Phenylsulfonic acid functionalized carbon quantum dots based biosensor for acetylcholinesterase activity monitoring and inhibitor screening

Article abstract of DOI:10.1039/c6ra18978d

Phenylsulfonic acid functionalized carbon quantum dots (PSA-CQDs) were prepared and used to construct a convenient and reliable fluorometric biosensor for acetylcholinesterase (AChE) activity and inhibitor screening. Effective static quenching process occurred owing to the formation of nonfluorescent complex when copper(ii) ions were added to PSA-CQDs, which results in its fluorescence quenching due to strong coordination of copper ions to sulfonic groups of PSA-CQDs. As a result, the fluorometric assay for AChE was established based on the stronger affinity between copper and thiocholine hydrolyzed from acetylthiocholine under the hydrolysis of AChE which can induce fluorescence recovery. Meanwhile, the inhibition effect of tacrine to AChE was assessed because its effective inhibition effect to AChE leads to the prevention of hydrolysis of acetylthiocholine and the following occurrence of PET process which causes the quenching of fluorescence. This detection method is convenient and efficient, which has high sensitivity with detection limit as low as 2.6 U L^-1 and a broad linear scope ranging from 3.8 to 80.0 U L^-1. This work proposed a competitive assay strategy among the sulfonic acid modified probe, copper cation and thiocholine to quantify AChE activity using functional carbon quantum dots, and can be utilized to design detection methods for other thiol-containing targets of interest.

Full text of DOI:10.1039/c6ra18978d

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Phenylsulfonic acid functionalized carbon quantum dots based
biosensor for acetylcholinesterase activity monitoring and
inhibitor screening
Received 00th January 20xx,
Accepted 00th January 20xx
Fengqi Zhou,† Hui Feng,† Yafen Fang, Qian Sun, and Zhaosheng Qian*
DOI: 10.1039/x0xx00000x
Phenylsulfonic acid functionalized carbon quantum dots (PSA-CQDs) were prepared and used to construct a convenient
and reliable fluorometric biosensor for acetylcholinesterase (AChE) activity and inhibitor screening. Effective static
quenching process occurred owing to the formation of nonfluorescent complex when copper (II) ions were added to PSA-
CQDs, which results in its fluorescence quenching due to strong coordination of copper ions to sulfonic groups of PSA-
CQDs. As a result, the fluorometric assay for AChE was established based on the stronger affinity between copper and
thiocholine hydrolyzed from acetylthiocholine under the hydrolysis of AChE which can induce fluorescence recovery.
Meanwhile, the inhibition effect of tacrine to AChE was assessed because its effective inhibition effect to AChE leads to the
prevention of hydrolysis of acetylthiocholine and the following occurrence of PET process which causes the quenching of
fluorescence. This detection method is convenient and efficient, which has high sensitivity with detection limit as low as
2.6 U/L and a broad linear scope ranging from 3.8 to 80.0 U/L. This work proposed a competitive assay strategy among the
sulfonic acid modified probe, copper cation and thiocholine to quantify AChE activity using functional carbon quantum
dots, and can be utilized to design detection methods for other thiol-containing targets of interest.
www.rsc.org/
Several methods have been applied to the monitor the activity of
AChE and to screen its potential inhibitors, such as colorimetric,
Introduction
electrochemical,
interferometric,
chemiluminometric,
and
Acetylcholinesterase (AChE) is a key enzyme in the human
central nervous system, which catalyzes the hydrolysis of
acetylcholine (ACh) to choline to maintain the level of
neurotransmitter acetylcholine.1 Researches have indicated that in
the athogenesis of Alzheimer’s disease (AD), AChE plays an
important role because hydrolytic decomposition of ACh in the
nervous system by AChE can accelerate the assembly of amyloid β
peptides into amyloid fibrils.2 As a result, fast and effective
detection of AChE is important in diagnosis of Alzheimer’s disease.
