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New Journal of Chemistry Accepted Manuscript

L-cysteine-capped CdTe quantum dots as a fluorescent probe for
sequential detection of lysozyme and trypsin
Published on 18 April 2017. Downloaded by Fudan University on 26/04/2017 03:41:29.

Received 00th January 20xx, a b a*

Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Fanping Shi, Siyu Liu and Xingguang Su
In this work, we designed a simple and sensitive fluorescence probe for sequential detection of lysozyme and trypsin.
Firstly, L-cysteine was chosen as stabilizer to obtain L-cysteine-capped CdTe QDs. Lysozyme with positive charges can
interact with L-cysteine capped CdTe quantum dots (QDs) via the special assembly between lysozyme and L-cysteine,
resulting in the quenching of the fluorescence of CdTe QDs. Lysozyme can be hydrolyzed into small fragments in the
presence of trypsin, and the interaction between L-cysteine-capped CdTe QDs and lysozyme would be inhibited, which
could be used for trypsin quantification. There was a linear relationship between the fluorescence intensity of CdTe QDs
and lysozyme concentration in the range of 75-1000 ng mL-1 and another linear relationship between the fluorescence
intensity of CdTe QDs and the logarithm of trypsin concentration in the range of 10-500 ng mL-1. The detection limits was
28.33 ng mL-1 for lysozyme and 8.35 ng mL-1 for trypsin, respectively. The established method showed a good selectivity for
lysozyme and trypsin detection, respectively.

detection of hydrolases is desirable. Being the most important

Introduction
Quantum dots (QDs) ，as a new class of fluorescent probe has
drawn much attention in recent years. Compared with
conventional organic fluorescent dyes and fluorescent
proteins, QDs have higher quantum yield, tunable size-
dependent photoluminescence and narrow emission peaks1.
All these characters gained them rising status in biological
fields, such as bio-analysis, environmental analysis and clinical
or medical detections2-4. Surface capping ligands of QDs is an
important factor affecting the properties of QDs, and the
fluorescence of QDs is extremely sensitive to their surface
states. Different stabilizers of thiol-acid or thiol-amine have
been used in the synthesis to obtain water-soluble QDs with a
shell of negative or positive charge around them for
bioconjugations5. Some sensors are established based on the
surface interaction between biothiol-capped QDs and special
detected targets6.
Biological hydrolases are involved in the control of multiple
biological processes. They catalyze the hydrolytic cleavage of
specific hydrolysis site in target7. Biological hydrolases activity
is associated to numerous pathological conditions8. Thus, the
development of effective methods for the quantitative
enzymes in physiology and pathology, lysozyme and trypsin
were chosen as representatives for research in this work.
Lysozyme is a special small protein with high isoelectric point
(pI) and electropositivity at neutral pH. As a kind of body’s own
antibiotic, it is widely distributed in body tissues and secretions
9
(blood, urine and saliva) . It catalyzes the hydrolyzation of the
β-linkage between N-acetylglucosamine and N-acetylmuramic
acid of mucopolysaccharides in the bacterial cell wall to
10
achieve bactericidal action . Clinical studies indicate that the
changes of lysozyme concentrations in urine and serum are
associated with leukemia, tuberculosis, renal diseases,
11
meningitis and acute bacterial infection . Up to now, a variety
of strategies have been reported for the determination of
lysozyme. Traditional methods for lysozyme assay include
12
high-performance liquid chromatography (HPLC) , enzyme-
13
linked immunosorbent assay (ELISA) , resonance Rayleigh
14 15 16
scattering (RRS) , voltammetric and colorimetric . Recently,
the establishment of lysozyme aptamer-based biosensor has
attracted more attention. The aptamer-modified electrodes
and the combination of quantum dots and lysozyme aptamer
17, 18
both show selective detection for lysozyme .
Trypsin is a critical important digestive enzyme originating
from the pancreatic cells. It can cleave proteins on the c-
19
terminal side of arginine and lysine residues . As a kind of
serine protease, it is more active under slightly alkaline
20
conditions . Trypsin plays a key role in controlling the
pancreatic exocrine function. Its level changes in human body
relate to pancreatic diseases. For instance, up-regulated
activity of trypsin causes pancreatitis that carries a high risk of
21
pancreatic cancer . Besides, disordered trypsin concentration
22
has an impact on meconium ileus and cystic fibrosis .

