International Journal of Environmental Analytical

Chemistry

ISSN: 0306-7319 (Print) 1029-0397 (Online) Journal homepage: http://www.tandfonline.com/loi/geac20

The dual-modes of QCM immunosensing for

estradiol analysis via dynamic or round-off
calibration

Dandan Chen, Xiuhua Wei, Ya Yang & Yifeng Tu

To cite this article: Dandan Chen, Xiuhua Wei, Ya Yang & Yifeng Tu (2016) The dual-
modes of QCM immunosensing for estradiol analysis via dynamic or round-off calibration,
International Journal of Environmental Analytical Chemistry, 96:14, 1389-1401, DOI:
10.1080/03067319.2016.1264582

To link to this article: http://dx.doi.org/10.1080/03067319.2016.1264582

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INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY, 2016
VOL. 96, NO. 14, 13891401
http://dx.doi.org/10.1080/03067319.2016.1264582

The dual-modes of QCM immunosensing for estradiol

analysis via dynamic or round-o calibration
Dandan Chena, Xiuhua Weia,b, Ya Yanga,c and Yifeng Tua
a
Department of Chemistry, Institute of Analytical Chemistry, Dushu Lake Campus, Soochow University,
Suzhou, P. R. China; bCollege of Chemistry and Chemical Engineering, Shangqiu Normal University,
Shangqiu, P. R.China; cDepartment of Forensic Medicine, Dushu Lake Campus, Soochow University,
Suzhou, P. R. China

ABSTRACT ARTICLE HISTORY

In this work, a piezoelectric immunosensor is developed for the Received 8 June 2016
detection of estradiol (E2). The gold-coated chip of quartz crystal Accepted 15 November 2016
microbalance was modied by a 3-mercaptopropionic acid self- KEYWORDS
assembly monolayer, activated by carbodiimide/n-hydroxylsuccini- Quartz crystal microbalance;
mide, and then to immobilise the E2 antibody via the interaction self-assembly monolayer;
with their amine groups. The resonance frequency change electrochemical
resulted by the binding of E2 on immobilised antibody diers immunosensor; dynamic
from that of a blank (F), was correlated to the antigen concentra- calibration; estradiol
tion. The construction of the sensing interface was conrmed by
electrochemical impedance spectroscopy. Both round-o or
dynamic modes were explored as analytical detection strategy in
this work. Using round-o mode, the output F responds to the
concentration of E2 with a detection limit of 0.04 g mL1 and a
linear range from 0.1 to 10 g mL1. Meanwhile, using a dynamic
mode, the presence of E2 induced in a real-time sensing output
along with the incubation of antigen. The slope of sensing output
is in linear relation with the concentration of E2 with a detection
limit of 0.06 g mL1. Thus, a faster immunesensing technique is
established for rapid analysis requiring only several seconds other
than the round-o mode which needs a complete incubation
period for at least 60 min. This nding will greatly promote the
applicability of immunosensor for rapid, real-time or in-site assays.

1. Introduction
Biosensor is a category of sensitive and selective analytical devices for chemical or
biological substances to convert the information of concentration into an electrical
signal [1]. Among those biosensors, the immunosensor oers high selectivity on account
of the immuno-recognition, which is also a kind of eective devices with low detection
limits [2,3]. In this subset, electrochemical immunosensors have been widely applied in
health care [46], food safety [7,8], environmental monitoring [9,10] etc.
Quartz crystal microbalance (QCM), a very sensitive quantifying technic based on the
piezoelectric eect of quartz chip, linearly converts the mass change on its surface into a
frequency change as the output signal according to the Sauerbrey equation [11]. QCM-