On the other hand, AChE inhibitors had proven to be the principal
drugs in the symptomatic treatment of Alzheimer’s disease.3 Thus
seeking the potential inhibitors and screening of their inhibition
effect have great significance in the drug development for AD
treatment. Due to the wide range fields of pharmaceutical and
health care application, there is an urgent demand for convenient
and sensitive methods for real-time AChE activity monitoring and
AChE inhibitor screening.
fluorometric approaches.4 Among these methods, two traditional
methods including colorimetric method by using Ellman’s reagent5
and the detection of hydrogen peroxide produced by oxidation of
the AChE-induced choline6 8 are widely applied in detection of AChE
–
activity. However, both the two traditional methods have
disadvantages owning to the facts that they lack sufficient
sensitivity or require time-consuming procedures, and furthermore
Ellman’s reagent might result in false-positive effect. In order to
enhance the sensitivity, many methods such as chemiluminometric
or fluorometric approach based on organic compounds have been
employed to establish assays of AChE activity and AChE inhibitors
12
–
evaluation,9
which provide higher sensitivities and lower
detection limits. However, dye molecules have their inherent
disadvantages including rapid bleaching, insufficient emission
intensities and instability. In recent years, several new fluorescent
materials such as fluorescent conjugated polymers or proteins,13
15
–
inorganic quantum dots16,17 and noble metal nanoparticles18 22 have
been developed and employed for AChE activity and inhibitor
monitoring. Although these fluorescent materials with high
fluorescence quantum yields, strong absorption and size-tunable
emissions, have shown good performance in AChE activity
monitoring, simple low-cost, label-free and sensitive assays for
AChE activity and inhibitor monitoring in biological samples and for
–
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discovering new drugs are still in demand.
Carbon quantum dots (CQDs), as
mixture of concentrated sulfuric acid (180 mL) and nitric acid (60
kind of luminescent mL). The mixture was heated at 80◦C for 5 h. The mixture was
a
nanomaterial, have shown their distinctively optical properties and cooled and diluted with deionized water (800 mL). The dark CQDs
outstanding performance in photovoltaic devices, photocatalysis solution was neutralized with sodium hydroxide. The final product
25
and bioimaging23
due to their outstanding advantages over solution was dialyzed in a dialysis bag (1000 Da) for 3 days, and then
–
organic dyes, semiconductor quantum dots and noble metal the resultant solution was further treated with the dialysis bag
nanoclusters, such as stable light emitting, high quantum yield, (50,000 Da) to remove large non-fluorescent materials.
good photostability, easy modulation, low toxicity and excellent
The synthesis of PSA-CQDs was as follows: CQDs powder (0.05 g)
biocompatibility. Recently, carbon quantum dots as an excellent was dissolved in deionized water (30 mL), and then a certain
fluorophore were utilized to design novel fluorescent amount of EDC and NHS (0.5 g) were added to activate the carboxyl
chemo/biosensors in vitro and in vivo,26 and begun to be applied groups on the surface of CQDs for 1 h. An excessive amount of 4-
28
–
to the detection of enzymes activity evaluation.29-33
amino-phenylsulfonic acid (0.5 g) was added to the preceding
In this study, a fluorometric assay for acetylcholinesterase activity solution after the pH was adjusted to 7. This reaction would
monitoring and inhibitors screening was developed based on a proceed for over 7 days at room temperature to obtain PSA-CQDs
competitive interaction strategy among functionalized carbon nanoprobe with excellent optical property. In order to accelerate
quantum dots (CQDs), copper ion and thiochioline. Carbon quantum the formation of amide bonds between CQDs and 4-amino-
dots abundant in carboxyl group were used to prepare phenylsulfonic acid, the resulting solution was heated at 90 °C for 3
phenylsulfonic acid functionalized carbon quantum dots (PSA-CQDs). days. Finally, as-prepared PSA-CQDs nanoprobe was purified with
The phenylsulforic acid groups on the surface of PSA-CQDs function microporous filter (0.22 ꢀm) and a dialysis bag (1000 Da) for 48 h to
as the functional unit to coordinate with metal ions. The remove molecular substance including 4-amino-phenylsulfonic acid,
fluorescence of PSA-CQDs was found to be quenched in the EDC and NHS. As-prepared PSA-CQDs nanoprobe was obtained and
presence of copper ions (Cu2+) owing to the formation of further used in the following detection.
nonfluorescent PSA-CQDs/Cu(II) complex. Acetylthiocholine (ATCh)
was used as the substrate of AChE, and can be hydrolyzed to
thiocholine under the catalysis of AChE. The generated thiocholine
has stronger affinity to copper ion than phenylsulfate ion, and thus
can bind to copper ion to form a more stable complex. As a result,
the quenched fluorescence can be recovered in the presence of
ATCh and AChE . By taking advantage of competitive strategy among
copper ion, carboxyl group on the surface CQDs and thiocholine,
and specific catalytic hydrolysis of ATCh into thiocholine by AChE,
the fluorescent assay for AChE was established. Moreover, the
function of inhibitors screening for AChE was further assessed using
tacrine as an example.