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Traditional methods for trypsin detection involve gel nm and the PL intensity referred to the maximum emission of
23 24 25
electrophoresis , HPLC and electrochemistry . Currently, QDs-DA at 545 nm. UV-vis absorption spectra were obtained
fluorescence assay has been proved an excellent method for
trypsin activity assay. A series of fluorescent nanocomposites
such as conjugated polyelectrolytes, self-assembled graphene
quantum dots, Mn:ZnSe d-dots and Bovine Serum Albumin
(BSA-)stabilized gold nanoclusters were used as sensitive

New Journal of Chemistry Accepted Manuscript

26-29
fluorescent probes for trypsin activity study . Nevertheless,
these methods suffer from the drawbacks of sophisticated
instrumentation, rigorous conditions, time-consuming and
synthesis difficulty. Thus, a novel and straightforward methods
for trypsin detection has been attracting much more interest.
Published on 18 April 2017. Downloaded by Fudan University on 26/04/2017 03:41:29.

Herein, we presented a simple and sensitive fluorescent

method for sequentially detecting lysozyme and trypsin.
Firstly, we chose L-cysteine as stabilizer to obtain L-cysteine-
capped CdTe QDs. Since L-cysteine-capped QDs were
synthesized in aqueous solution, there were certain trap sites
on the surface of QDs. L-cysteine and lysozyme could generate
a kind of complex on the QDs surface due to the special
assembly, and facilitated an effective electron transfer process
between L-cysteine QDs and lysozyme, resulting in the
quenching of CdTe QDs fluorescence. When trypsin was Scheme 1 (A) The surface status of L-cysteine capped CdTe QDs;(B) A schematic
added, lysozyme would be cleaved into smaller fragments on representation of the fluorescent sensing mechanism for the detection of lysozyme and
the C-terminal side of arginine and lysine residues. Since the trypsin based on the L-cysteine capped CdTe QDs.

binding condition of lysozyme to lcys-CdTe QDs was destroyed,

the sensitive and selective detection of trypsin was realized.
The design principle for sequential detection of lysozyme and
trypsin based on L-cysteine-capped CdTe QDs is schematically
illustrated in Scheme 1.
Experimental
Reagents
All chemicals used were of analytical reagent grade without
further purification. The water used in all experiments has a
-1
resistivity higher than 18MΩ cm . Tellurium powder (~200
mesh, 99.8%), cadmium chloride (99%) NaBH4 (99%), alkaline
phosphatase (ALP), glucose oxidase (GOx), urease, horseradish
peroxidase (HRP), trypsin and pepsin (Pep) were purchased
from Sigma-Aldrich Corporation (http: //www. sigmaaldrich.
com). NaCl, KCl, CaCl2, Mg(NO3)2 and Zn(NO3)2·6H2O were
purchased from Beijing Chemical Works. Lysozyme was
purchased from Sangon Biotechnology Co., Ltd. L-cysteine,
glycine, arginine, phenylalanine, threonine, glutamic,
tryptophan, L-tyrosine, glucose, urea, glutathione was
purchased from Beijing Dingguo Biotechnology Co. Ltd
(http://www.dingguo.com). The human serum was obtained
−1
as a gift from the university hospital. The 50 mmol L Tris–HCl
buffer solution was used as the medium for detection process.
The above solutions were all stored at 0~4 °C and diluted with
ultrapure water to the concentrations used in the experiment.
Apparatus
All fluorescence measurements were carried out in a 1 cm
path length quartz cuvette with a Shimadzu RF-5301 PC
spectrofluorophotometer equipped with xenon lamp
usingright-angle geometry. The excitation wavelength was 360
using a Varian GBC Cintra 10e UV-vis spectrometer. All the
optical measurements were carried out at room temperature
under ambient conditions. All pH measurements were made
with a Starter-2C pH meter obtained from Ohaus Instruments
Co. Ltd. (http: //asiapacific.ohaus.com), Shanghai, China. High
Resolution Transmission electron microscopy (HRTEM) images
were obtained with a JEOL-3010 electron microscope
operating at 300 kV. HRTEM samples were prepared by
dropping the aqueous L-cysteine CdTe QDs solution onto
carbon-coated copper grids and allowing the excess solvent to
evaporate. The fluorescence image of the cells was taken by an
inverted fluorescence microscope (Olympus FV1000 IX71)
equipped with a multispectral imaging system (Nuance, CRI,
Woburn, MA, USA).
Preparation of L-cysteine-capped CdTe QDs
Synthesis of L-cysteine-capped CdTe QDs (lcys-CdTe QDs) in
this study was carried out following the general procedure
4
described in our previous work . Briefly, the precursor
solution of lcys-CdTe QDs was formed in water by adding fresh
−3 −1
NaHTe solution to 1.25× 10 mol L N2 -saturated CdCl2
solution at pH 10~11 in the presence of L-cysteine as
2+ −
stabilizing agent. The molar ratio of Cd / L-cysteine /HTe was
charged at 1: 2.4: 0.5. The precursor solution was subjected to
reflux at 100° C under open-air conditions with condenser
attached and 35 minutes later lcys-CdTe QDs were obtained.
Ethanol was added to the stock solution to obtain QDs
precipitate, and the precipitation process was repeated three
times. The unreacted residues were removed by the cycled
washing. The purified QDs were dissolved in ultrapure water
and stored in the darkroom. The fluorescence emission
wavelength of lcys-CdTe QDs used in the present experiments