CONTACT Yifeng Tu tuyf@suda.edu.cn; Ya Yang yangya@suda.edu.cn

2016 Informa UK Limited, trading as Taylor & Francis Group
1390 D. CHEN ET AL.

based sensors have wide applications in real-time monitoring of environmental con-

taminants, rapid and specic detection of virus, bacterial or the determinations of
hydrocarbons in aqueous or gas phase environments etc. [1216]. To construct the
QCM chips, the surface of quartz chip is coated with Au, Ag, Pt or other metals in a
certain pattern to perform the piezoelectric eect and thus provides a good platform for
building sensing matrix on them.
Self-assembly modication (SAM) is a simple and multifunctional surface modifying
strategy which was successfully applied in the development of numerous biosensors
[1720]. Those sensors exhibit advantages including fast response, good reproducibility
and enhanced sensitivity based on the ordered and reproducible molecular interface of
SAM for biomolecular binding [21]. Moreover, the preparation of SAMs is relatively easy
and various SAMs are available for dierent surface chemistry [22]. Therefore, it provides
a desired substrate for biosensor constructing.
This study combines the advantages of high sensitivity of the piezoelectric oscillation
of quartz chip upon the surface loading and highly specic immune recognition, to
develop a QCM-based immunosensor via the SAM strategy. As illustrated in Figure 1(a), a
3-mercaptopropionic acid (3-MPA) SAM layer was obtained by their attachment on gold
surface of QCM chip via the thiol group, leaving the carboxyl groups outward for further
surface functionalisation. Then, after the activation with 3-(3-dimethylaminopropyl)-1-
ethylcarbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), the antibody
was linked with those carboxyl groups via the amide bonds to be immobilised. After

Figure 1. (A) The schematic diagram of the fabrication of the immunosensor; (B) the EIS curves of (a)
bare chip; (b) modied by 3-MPA; (c) activated with EDC/NHS and bound with (d) 20 L of or (e)
enough E2 antibody; (C) the frequency output of (a) bare chip; (b) modied by 3-MPA; (c) activated
with EDC/NHS and (d) bound with enough E2 antibody.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1391

incubated with antigen, the performance of the sensing chip was evaluated by detecting
dierently concentrated antigens. Here, two detection modes were employed. The rst
one, called round-o mode, means the approach to acquire the result after the
accomplishment of immune-reaction. The second one, a dynamic mode, means the
realisation of measurement during the process of immuno-reaction before the achieve-
ment of equilibrium.
Although the immunosensor with round-o mode for detection exhibits high sensi-
tivity, its operation requires a long incubation time (approximately 1 h) to ensure the
nish of anity interaction between antibody and antigen. However, in clinical or other
related cases, a fast assay is expected to ensure the real-time quantifying within a few
minutes even seconds. The dynamic mode is promising to provide a rapid and sensitive
detection. The results showed that the measurement time could be greatly shortened to
several seconds based on the relationship between the responding slope and antigen
concentration, prophesying a novel approach for obtaining a time-saving immunosen-
sing manner. Thus, the meaning of our research is not only to develop an innovative
immunosensor, but also to open a new way to instantaneous immunosense.
As a model analyte, the estradiol, 17- estradiol (E2) [2325], was selected for
feasibility testing of the developed immunosensor. The E2 will be accumulated in
ecosystem through the food chain and then nally deposited into human body in fat
tissues, result in some long-term toxicity. Its hazards on biosomes, mostly on aquatic
organisms, are mainly in the reproductive system. It leads to accelerated synthesis of
vitellogenin in body, hence to induce the variation of sexual organs and the feminisa-
tion, and so on. Therefore, its level in female body, the addition in foods, cosmetics etc.,
is very signicant for current physiological, pharmaceutical or industrial researches.
Compared to currently employed methods for E2 testing including gas chromatogra-
phymass spectrometry [26], high performance liquid chromatograph [27], immunoas-
say [2830] or electrochemical methods [3133], the QCM immunosensor reported in
this paper oers advantages such as high sensitivity and selectivity, short sensing time,
small size, portability and low cost and simpler operation than part of other electro-
chemical methods.