Condition optimization for fluorescence quenching and recovery
of PSA-CQDs
For optimization of incubation time for fluorescence quenching
by copper (II) ions, 2.0 μL of copper(II) ion solution (10.0 mM) was
added into 2.50 mL of PSA-CQDs solution (HMT, pH 7.0). Under the
optimum incubation time, fluorescence quenching of PSA-CQDs was
assessed with continuous addition of copper ions. The fluorescence
intensity of the mixtures containing PSA-CQDs and varying amounts
of Cu2+ was monitored using fluorescence spectrometer at the
optimal excitation wavelength. For optimization of incubation time
for catalytic hydrolysis of acetylthiocholine by AChE, 370.0 μL of
acetylthiocholine (10.0 mM) and 15.0 μL of AChE (100.0 U/mL) were
incubated for 10, 20, 30, 40, 50, 60, 70 and 80 min respectively, and
then mixed with PSA-CQDs/Cu(II) solution, and finally its
fluorescence was recorded after 15 min of incubation time. For
optimization of incubation time for fluorescence recovery by
thiocholine generated from catalytic hydrolysis of acetylthiocholine
by AChE, the fluorescence of PSA-CQDs was first quenched by
adding 3.0 μL of copper(II) solution (10.0 mM) into 2.50 mL of PSA-
CQDs solution (HMT, pH 7.0). Then, a certain amount of the mixture
of ATCh and AChE which has been incubated for 1 h was introduced
to the above mixture.
Experimental
Materials and reagents
Triple-distilled water was used throughout the experimental
process. Activated carbon, copper(II) nitrate (Cu(NO3)2),
acetylthiocholine (ATCh), 4-amino-phenylsulfonic acid (APSA), and
hexamethylenetetramine (HMT) were purchased from Aladdin Ltd.
(Shanghai, China). Acetylcholinesterase (AChE, EC 3.1.1.7, 2 kU),
alkaline phosphatase (ALP, EC 3.1.3.1) from bovine intestinal
mucosa, acid phosphatase (ACP, EC 3.1.3.2) bovine serum albumin Fluorescent AChE assay
(BSA), and immuneglobulin G (IgG) were bought from Sigma-Aldrich
(Shanghai, China). The concentration of hexamethylenetetramine
buffer solution (HMT, pH 7.0) was 10.0 mM. All reagents were of
analytical grade and without any further purification.
For the AChE assay, different levels of AChE ranging from 0.0 to
200.0 U/L were added into the mixture containing 2.50 mL of PSA-
CQDs solution (HMT, pH 7.0), Cu2+ (12.0 μM) and 370.0 μL of ATCh
(10.0 mM), and then the fluorescence spectra of the mixture were
recorded respectively after an incubation time of 10 min. Under the
optimal conditions, the selectivity of the assay toward AChE was
evaluated. Four biological species including bovine serum albumin
(BSA), immune globulin G (IgG), acid phosphatase (ACP) and alkaline
phosphatase (ALP) were selected to assess the selectivity of the
Synthesis of phenylsulfonic acid functionalized carbon quantum
dots (PSA-CQDs)
A brief procedure for synthesis of carbon quantum dots is
described as follows: activated carbon (2.0 g) was added into a
2 | J. Name., 2012, 00, 1-3
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sensing system to AChE. A certain amount of copper ion (5.0 μL, and Cu(II) possesses non-fluorescent nature due to static quenching
10.0 mM) was first added into 2.5 mL of PSA-CQDs solution. The mechanism, which is utilized to design the fluorescence Turn-on
fluorescence of the resulting mixture was regarded as the blank. assay for AChE activity. Acetylthiocholine (ATCh) was adapted as the
Then, each of the following species including AChE (15.0 μL, 100.0 substrate of AChE because it has been proved that ATCh can be
U/mL), BSA, ACP, IgG and ALP (15 μL, 100.0 U/mL) was separately effectively hydrolyzed to thiocholine and acetate under the catalysis
incubated with 370 μL of ATCh(10.0 mM) for 60 min, and then of AChE.9,14 Scheme 1 shows the schematic illustration of detection
mixed with PSA-CQDs/Cu(II) solution. The resultant mixture was strategy for AChE activity and its inhibitor screening on the basis of
monitored by fluorescence spectroscopy at the excitation PSA-CQDs nanoprobe mediated by copper ion. PSA-CQDs
wavelength of 425 nm after 20 min incubation.
nanoprobe exhibits strong green fluorescence, but the introduction
of copper ion leads to severe fluorescence quenching due to the
formation of non-fluorescent complex between PSA-CQDs and Cu(II).