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−6
was 545 nm and the concentration of QDs was 2.45×10
−1
L .
Determination of lysozyme and trypsin
To study the fluorescence quenching effect of lysozyme on
lcys-CdTe QDs, Tris-HCl buffer solution (100 µL, pH 7.0), lcys-
-8 −1
CdTe QDs (30 µL, 3.68×10 mol L ) and different
concentrations of lysozyme solution were successively added
into a 2.0 mL calibrated test tube, then diluted to the mark
with ultrapure water at room temperature and shaken
thoroughly until the solution was fully mixed before
fluorescence measurement.
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For trypsin detection, certain concentration of lysozyme (200
µL, 1 µg mL-1) was mixed with different concentrations of
trypsin in 2.5 mmol L-1 Tris-HCl buffer (pH 8.2). After the
incubation at room temperature for 1h, the reaction mixture
was added to the calibrated test tube with 30 µL lcys-CdTe
QDs and 100 µL Tris-HCl buffer (pH 7.0) in it and diluted to the
mark with ultrapure water. Thoroughly mix the above mixture
before subsequent fluorescence measurements.
In the experiments, all the PL measurements were performed
under the same conditions: the slit widths of the excitation
and emission were both 10 nm and the excitation wavelength
was set at 360 nm. The fluorescence spectra were recorded in
the wavelength range of 450~650 nm.
Human serum samples detection
For serum samples detection, drug-free human blood samples
were collected from the university hospital. All the blood
samples were obtained by venipuncture and centrifuged at
10,000 rpm for 10 min at room temperature. Due to the high
protein bonding in the blood plasma, some pretreatments
were carried out to eliminate the interferences and improve
the recovery according to previous reports.4 The supernatant
was separated and deproteinized by adding acetonitrile
(CH3CN) in samples (CH3CN: serum=1:1). After vigorously
shaking for 2 minutes, the mixture was centrifuged at 10,000
rpm for 10 min at 4°C. The obtained supernatant was filtered
twice. The filtrate was adjusted to neutral pH and then diluted
by 25 times with deionized water. Different concentrations of
trypsin were added to the diluted serum samples to prepare
the spiked samples. Real samples detection was carried out
using the procedure described above. All experiments were
performed in compliance with the relevant laws and
institutional guidelines, and the relevant institutional
committees have approved the experiments.
Results and discussion
Characteristics of Lcys-CdTe-QDs
The morphologies of the as-prepared lcys-CdTe QDs were
confirmed by transmission electron microscopy (TEM). From
the TEM images, it can be seen that uniform quantum dots
have been formed through the synthesis processes. Their
lateral size distribution is mainly in the range of 1~4 nm with a
narrow size distribution and an average diameter of 3.5 nm,
suggesting that the QDs are mostly uniform in size. (Inset 1 of
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ARTICLE
mol Fig. 1a) The high-resolution TEM (HRTEM) image shown in the
inset 2 of Fig. 1a clearly revealed that individual lcys-capped
New Journal of Chemistry Accepted Manuscript
Fig.1 (a) TEM images of GQDs. The inset 1 of (a) is the size distributions of GQDs. The
inset 2 of (a) is the HRTEM image of GQDs; (b) FT-IR spectra of the free L-cysteine and
lcys-CdTe QDs; (c) The photograph of lcys-CdTe QDs aqueous solution prepared by
various reaction times under UV irradiation conditions (λex=365nm) by a digital camera;
Normalized fluorescence emission spectra (d) and UV-vis absorption spectra (e) of lcys-
CdTe QDs with controllable maximum emission wavelengths ranging from 525 to 585
nm under various reaction times (5 min, 10 min, 35 min and 50 min). The samples were
directly extracted from the original solution right after reaction without further
−6 −1