2. Experimental
2.1. Materials and apparatus
E2 was purchased from Aladdin Biochemical Technology Co. Ltd (98%, Shanghai, China).
The E2 antibody was purchased from FengShou Industrial Co. Ltd (2 mg mL1, Shanghai,
China). 3-MPA (>99%) and bovine serum albumin (BSA) were obtained from Alfa Aesar
(Tianjin) Chemicals Co. Ltd (Beijing, China). In our work, 0.01 mol L1 phosphate buered
saline (PBS) of pH 7.0 was used to dilute all of the solutions. EDC was obtained from
Sigma Aldrich Co. (98%, St. Louis, USA). NHS was purchased from Fluka (98%, Buchs,
Switzerland). The ultrapure water was used throughout and all reagents were used
without further purication.
A CHI400A Electrochemical QCM Workstation (in conjunction with a 13.7-mm dia-
meter, 7.995 MHz AT-cut quartz crystal wafer with gold discs on its surfaces bilaterally in
1000 of thickness and 5.1 mm of diameter, and a 3-mL Teon detection cell) was
1392 D. CHEN ET AL.

obtained from Chenhua Instruments Co. Ltd. (Shanghai, China). The QCM tests were
carried out on an electromagnetic shielded and shock proofed platform. The electro-
chemical impedance spectroscopy (EIS) detections were carried on with an RST5200
Electrochemical Workstation (Suzhou Risetest Instrument Co. Ltd., Suzhou, China). All of
the experiments were carried out with a three-electrode system, with QCM chip as
working electrode, a platinum wire as counter electrode and a saturated calomel
electrode as reference electrode.

2.2. Preparation of the immunosensing chip

The chip was pretreated with Piranha solution (30% H2O2: 98% H2SO4 = 1:3, v/v) for
1 min. Thereafter, it was rinsed with ultrapure ethanol and water successively, followed
by drying with nitrogen blowing.
The cleaned QCM chip was then immersed in the PBS solution of pH 7.0 contain-
ing 10 mmol L1 of 3-MPA for 12 h to form a self-assembled monolayer. After
washing it with ultrapure water to remove excess free 3-MPA molecules, the modied
chip was then placed in a mixture of EDC and NHS in PBS for 5 h to activate the
carboxylic groups of 3-MPA. Following this, 100 L of a 160 times diluted E2 antibody
solution was dropped onto the modied gold disc on one side of the chip. This
assembly was then kept in a wet environment at 4C overnight to facilitate the
immobilisation of E2 antibody. The residual unbound antibody was washed o by
water. To block the unreacted succinimidyl groups to prevent the non-specic
adsorption of impurity proteins, the BSA solution was dropped on it, standing for
half hour, then washed o with water. After drying in a nitrogen stream, the sensing
chip was stored at 4C.

2.3. The characterisation and implementation of sensor performance

EIS was used to conrm the sensor construction based on the changes of the surface
impedance. All experiments were conducted in PBS containing 5 mmol L1 of
1
FeCN3
6 and 0.1 mol L of NaCl within the frequency range from 1 Hz to 100 kHz
(at open-circuit potential, 0.01 V of AC perturbative potential). The FeCN3
6 was used
as the redox probe because of its excellent reversibility. A ZSimpWin software was
used for the data tting. The electron transfer resistance (Ret) estimated by the
semicircle diameter in the high-frequency region of Nyquist plot was used to verify
the surface changes of the chip.
Two sensing modes were carried on for the detection of E2: (1) The round-o mode.
In which the background resonance frequency (F0) was measured using a blank sensing
chip, then the sensing output was measured again as F1 after it was incubated in 40 L
of an E2 standard solution placed in a humid environment at 35C for 1 h, rinsed with
water and dried by nitrogen to remove any adhering E2. In this way, the frequency shift
(F), arising from antigen binding, was obtained as the dierence of F0 to F1. (2) The
dynamic mode. The sensing signal was acquired immediately when the chip was placed
in either 30 L of PBS or 30 L of a known E2 standard solution. There is a dynamic
output of resonance frequency along with time; thus, a rapid quantication of E2 was
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1393

achieved by estimating the slope of the rst linear segment of sensing signal versus
time, related to the E2 concentration in a rst-order anity interaction.

