were collected, chopped, and crushed to
homogenate. Then 40 mL of acetonitrile was added, and the
resulting mixture was filtered through a 0.22 μm membrane to
remove the insoluble materials. The obtained filtrate was dried
on a water bath. After that, methanol was added to a final
volume of 5 mL (directly analyzed by high-performance liquid
chromatography (HPLC)). One mL of the resulting solution
To obtain a better sensing performance of H GFP for Cu ,
39
some important issues were investigated. The fluorescence
intensities at different incubation times were recorded and
investigated as the dynamic monitoring of the interaction
2+
between Cu and H GFP. As shown in Figure S-2, Supporting
39
Information, the fluorescence of H GFP decreases quickly in
39
the first 1 min (decreased ∼87%) and then gradually decreases
with the incubation time increasing and tented to a saturation
point at 5 min. This quick and high-efficiency fluorescence
quench indicated that the proposed method was rapid and
sensitive for Cu2+ detection. Additionally, the effect of pH was
also examined. Fluorescence of H GFP and quenching
was dried again, and H O was added to a final volume of 1 mL.
2
Ten μL of the solution was incubated with AChE (0.2 mU) in a
final volume of 19 μL (1× buffer) at 37 °C for 10 min. The
following procedure was the same as described in the Inhibitor
Screenings section.
3
9
efficiency kept stable as the pH of the buffer solution increased
from pH 6.0 to pH 8.0 (Figure S-3, Supporting Information),
showing that the proposed method can work well in the buffer
with neutral pH. Thus, the following experiments were
RESULTS AND DISCUSSION
Fluorescence Quenching of H GFP by Cu . H GFP
was designed, cloned, expressed, and then purified from E. coli,
■
2+
39
39
performed at pH 7.0 with an incubation time of 5 min.
9
2+
according to the published H GFP sequence. As shown in
Cu Sensing Based on H GFP. Figure 1C shows the
3
9
39
2+
Figure 1A and Figure S-1, Supporting Information, there are 39
mechanism for Cu detection based on H GFP. The proximal
39
histidine residues on the surface of H GFP provide multiple
3
9
2+
2+
binding sites for Cu . After binding of Cu on the surface, the
fluorescence emission of H GFP is quenched by Cu . On the
basis of this principle, detection of Cu in aqueous solution
was studied under preoptimized conditions. When the
concentration of Cu2+ increased from 0.05 to 15 μM, the
fluorescence of H GFP decreased continually and was almost
2
+
39
2
+
39
2+
completely quenched by 10 μM Cu (Figure 2A). According
to the Cu2+ concentration-dependent quenching curve, the
dissociation constant value (K ) of 16.2 nM between H GFP
d
39
and Cu2+ could be estimated by least-squares curve fitting
34
analysis (details are shown in Supporting Information). By
2
+
comparison, the FP-based sensors with a single Cu -binding
site show lower fluorescence quench rates (<75%) and much
lower affinity with Cu2+ (as shown in Table 1). From the Cu
concentration-dependent calibration curve, a good linear
2+
2
relationship (R = 0.993) in the concentration ranging from
0
.05 to 2.0 μM was obtained (inset in Figure 2B), and the
lowest detection limit was 50 nM. The LOD of the proposed
Figure 1. (A) Overall structure of H GFP. (B) Fluorescence spectra
assay is much lower than those of many of the previous
3
9
2
+
2+
11−13,16
of H GFP (curve 1), H GFP−Cu (curve 2), and H GFP/Cu /
reported FP biosensors (Table 1),
which could be
3
9
39
39
2+
EDTA (curve 3) in the buffer solution. [H GFP] = 100 nM, [Cu ] =
0 μM, and [EDTA] = 20 μM. (C) Principle of H GFP-based Cu
39
attributed to the multivalent binding sites and the correspond-
2+
1
2+
39
ing higher affinity of H GFP toward Cu .
39
sensor.
To examine the specificity of the sensor, the fluorescence
2
+
responses of H GFP toward various metal ions including Cu ,
3
9
2+
2+
2+
+
2+
2+
3+
2+
2+
2+
3+
histidine residues in the primary sequence of the H GFP
Zn , Co , Mn , Ag , Cd , Pb , Al , Ni , Ca , Fe , Fe ,
and Hg2+ were studied under the same conditions; the final
concentration of the ions was 10 μM. It can be seen that only
39
molecule, and most of them are proximal to the surface of the
H GFP molecule. It is known that histidine has a strong
3
9
2
+
2+
2+
2+
tendency to chelate Cu , and Cu is one of the best known
fluorescence quenchers, which could induce the fluorescence
quenching of organic fluorescent dyes and FPs through
electron transfer.