treatment. The concentrations of all the QDs were 2.45×10 mol L , and fluorescence
emission spectra were recorded at λex=365 nm.
CdTe QDs appeared as roughly spherical particles and
possessed a relatively good monodispersity and crystallinity.
To verify the existence of L-cysteine on the surface of the
prepared QDs, we compared the FT-IR spectra of free L-
cysteine and L-cysteine capped CdTe QDs (Fig.1b). Compared
to free L-cysteine, only the characteristic peaks of the carboxyl
and amino groups were observed on the lcys-CdTe QDs. The
-1
characteristic peaks of thiol group observed at 2550~2670 cm
in free L-cysteine disappeared because of the covalent affinity
2+
of Cd to the -SH group of L-cysteine. By changing the reaction
time under the optimal conditions, we got lcys-CdTe QDs
original solution with different emission wavelengths from 525
to 585 nm. The different colours from green to orange could
be observed under UV light. (Fig. 1c) The fluorescence
emission and UV-vis absorption spectra of lcys-CdTe QDs
prepared at different reaction times were shown in Fig.1d and
Fig.1e. With the extension of reaction time, the red-shift of the
fluorescence emission peaks of the quantum dot was
occurred, accompanied with the gradual rise of UV
absorption peak.
The fluorescence emission and UV-vis absorption spectra of
lcys-CdTe QDs, lcys-CdTe-QDs-lysozyme and lcys-CdTe-QDs-
lysozyme-trypsin system were shown in Fig.2. It can be seen
from Fig.2a that the lcys-CdTe QDs possessed an absorption
maximum at 524 nm and an emission maximum at 550 nm
upon excitation at 360 nm. Compared with the original
fluorescence spectra of lcys-CdTe QDs, the fluorescence
spectra of lcys-CdTe QDs-lysozyme system exhibits an obvious
blue shift around 5 nm indicating the change in surface nature
of lcys-CdTe QDs upon binding with lysozyme. In the presence
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of trypsin, a remarkable fluorescence recovery is observed for
the lcys-CdTe QDs-lysozyme-trypsin system. The presence of
large amounts of guanidino on lysozyme surface makes
30
lysozyme positively charged surface states . The surface of
lcys-CdTe QDs shows electronegative in neutral condition.
According to the pI of L-cysteine (5.8) and lysozyme (11.0), it
seems that the electrostatic effect might be the driving force
for assembly of L-cysteine and lysozyme. The blue shift could
be explained by the decrease of water molecular polarizability
caused by the combination of lysozyme and lcys-CdTe QDs,
31
leading to reductive Stokes shift. From Fig.2b we can see
that there was an obvious spectral change when compared the
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absorption spectra of Lcys-CdTe QDs with that of Lcys-CdTe
QDs-Lysozyme system, implying the formation of lysozyme and
lcys-CdTe QDs complex. When trypsin is induced to the
system, the absorption spectrum gives a blue shift, meaning
the tendency to return to the original state. So, it is reasonable
for us to assume that the fluorescence quenching is ascribed
to the binding of lysozyme and L-cysteine on the surface of
CdTe QDs. The quenching of lcys-CdTe QDs should be ascribed
to the selective assembly of L-cysteine and lysozyme.
Fig.2 (a) The photoluminescence emission spectra of (1) lcys-CdTe QDs, (2) lcys-CdTe
QDs-lysozyme, (3) lcys-CdTe QDs-lysozyme-trypsin in 2.5 mmol L-1 Tris-HCl buffer
(pH=7.0). The concentration of lcys-CdTe QDs, lysozyme and trypsin were 36.8 nmol L-1,
1 µg mL-1, and 0.5 µg mL-1, respectively. (b) The UV-Vis absorption spectra of lcys-CdTe
-1
QDs, lcys-CdTe QDs-lysozyme, lcys-CdTe QDs-lysozyme-trypsin in 2.5 mmolL Tris-HCl
buffer (pH=7.0)
Detection of lysozyme and trypsin
L-cysteine has a different charge under different pH conditions
due to the isoelectric point. So acid and alkali environment
changes lead to the different surface status of lcys-CdTe QDs,
32
and the PL intensity of lcys-CdTe QDs are pH-dependent . The