2.4. The pretreatment of real samples for sensor behaviour evaluating

In evaluating the practical applicability of the developed sensing chip, a facial cleanser
and a skin milk were used as samples. According to the detailed list of carcinogenic
hormones in cosmetics, it is warning that the probably illegal addition of E2 will induce
the risk of carcinogen from those cosmetics with the eect of eliminating acne, whiten-
ing skin or reducing wrinkles. It has been prohibited in our country since the strength-
ening management of government, but some negative news still be exposed
occasionally. In our work, accurately weighted 0.4 g of a sample was ultrasonically
mixed with 4 mL of ethanol. The supernatant was collected after the solution was
centrifuged at 3000 rpm for 5 min. After being evaporated to dryness, the residue was
dissolved in an ethanolTriton XPBS (1:1:8 v/v) mixture.

3. Results and discussion

3.1. Conrmation of sensor construction
EIS was used to conrm the construction of sensing matrix on gold-coated quartz chip.
As shown in Figure 1(b), with FeCN3 6 as electrochemical probe, the EIS showed the
change of insulation of chip surface. In Nyquist diagram, there is a semicircle corre-
sponding to Ret (a most important parameter to conrm the surface change), followed
by a linear portion (Warburg impedance, reecting the semi-innite linear diusion of
redox probe in solution). The single-capacitance arcing means a typical RC equivalent
circuit (approximately Rs(Q(RetW)), as inserted). Here, the Q represents the constant
phase element, which implies a non-intuitive capacitance of sensing matrix other than
the double layer due to surface roughness, distributed reaction rates, varied thickness or
composition, and non-uniformed current distribution.
From the diameter of the semicircle in the Nyquist plot, the Ret of a bare chip (curve
a) was estimated to be 194 , indicating a rather fast electron transfer. As a result of
electrostatic repulsion by the carboxyl group (COO) on a 3-MPA-modied chip surface
towards FeCN3 6 , the diameter of the corresponding semicircle (curve b) has increased
(853 ). This result supports the formation of the 3-MPA SAM on the chip surface. After
activating the modied chip with EDC/NHS, the diameter of the semicircle (curve c)
increased more (1634 ), again demonstrating the diculty in electron transfer at the
chip/electrolyte interface. After attaching the antibody on the sensing chip, the semi-
circle diameter (curve d) further increased (3220 ), owing to the large resistance for
electron transfer from those giant biomolecules. Curve e was obtained when the sensing
surface was saturated with the antibody.
The QCM is also a good tool to characterise the formation of sensing interface. As
shown in Figure 1(c), the resonance frequency decreased along with the 3-MPA assem-
bling, EDC/NHS activation and antibody immobilisation for 340, 151 and 304 Hz, clearly
demonstrated the change of surface loading quantity on quartz chip during the proce-
dure of surface functionalisation.
1394 D. CHEN ET AL.