Cu with H GFP will result in Cu binding with the histidine
Cu and Hg caused dramatic fluorescence quenching, and
there were no obvious fluorescence changes for the other metal
ions (Figure 3A). Comparatively, the fluorescence quenching
induced by Hg is much weaker as compared with that by
Cu , and the fluorescence response to Hg can be masked
3
1−33
2+
Therefore, it is expected that mixing
2
+
2+
2+
2+
39
−
residues on the surface of H GFP and subsequently quenching
upon the use of Cl as a coadditive. Moreover, the coexistence
3
9
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2
+
Figure 3. (A) Selectivity of the H GFP-based Cu sensing system.
39
2+
The concentrations of Cu and other metal ions were 10 μM, and the
concentration of Cl was 50 mM. (B) Visual detection of Cu (0, 5,
0, and 20 μM Cu ) using H GFP-containing agarose hydrogels
−
2+
2+
1
39
under visible light (I) and UV light (II).
Visual detection by naked eyes is preferable for practical
Figure 2. (A) Fluorescence spectra of H GFP (100 nM) in the
applications. It was reported that this H GFP can refold and
39
39
presence of Cu2 with varying concentrations. (B) Fluorescence
+
9
recover its fluorescence when boiled and cooled at pH 4−5.
We found that H GFP kept 89% of its fluorescence after 9
2+
responses of H GFP to Cu with varying concentrations. F and F0
39
39
are fluorescence intensities of H GFP in the presence and absence of
3
9
months at −20 °C (data not shown). Such exceptional
2+
2+
Cu , respectively. Inset shows the linear responses to Cu ranging
solubility and stability of H GFP gave us the chance to exploit
3
9
from 0.05 to 2 μM.
2
+
the application of the sensor for visual detection of Cu .
Immobilizing H GFP into hydrogels is a reasonable solution
39
Table 1. Comparisons between H GFP-Based Sensor and
3
9
2
+
because these materials have high loading capacity and low
Other FP-Based Biosensors in Cu Detection
background. The large molecular weight (∼27.4 kDa) of
a
probe
Kd/μM
LOD/μM
QE
ref
H GFP could restrict the diffusion and prevent the release of
39
mEmerald-3H
0.2
0.1
0.5
0.1
1.0
0.05
∼75%
∼15%
∼40%
∼35%
∼98%
11
12
13
16
H GFP from the hydrogels, and the accumulative properties of
39
His GFP
17
functional hydrogels allow it to be used for visual detection of
6
2+
Rum13
10.9
5.6
Cu . To achieve this purpose, H GFP-dropped agarose
39
GFPdopa
H GFP
hydrogels were prepared. Because of the superb stability of
9
0.0162
this work
H GFP (e.g., the ability to refold after boiling ) and low
39
39
a
Fluorescence quenching rate in the presence of 10 μM Cu2+.
melting temperature of low-melting-point agarose, the gelation
process has no effect on the optical properties of H GFP, and
3
9
the transparent agarose hydrogels emitted strong green
fluorescence under UV irradiation, indicating that the
H GFP-containing agarose hydrogels were successfully pre-
of other metal ions showed no obvious interference for Cu2+
assays (Figure S-4, Supporting Information). Metal ions, such
as Ni , Zn , and Co could also chelate histidine residue and
39
2+
2+
2+
pared. The fluorescence of hydrogels under UV lamp grew
are widely used in immobilized metal affinity chromatog-
fainter with the increase of Cu2+ concentration and disappeared
3
5
2+
raphy. However, the binding affinity between histidine
after the treatment with 20 μM Cu (Figure 3B). The United
2+
2+
residue and Cu is much stronger than with these metal
States defined maximum contaminant level for Cu in tap
3
6
2+
ions, and only Cu shows remarkable fluorescence
water is 20 μM; therefore, these H GFP-containing agarose
39
2
+
2+
2+
2+
quenching effect. Thus, these ions (Ni , Zn , and Co ) do
not cause interference to Cu detection in the H GFP-based
hydrogels are suitable for instrument-free visual Cu detection
and water quality control under practical conditions.