pH value did not only affect the PL intensity and stability of
original lcys-CdTe QDs, but also the subsequent fluorescence
quenching by lysozyme. We systematically studied the pH
effect on the the fluorescent probe. As shown in Fig.3a, the PL
intensity of lcys-CdTe QDs solution increased with the
increasing of pH value and reached maximum at pH 7.0, then
decreased gradually from pH 7.0 to 9.0. When lysozyme was
added into the lcys-CdTe QDs solution, the fluorescence
quenching efficiency at neutral or acid pH is significantly better
than that in alkaline conditions, indicating that quenching
effect is related to the surface status of lcys-CdTe QDs.
Investigation of the pH dependence of this assay for lysozyme
detection also seems to confirm the conclusion that the
quenching of lcys-CdTe QDs should be attributed to the special
assembly of lysozyme and the l-cysteine on QDs surface.
Therefore, in order to obtain a better recovering condition, pH
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7.0 was chose in the further lysozyme quenching experiments.
33
As for trypsin, it has significant activity at pH 8.2 . So, pH 8.2
was chose in the trypsin enzyme digestion experiments. Under
the optimal pH, the reaction time for lysozyme quenching on
lcys-CdTe QDs was investigated. As shown in Fig. 3b, the
quenching effect of lysozyme on the PL intensity of lcys-CdTe
QDs was finished in 1 minute. So, we chose 1 min as
New Journal of Chemistry Accepted Manuscript
stabilization time before the fluorescence measurements in
the following experiments.
Fig. 3 (a)The effect of pH on the PL intensity of lcys-CdTe QDs in the absence and
-1
presence of 1µg mL lysozyme; (b) Effect of incubation time on the PL intensity
of lcys-CdTe QDs in the presence of 1µg mL-1 lysozyme in Tris-HCl buffer (2.5
mmolL-1, pH=7.0)
In order to investigate the quenching ability of lysozyme on the
PL intensity of lcys-CdTe QDs, the fluorescence spectra of lcys-
CdTe QDs in the presence of a series of lysozyme
concentrations were recorded (Fig. 4). PL intensity of lcys-CdTe
QDs decreased gradually with the increasing concentration of
lysozyme (from 75 to 2000 ng mL -1). Inset of Fig.4 showed a
good linear relationship between the PL intensity ratio I/I0 (I0
and I was the PL intensity of the lcys-CdTe QDs in the absence
and presence of lysozyme) and the lysozyme concentration in
the range of 75 ~1000 ng mL -1 or 5.36~71.43 nmol L-1. The
linear regression equation is I/I0= (1.011±0.008) + (-6.920×10-
4
±1.633×10-5) CLysozyme, ng mL-1. The corresponding regression
coefficient was 0.995. The limit of detection (LOD) was found
to be 28.33 ng mL -1 or 2.02 nmol L-1using the criterion of three
times the standard deviation of the blank signal. The standard
deviation for nine replicate measurements of 375 ng mL -1
lysozyme was 2.08%.
In the presence of trypsin, the arginine-rich lysozyme could be
broken up into smaller fragments due to the hydrolysis,
causing the recovery of PL intensity of lcys-CdTe QDs-lysozyme
system. As shown in Fig. 5, the PL intensity of the lcys-CdTe
QDs-lysozyme system increased with the increase of the
trypsin concentration. The inset in Fig. 5 showed that there a
good linear relationship between the PL intensity ratio (Ir/I0) (I0
was the PL intensity of lcys-CdTe QDs in the absence of
lysozyme and trypsin, and Ir presents the PL intensity of lcys-
CdTe QDs-lysozyme system in the present of trypsin) and the
logarithm of trypsin concentrations in the range of 10- 500 ng
-1
mL . The linear regression equation was Ir/I0= (0.268±0.017) +
-1
(0.263±0.009) log CTrypsin, ng mL . The corresponding
regression coefficient was 0.995. The limit of detection (LOD)
-1 -1
was found to be 8.35 ng mL (0.348 nmol L ). The standard
-1
deviation for nine replicate measurements of 200 ng mL
trypsin was 4.74%.
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more quickly and more completely in the presence of high