3.2. Optimisation of experimental conditions and properties of sensing chip

The 3-MPA SAM is the basic of whole process of sensing matrix construction, while the
period of this operation is very important to ensure an excellent foundation. As shown in
Figure 2(a), Ret gradually increased along with the time till 12 h. After that, Ret exhibited
a plateau, indicating a full coverage of 3-MPA on the gold surface. Therefore, a 12-h
period appears to be adequate in assembling of 3-MPA. Besides, the activation of EDC/
NHS is also an important factor for sensing property because it would aect the
eciency of antibody loading. As shown in Figure 2(b), Ret increased when the activation
time was prolonged to 5 h, beyond which Ret was observed to begin decreasing. This is
likely to be caused by the hydrolysis of the intermediate, which regenerated the
carboxyl and released an N-substituted urea [34]. Therefore, 5 h was chosen as the
optimal activation time. Meanwhile, the concentration ratio of EDC to NHS was also
studied, and the results were displayed in Figure 2(c). Ret increased when the ratio of
EDC to NHS was gradually increased to 8:1, over which Ret began to decrease due to the
hydrolysis of EDC, which produces the regenerated carboxyl group. Undoubtedly, the
concentration ratio of EDC to NHS for 8:1 is the best choice. The quantity of antibody
loading on sensor surface plays a core role in the detection of antigen. As shown in

4000
a
3000 b

3000
2500
Ret /
Ret /

2000 2000

1500
1000
6 8 10 12 14 16 2 3 4 5 6
Time/h Time/h

d
c 90
4500
Ret / 103

60
Ret /

3000
30

0
1500
2:1 4:1 5:1 6:1 8:1 10:1 0.0 0.2 0.4 0.6 0.8 1.0
CEDC:CNHS m /g

Figure 2. The optimised experimental condition of (A) the time of MPA self-assembly; (B) the
activation time of EDC/NHS; (C) the concentration ratio of EDC to NHS and (D) the loading amount
of antibody.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1395

Figure 2(d), the Ret increased from 3223 to maximal 84470 , and then, it did not
increase any more. It is reasonable that sucient antibodies were bonded on the
sensing chips.
As generally acknowledged, to block the unreacted succinimidyl group by BSA is a
good way to avoid non-specic conjunction of impurity proteins on sensor surface. We
had checked the dierence of frequency output before and after the blocking; it
presented a dierence less than 1%, which means almost fully saturated covering of
E2 antibody on chip surface. This result supports the verdict of excellent specicity of
resultant sensor.
Under these optimised experimental conditions, as shown in Figure 3(a), a constant
frequency of ~7991,223 Hz was gained at the resultant sensor (curve a), and this output
kept the same (~7991,222 Hz) after the sensor was placed in blank PBS for 1 h of
incubation (curve b). Then, the frequency output decreased to ~7991,208 Hz if the
sensing chip was incubated in an E2 solution (curve c). In this way, a calibration plot
was constructed based on the decrease of frequency obtained in dierent standard E2
solutions, as shown in Figure 3(b). The regressive equation is F 4:16 5:86CE2

Figure 3. (A) The frequency output of sensing chip for (a) background; (b) incubated in blank PBS
and (c) incubated in E2 solution (2 g mL1); (B) the calibration curve for E2; (C) the frequency output
of sensing chip during 7 days and (D) the frequency output of sensing chip with the interference of
coexisting substances (20 g mL1) for the determination of E2 (2 g mL1).
1396 D. CHEN ET AL.