No change can be observed in the presence of mere ATCh in PSA-
CQDs/Cu(II) complex solution. However, the introduction of both
ATCh and AChE into the preceding solution brings about substantial
fluorescence recovery, which is achieved by the fact that thiocholine
generated from ATCh under catalytic hydrolysis of AChE takes
copper ions away from PSA-CQDs/Cu(II) complex due to its stronger
affinity to copper ion, and this competitive binding causes
breakdown of PSA-CQDs/Cu(II) nanoassembly and resets CQDs in
their free state. The enhanced fluorescence correlates to varying
levels of AChE, and thus the assay for AChE level assessment can run
in this way. When its inhibitor tacrine is present, AChE activity
would be inhibited to loss its capability to catalytically hydrolyze
ATCh into thiocholine, and consequently the fluorescence of the
system remains quenched due to the absence of thiocholine. The
inhibition effect of tacrine on AChE activity can be assessed by
fluorescence quenching degree of the standard assay.
Inhibitor screening
For tacrine, different concentrations of tacrine from 0.0 μM to
10.0 μM was added into the mixture containing 2.50 mL of PSA-
CQDs solution (HMT, pH 7.0), Cu2+ (12.0 μM) and AChE (600.0 U/L),
incubating for 60 min. And then 370.0 μL of ATCh (1480.0 μM) was
added into the system. After an incubation time of 20 min, the
fluorescence spectra of the mixture were recorded respectively.
Characterization methods
The morphologies of all samples were characterized by
transmission electron microscopy (TEM), which was performed on a
JEOL-2100F instrument with an accelerating voltage of 200 kV. The
X-ray photoelectron spectroscopy analyses were conducted using a
Kratos Axis ULT RAX-ray photoelectron spectrometer with a 165 nm
hemisphereical electron energy analyzer. The UV–vis spectra were
recorded on
a PerkinElmer Lambda 950 spectrometer. The
photoluminescence spectra were conducted on a PerkinElmer LS-55
fluorescence spectrometer.
Synthesis and characterization of PSA-CQDs nanoprobe
The synthesis and characterization of CQDs were reported in our
previous paper.31,32 CQDs were prepared by means of chemical
oxidation of activated carbon powder using concentrated sulfuric
and nitric acids, and further purified by dialysis which retains CQDs
in size from 1 kDa to 50 kDa. As-prepared CQDs act as the starting
materials to synthesize the following nanoprobe. 4-amino-
phenylsulronic acid was covalently linked to the surface of CQDs via
EDC/NHS coupling reaction. PSA-CQDs nanoprobe shows bright
green fluorescence under the UV lamp. TEM images in Figure S1
indicate that PSA-CQDs nanoprobes have close size to CQDs in the
range of 2-5 nm, and also same crystalline structure to CQDs.
Comparison in XPS spectra between PSA-CQDs and CQDs in Figure
S2 illustrates that phenylsulfonic acid has covalently bonded to the
surface of CQDs due to the new signals at 398.7 eV, 167.6 eV and
230.7 eV from N 1s, S 2p and S 2s for PSA-CQDs with respect to that
of CQDs. High-resolution S2p XSP spectrum in Figure S2 further
prove the appearance of S element in the nanoprobe. IR spectra of
CQDs and PSA-CQDs in Figure S3 showed four new absorption peaks
at 2927.6 cm-1, 1603.9 cm-1, 1035.9 cm-1 and 696.6 cm-1 originated
from vibrations of N-H bond, C=O bond, S-O bond, and benzene ring
for PSA-CQDs with respect to that of CQDs, which further verifies
the covalent linkage between phenylsulfonic acid and CQDs. Figure
1A displays the fluorescence spectra of CQDs and PSA-CQDs
nanoprobe, respectively. A fluorescence emission peak at 500 nm
was recorded for PSA-CQDs nanoprobe when exited at the optimum
wavelength of 425 nm, which is slightly blue-shifted with respect
Scheme 1. Schematic illustration of detection strategy for AChE activity monitoring and
its inhibitor screening based on a competitive strategy.
Results and discussion
Principle of AChE activity monitoring and its inhibitor screening
based on PSA-CQDs
Carbon quantum dots were synthesized using cheap activated
carbon as raw materials through chemical oxidation approach
according to our previous papers.31,32 4-Amino-phenylsulfonic acid
was used to functionalized on the surface of carbon quantum dos to
prepare PSA-CQDs nanoprobe. It is found that phenylsulfonic acid
can coordinate to copper ions, and the complex between PSA-CQDs
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