trypsin concentrations, which implies that an increase in the
amount of trypsin gave rise to high initial rates of the catalytic
reaction.

New Journal of Chemistry Accepted Manuscript

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Fig. 4 Fluorescence spectra of lcys-CdTe QDs upon the addition of different

concentration of lysozyme. The curves represent the concentrations of lysozyme 0, 75,
150, 250, 375, 500, 600, 750, 1000, 1500, 2000 ng mL-1, respectively. The inset
represents plot of photoluminescence intensity ratio (I/I0) of the fluorescent probe
versus the concentration of lysozyme. The lcys-CdTe QDs-lysozyme system is incubated
-1
in Tris-HCl buffer solution (2.5 mmol L , pH 7.0) for 1 minute. I0 and I are the PL
Fig.6 Effect of the hydrolysis reaction time on the PL intensity of lcys-CdTe QDs-
intensity in the absence and presence of lysozyme.
lysozyme-trypsin system at different trypsin concentrations.

Interference and selective study

We further studied various coexistence substances on the
detection of lysozyme. Table 1 showed the effect of some
inorganic ions, amino acids and small biological molecules on
the determination of lysozyme. Tolerable concentration was
defined as the concentrations of coexisting substances causing
less than ±7.0% relative error. The tolerable concentration
-1 -
ratios of coexisting substances to 1.0 μg mL or 71.43 nmol L
1 + + 2+
lysozyme was over 200-fold for Na , K , Mg , 100-fold for
glycine, arginine, phenylalanine, threonine, glutamic acid,
tryptophan, L-tyrosine, glucose and urea, 10-fold for human
serum albumin (HSA) and hemoglobin, and 500-fold for
glutathione. The results showed that there was little
interference from commonly existing substances.

Fig. 5 Fluorescence spectra of lcys-CdTe QDs-lysozyme system upon the addition of Table 1 The effect of coexisting substances on the determination of lysozyme.
different concentration of trypsin. The curves represent the concentrations of trypsin 0,
10, 20, 50, 100, 200, 375, 500 ng mL-1, respectively. The concentration of lysozyme is 1
µg mL-1. The inset represents the plot of photoluminescence intensity ratio (Ir/I0) (I0 was
the PL intensity of lcys-CdTe QDs in the absence of lysozyme and trypsin, and Ir
presents the PL intensity of lcys-CdTe QDs-lysozyme system in the present of trypsin)
versus the logarithm of trypsin concentrations.

The linear range and detection limits of some reported

methods for the detection of lysozyme and trypsin are listed in
Table S1 and Table S2 for comparative purpose. It can be seen
that our present method offered a comparable detection limit
and dynamic range.
To demonstrate the potential of the lcys-CdTe QDs-lysozyme
system for monitoring the activity of trypsin, the fluorescence
quenching of lcys-CdTe QDs at 550 nm was studied as a
function of the trypsin concentration. Fig. 6 shows time curves
of the PL quenching of lcys-CdTe QDs at a fixed concentration
of lysozyme with various trypsin concentrations from 50 to 500
-1
ng mL . It is observed that the PL intensity was recovered

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Coexisting substance Tolerable Molar ratio or ΔI /I Real sample detection

concentration Quality Ratio (%)
In order to evaluate the feasibility of the proposed method for
Na+ 14 μmol L-1 200 2.2 trypsin detection in real samples, the developed fluorescence
K+ 14 μmol L-1 200 6.4
probe was applied to the determination of trypsin in human
Mg2+ 14 μmol L-1 200 -2.7
serum samples. The results obtained by the standard addition
Glycine 7 μmol L-1 100 4.2
Arginine 7 μmol L-1 100 3.1 method were shown in Table 2, and the accuracy of the