(R2 = 0.9934) within a linear range of 0.110 g mL1 and a detection limit (LOD) of
0.04 g mL1 (according to IUPAC rule, after the detections of 20 times for blank
solution, the standard deviation [SD] could be calculated). Thus, the limit of detection
can be calculated from it and the sensitivity of the sensor (slope of the calibration curve,
S): LOD = 3 SD/S).
The reproducibility of sensor preparation was evaluated with ve parallel chips for
3.3% of RSD. Also, the output of the sensing chip does not show remarkable change
over a 7-day period (Figure 3(c)) by storing at 4C between every two tests, supporting
its good stability. Selectivity is an extremely important evaluation criterion of the
performance of immunosensor. Figure 3(d) shows the disturbance of some possible
coexistent compounds in cosmetic products on the determination of 2 g mL1 E2
including 1,3-propanediol, glycerol, polyethylene glycol, sodium dodecyl sulphate. The
values of F by E2 or doped with 10 times concentrated impurities had no dierence,
indicating that there was no interference from possible coexistent compounds with a
tolerance of at least 300.
As previously described, the sensing chip needed to be incubated in sample solution
for at least 1 h before the determination. Thus, there is a severe limitation in terms of the
timeliness of target assay. Alternatively, we successfully achieved to gain the sensing
output with a dynamic mode, fast and credible. While 30 L of solution was dropped on
the QCM sensor, it merely covers the chip as a thin liquid lm, the mass-transfer
eciency is greatly promoted by the high-frequency oscillation of quartz chip to rapidly
obtain an instantaneous response. The results are shown in Figure 4(a). In these experi-
ments, a proportionally decrease in frequency output with E2 concentration was
obtained in tens of seconds after the introduction of an antigen solution into the cell.
There was no apparent frequency change obtained in a blank solution (curve a); mean-
while, the frequency decreased accordingly as curve b (0.2 g mL1) to f (1 g mL1)
upon the increased concentration of E2. In this mode, a calibration was established
between obtained F in those standard E2 solutions, as shown in Figure 4(b). At the
same time, we found that the slope of the rst linear segment of sensing curves in
Figure 4(a) also increased along with the arising E2 concentration. When the slope of
each curve was calculated within the rst 5 s in Figure 4(a), a linear calibration plot is
obtained as shown in Figure 4(c). The regress equation is k 0:135CE2  0:0502,
r = 0.989 (here, the k represents the slope of output signal of the rst 5 s, F/t), with
a detection limit of 0.06 g mL1, only a little attenuation to 0.04 g mL1 estimated
using the round-o mode. On the whole, it is accordance with the rule of rst-order
reaction (obeys to Langmuir equation [35]); so, there is a constant reaction rate during
the early stage of incubation then gradually reduced. As we all know, this reaction rate is
linearly proportional to the reactant concentration in solution, thus to be picked up from
sensing output to quantify the antigen concentration.
There are already many reports about the detection of estradiol, as listed in Table 1.
Compared to them, our sensor exhibits a medial sensitivity but wider linear range and
better reproducibility. In report of that QCM sensor [36], the E2 antigen (regarded as a
hapten) required to be coupled with the protein to form the holoantigen, via a complex
pre-derivation, thus to promote the sensitivity of mass-sensing. As the case of direct
sensing for hapten in our work, although the sensitivity is somewhat lower, but our
sensor provides a second rate fast response with dynamic mode detection, especially
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1397

a f b
18 20
e
d 15

F/ Hz
F/ Hz

12
c
10
6 b

5
0 a
2 4 6 8 10
0 40 80 120
-7 -1
time/s CE2 10 g/mL

c
1.2
k / Hz/s

0.8

0.4

0.0

0 2 4 6 8 10
-7 -1
CE2 10 g/mL

Figure 4. (A) The frequency output of sensing chip with a dynamic mode for 30 L of (a) PBS, (bf)
2.0, 4.0, 6.0, 8.0 and 10 107 g mL1 of E2; (B) the calibration plot of f upon E2 and (C) the
calibration plot of the responding slope in rst 5 s versus E2.

adaptable for point-of-care usage. It meet the requirements of The hygienic standard
for cosmetics of China for detection of estrogen (in this standard, the detection limit of
E2 by HPLC is 40 g g1) [37]. On other hand, even if there is the requirement of
detection for those lower concentrated samples, the sensor is still useful by the jointing
with suitable enrichment technic such as SPME, LPME and so on. Compared with other
electrochemical methods, the sensor we reported here does not need any electroche-
mical reactive probe and, meanwhile, can be implemented with simplest operation and
fastest achievement.
The feasibility of acquired sensing response within several seconds would allow the
application of developed immunosensor for rapid, real-time or in situ uses. As we all
know, the most remarkable advantage of immnosensing technique is the specicity
from the anity reaction between antigen and antibody. Also, the high sensitivity of the
QCM technique will enhance the sensing capability based on high-performance mass
transduction. But, the shortage of detection time is a crucial obstacle for its real
application. From our research, it is possible that we can seek a balance between
them to achieve a high-quality immunosensor with good specicity, fast response and
ample sensitivity.
 1398
D. CHEN ET AL.