2
+
3
9
assay. Furthermore, to evaluate the practical application of this
AChE Activity Detection Based on H GFP. The
3
9
2
+
new assay, the response of H GFP to Cu in urine (10%, v/v)
function of H GFP as a biosensing platform was further
3
9
39
was investigated, and it was found that fluorescence response
expanded by exploring its application in enzymatic activity
2
+
was linearly associated the concentration of Cu ranging from
.1 to 10 μM (Figure S-5, Supporting Information). The
analysis. Taking advantage of the fact that the fluorescence of
2
+
0
H GFP can be effectively quenched by Cu , a novel
39
2
+
concentrations of Cu in tap water and urine samples were
detected by applying a standard addition method, and
satisfactory accuracy and precision were achieved (as shown
in Table S-1, Supporting Information).
fluorescent method for AChE activity assay was proposed.
The sensing mechanism is shown in Figure 4A. Acetylthiocho-
line (ATCh), an analogue of acetylcholine (the natural
substrate of AChE), can be hydrolyzed by AChE into
D
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Article
2+
Figure 4. (A) Mechanism of the H GFP/Cu -based fluorometric
39
assay for AChE activity detection. (B) Fluorescence spectra of
2
+
2+
H GFP/Cu (curve 1), H GFP/Cu with ATCh (curve 2),
3
9
39
Figure 5. (A) Fluorescence spectra of H GFP/Cu2+ at various
2+
2+
H GFP/Cu with AChE (curve 3), and H GFP/Cu with both
39
3
9
39
−1
concentrations of AChE. Inset A shows the fluorescence spectra of
ATCh and AChE (curve 4). [ATCh] = 50 μM, [AChE] = 5 mU mL .
2
+
H GFP/Cu at low concentrations of AChE. (B) Relationship
39
2+
between the fluorescence intensity of H GFP/Cu and the AChE
39
thiocholine (TCh). TCh, a kind of mercaptans, can react with
concentration. Inset B shows the linear responses to AChE ranging
2+
Cu to form the Cu(I)−TCh complex by the well-known
−1
from 0.025 to 2 mU mL . F and F are the fluorescence intensities of
0
37
reaction of copper with mercaptans. The formation of
2+
H GFP/Cu in the absence and presence of AChE, respectively.
39
2
+
Cu(I)−TCh complex prevents Cu from binding H GFP.
39
2
+
As a result, electron transfer between H GFP and Cu will not
3
9
occur and H GFP will emit intense fluorescence upon
of fluorescence response versus the concentration of AChE is
3
9
−1
irradiation. However, in the presence of AChE inhibitors, the
hydrolysis of ATCh is inhibited and TCh will not be generated,
shown in Figure 5B with a plateau point at 5 mU mL of
2
AChE. A good linear range (R = 0.992) from 0.025 to 2 mU
and Cu2 will interact with H GFP to form the H GFP−Cu
+
2+
−1
mL was obtained as the inset of Figure 5B shows. The assay
3
9
39
−1
complex, which leads to the quenching of H GFP’s
allowed the detection of AChE as low as 0.015 mU mL (S/N
= 3), which is much lower than many previously reported
AChE assays (detailed comparison is shown in Table S-2,
3
9
fluorescence. Therefore, AChE activity and its inhibitor can
be reflected by the fluorescence intensity change of H GFP.
39
2
9,38−41
Following the design, the feasibility of the proposed method
was investigated. Figure 4B depicts typical fluorescence
response of this AChE assay. After the incubation of AChE
Supporting Information).
To evaluate the specificity of
the proposed fluorometric assay toward AChE, other enzymes,
including horse radish peroxidase (HRP), carboxypeptidase Y
(CPY), and protein kinase A (PKA), were investigated as
control samples. Although the concentrations of these non-
specific enzymes were much higher than that of AChE, none of
them can induce remarkable fluorescence response in the assay
as AChE did (Figure S-7, Supporting Information). This result
2
+
with its substrate ATCh, Cu and H GFP were added for
3
9
detection. In comparison with the control sample (H GFP/
3
9
2
+
Cu ), the AChE-catalyzed reaction caused a significant
fluorescence enhancement of H GFP, which is ∼38-fold as
high as that of H GFP/Cu . However, either AChE or ATCh
3
9
2
+
39
2
+
caused negligible fluorescence responses relative to the control
clearly demonstrated that this assay based on H39GFP/Cu
2
+
sample (H GFP/Cu ). Thus, the hydrolysis product of ATCh
could be selective for the targeted detection of AChE activity.