New Journal of Chemistry Accepted Manuscript

Phenylalanine 7 μmol L-1 100 1.9 proposed method was evaluated by determining the average
Threonine 7 μmol L-1 100 -0.5 recoveries of trypsin in these samples. It can be seen that the
Glutamic 7 μmol L-1 100 1.9 RSD was lower than 7.05% and the average recoveries in real
Tryptophan 7 μmol L-1 100 5.1 samples were in the range of 98.9%-102%. The above results
L-Tyrosine 7 μmol L-1 100 -0.5 demonstrated the potential applicability of the lcys-CdTe QDs-
7 μmol L-1
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Glucose 100 -1.9 based fluorescence probe for the detection of trypsin in
Urea 7 μmol L-1 100 -5.7 human serum samples.
Glutathione 35 μmol L-1 500 2.7 Table 2 Measurements of trypsin in real human serum samples using
Human Serum Albumin 10 μg mL-1 10 4.8 the proposed probe.
Hemoglobin 10 μg mL-1 10 6.2 Sample Added Found
%RSD(n=3) %Recovery
ΔI=I0-I, where I0 and I are the photoluminescence intensity of lcys-CdTe- No. (ng mL-1) (ng mL-1)
QDs-lysozyme system in absence and presence of coexisting substances. 1 100 102.39 3.26 102
Here, the relative MW of lysozyme is calculated as 14000, the concentration 350 354.76 1.26 101
of lysozyme is 1.0 μg mL-1 or 71.43 nmol L-1. 2 100 98.89 6.92 98.9
350 357.60 0.24 102
To investigate the selectivity of lcys-CdTe QDs-based 3 100 99.92 7.05 99.9
fluorescence probe for lysozyme and trypsin, we studied the 350 358.18 0.73 102
fluorescence responses of the present probe to other enzymes The original concentration of trypsin in human serum samples were
at the concentration of 10 times of lysozyme (1 µg mL-1) or subjected to a 50-fold dilution with deionized water.
trypsin (0.5 µg mL-1), and the results were shown in Fig.7. It
can be seen that even the concentration of other enzymes
were 10-fold over that of lysozyme, they didn't cause obvious Conclusions
quenching effect, only lysozyme gave a satisfied response.
In this paper, L-cysteine capped CdTe QDs was employed as a
(Fig.7a). As discussed above, the selectivity should be ascribed
fluorescent probe for selective and sensitive determination of
to the selective assembly of l-cysteine and lysozyme. As shown
lysozyme and trypsin. Lysozyme with positive charges can
in Fig.7b, the fluorescence of lcys-CdTe QDs-lysozyme system
interact with lcys-CdTe QDs via hydrogen bonds and van der
have been enhanced a little by pepsin, alkaline phosphatase
Waals interactions, resulting in the quenching of the
(ALP), horseradish peroxidase (HRP) and glucose oxidase(GOx),
fluorescence of CdTe QDs. With the addition of trypsin,
which can be explained by the sensitization of protein to QDs.
lysozyme can be cleaved into smaller fragments and the lcys-
While the recovered PL efficiency by trypsin is as high as
CdTe QDs fluorescence was no longer quenched. Good linear
86.49%. The above results revealed good selectivity of the
relationships between the PL intensity and the concentration
present fluorescence probe for both lysozyme and trypsin
of lysozyme and trypsin are obtained under the optimum
detection.
conditions. The switch sensing mode, label-free property and
easy preparation of lcy-CdTe-QDs offer advantages for further
application.

Fig. 7 Selectivity study (a) The fluorescence quenching efficiency of the fluorescent
probe containing lcys-CdTe QDs in the presence of lysozyme and four other enzymes.
Lysozyme was used at 1µg mL-1 (about 69.4 nmol L-1), and other tested enzymes were
used at 10 µg mL-1. (b) The fluorescence recovery efficiency of the fluorescent probe
containing lcys-CdTe QDs -lysozyme in the presence of trypsin and four other enzymes.
Trypsin was used at 0.5 µg mL-1 (about 20.9 nmol L-1), and other tested enzymes were
-1
used at 5.0 µg mL . Quenching efficiency = (I0− Iq)/ I0 and Recovery efficiency = (Ir− Iq)/
(I0− Iq). I0 and I are the PL intensity of lcys-CdTe QDs in the absence and presence of
lysozyme or other enzymes, and Ir presents the PL intensity of lcys-CdTe QDs-lysozyme
system in the present of trypsin or other enzymes.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (No. 21075050, No. 21275063)
and the science and technology development project of Jilin
province, China (No. 20110334).
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