Table 1. The performance comparison of dierent methods for E2 detection.

Linear range Reproducibility RSD
Technique LOD (g mL1) (g mL1) Sensitivity (%) Reference
GC-MS 7.7 106 5 1062 104 [38]
MEKC 1.13 3.6100 [39]
CV 7.7 107 1.0 1062.5 104 7.17.4 (n = 5) [40]
DPV 9.3 105 1.0 1061.0 102 8.815.5 (n = 18) [41]
DPV 5.0 105 2.5 1055.0 104 [42]
Amperometry 2.5 107 2.44 1062.5 103 5.88.3 (n = 4) [43]
Amperometry 3.5 106 ~1.5 103 0.61 106 A g1 mL1 6.811.6 (n = 4) [44]
SWV EIS 1.8 105 ~1.2 103 3.7 106 A g1 mL1 [45]
2.6 105 ~1.0 103 5 106 k g1 mL1
QCM 0.05 0.55 [46]
QCM 1.7 107 1.0 1061.0 103 [36]
QCM (round-o mode) 0.04 0.110 5.86 Hz g1 mL1 3.3 (n = 5) This work
SWV: Square wave voltammetry; DPV: dierential pulse voltammetry; MEKC: Micellar electrokinetic chromatography; CV: cyclic voltammetry.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1399

3.3. Determination of E2 in real samples

The developed immunosensor was employed for detection of E2 in spiked cos-
metics to evaluate its feasibility. In round-o mode, as listed results in Table 2, the
recoveries of E2 are in the range of 92.6103%. In dynamic mode, as listed in
Table 3, the recoveries of E2 were in the range of 98.5116%. These results indicate
that the immunosensors response is satisfactory to be employed in real sample
assays.
The main source of E2 in environment may come from the emissions or unreason-
able uses of related production companies. It is once reported to be added in
cosmetics as facial cleanser or skin milk some times. The zeroed results of our
detections demonstrate the improved condition to restrict the illegal addition or
discharge of E2 under the strengthening of corporate governance from government
and industrial circles in our region. This ameliorative circumstance gives us the
condence to further perfect the quality and safety of foods, cosmetics, medicines
and the environment.

4. Conclusion
An immune QCM-sensing chip was developed to recognise E2 antigen by its antibody
based on anity interaction. The change of resonance frequency is related to corre-
sponding concentration of antigen. The results indicate that detection limit of the
sensing chip reached the level of 40 ng mL1 with good stability and reproducibility,
with estradiol as the model in round-o mode. Moreover, the results of detection in
dynamic mode with an LOD of 60 ng mL1 demonstrate that the sensing response time
could be shortened to several seconds to realise the rapid detection. Both detection
modes can be employed for targeted applications.

Table 2. The detected results in spiked samples with round-o

mode.
Sample Spiked (g g1) Found (g g1) Recovery (%)
1 5.0 4.81 96.2
2 10.0 9.21 92.1
3 20.0 20.4 102
4 40.0 39.2 98.0

Table 3. Results of E2 determination with dynamic mode.

Sample Spiked (g g1) Found (g g1) Recovery (%)
1 5.0 5.82 116
2 8.0 8.27 103
3 10.0 11.2 112
4 20.0 19.7 98.5
1400 D. CHEN ET AL.

Acknowledgements
We are grateful for the nancial supports of this research from National Natural Science
Foundation of China: (grant numbers 21175096 and 21375091).

Disclosure statement
The authors declare that there is no conict of interest regarding the publication of this article.

Funding
We are grateful for the nancial supports of this research from National Natural Science
Foundation of China (Grant Numbers: 21175096 and 21375091)..

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