AChE Inhibitors Screening. To further demonstrate the
potential application of this method in the inhibition assay, the
experiments were performed in the presence of the AChE
inhibitors. Tacrine, a well-known AChE inhibitor, which has
been used for the treatment of Alzheimer’s disease, was
selected. The inhibition efficiency (IE) was determined by the
following equation:
39
by AChE inhibited the binding of copper ion with H GFP,
39
resulting in a remarkable restoration of the fluorescence and
providing a “turn-on” detection of AChE. The optimal values
for the concentration of ATCh and hydrolysis times of AChE
were found to be 100 μM and 30 min, respectively (Figure S-6,
Supporting Information). Under the optimized conditions, the
dynamic response range of AChE in buffer solution was
recorded through fluorescence changes of H GFP. When the
3
9
IE (%) = 100 × (F − F)/(F − F )
(1)
−1
i
0
concentration of AChE increased from 0 to 10 mU mL , the
fluorescence intensity of H GFP was continually increasing
where F is the fluorescence intensity of the initial H GFP/
0 39
3
9
2+
(Figure 5A), implying a gradual hydrolysis of ATCh. The plot
Cu complex. F is the fluorescence intensity with AChE, and F
i
E
DOI: 10.1021/ac504390e
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
is that with both AChE and inhibitor. On the basis of the plot
of inhibition efficiency versus the concentration of tacrine
method can be successfully applied to detect pesticide residues
in real samples.
(
Figure S-8, Supporting Information), the corresponding IC50
value (the inhibitor concentration required for 50% inhibition
of the enzyme activity) was estimated to be 3.5 nM, which is
similar to those obtained by other AChE assays.
CONCLUSION
■
42
In conclusion, we have developed a simple and label-free
2
+
biosensor based on H GFP for the detection of Cu . The
39
Carbamate pesticides are also known as inhibitors of AChE.
Carbamate pesticides exhibit toxicity on human health and
distribute through their residues in agricultural products or in
drinking water. Carbaryl was employed to evaluate the viability
of this method to the detection of carbamate pesticides. The
relationship between inhibition efficiency and the carbaryl
concentration is shown in Figure 6A. The inhibition efficiency
binding of Cu2+ induces quick and dramatic fluorescence
quenching of H GFP, which makes this method rapid and
facile, proved by its “mix-and-read” property and fast detection
in 5 min, with excellent selectivity and high sensitivity (LOD =
39
5
0 nM). The H GFP-based sensor shows multifaceted
39
advantages over previously reported FP-based copper ion
sensors, such as higher binding affinity, better quenching
efficiency, and lower detection limit, because of its multivalent
2
+
binding of Cu . Furthermore, H GFP-containing agarose
39
2
+
hydrogels could be used for visual detection of Cu in tap
2+
water. Moreover, H GFP−Cu interaction is further utilized
39
2
+
to detect AChE activity based on the reaction of Cu with the
product of AChE-catalyzed ATCh hydrolysis. This homoge-
neous approach allows a low detection limit of AChE (0.015
−1
mU mL ) and is feasible for not only AChE inhibitor
screening but also for carbamate pesticides probing in
agricultural products. The present study provides a new
function of resurfaced FPs, as a versatile biosensing platform
for the label-free detection of metal ion and enzyme activity.
This work shows the prospect of the engineered proteins by
resurfacing as a promising molecular toolkit in bioanalytical
chemistry.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Additional information including extensive figures as noted in
text. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*
Tel.: +86-731-88821626; fax: +86-731-88821848; e-mail
address: huangyan.hnu@gmail.com.
Figure 6. (A) Inhibition efficiency versus the logarithm of the carbaryl
Notes
concentration. (B) Analytical results of carbaryl in apple, tomato, and
2+
The authors declare no competing financial interest.
cucumber by using H GFP/Cu -based method (black) and by using
3
9
HPLC (gray).
ACKNOWLEDGMENTS
■
was linearly dependent on the logarithm of the carbaryl
This work was financially supported by the National Natural
Science Foundation of China (nos. 21475037, 21190044,
1235002, and 21222507), the National Basic Research
Program of China (973 Program, no. 2011CB911002), and
the Foundation for Innovative Research Groups of NSFC
−8
−4
−1
concentration ranging from 1 × 10 to 1 × 10 g L , and the
corresponding linear equation is IE (%) = 21.644 log[carbaryl]
2
−1
2
(
g L ) + 181.109 (R = 0.993). The limit of detection (defined
as the concentration of inhibitor required to achieve 5%
4
3
−9
−1
inhibition ) was 7.03 × 10 g L (0.007 ppb), which is much
(
Grant 21221003).
lower than the maximum residue limits in the European Union
(
50 ppb) and United States (20 ppb) pesticides data set, as well
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■
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