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Ultrasensitive one-step rapid visual detection of bisphenol A in watersamples by label-free aptasensor

Zhanlong Mei a,1, Huaqin Chu a,b,1, Wei Chen a,n, Feng Xue a, Jian Liu a, Huaneng Xu b,Rui Zhang a, Lei Zheng a,n

a School of Biotechnology & Food Engineering, Key Lab of Food Nutrition & Safety of Anhui Province, Hefei University of Technology, Hefei 230009, PR Chinab School of Food Science & Technology, State Key Lab of Food Science & Technology, Jiangnan University, Wuxi 200234, PR China

a r t i c l e i n f o

 Article history:

Received 14 May 2012

Received in revised form

12 June 2012

Accepted 14 June 2012Available online 1 July 2012

Keywords:

Gold nanoparticles

BPA detection

Aptamer

Label-free

Food safety

a b s t r a c t

A simple, one-step, rapid method to detect bisphenol A (BPA) using a label-free aptasensor is presented.

A high selective anti-BPA aptamer was added to gold nanoparticles (GNPs) to prepare the label-free

aptasensor for BPA, which maintains good tolerance of GNPs under aqueous conditions with high salt

concentrations. With the presence of BPA in the aptasensor system, the GNPs would aggregate by

competitive binding of BPA and aptamer. Detection results can be visualized by the aggregation-induced

color change of GNPs without the use of any instrumentation. The limit of visual detection (LOD) was

found to be 0.1 ng/mL by naked-eye observation, which was competitive to some current rapid BPA

detection methods, even some instrumental based methods. Besides the obvious advantages, including

reduced detection time and operation procedures, the results of this method meet the various detection

requirements for BPA and are comparable to the traditional ELISA and instrument-based methods. The

proposed one-step, label-free method was successfully used to determine BPA in actual water samples.

Crown Copyright  &   2012 Published by Elsevier B.V. All rights reserved.

1. Introduction

Bisphenol A (BPA) is a widely used chemical and raw material for

epoxy resin, flame retardants and polycarbonate in plastic and paper

industries (Alonso-Magdalena et al., 2010; Bailin et al., 2008). Since

the first discovery of the release of BPA from polycarbonate bottle at

high temperature and pressures, greater attention has been paid to

research on BPA toxicity ( Jensen et al., 1995; Sharp et al., 1993).BPA is

currently regarded as an environmental endocrine disrupting chemi-

cal (EDC) that is potentially estrogenic. Data from multiple sources

indicate that the amount of PBA to which humans are exposed may

cause adverse health effects (Vandenberg et al., 2010). Moreover, in

adults, urinary BPA concentrations are positively associated with

cardiovascular disease, diabetes and impaired reproductive capacity.Recent in vivo and in vitro animal studies have documented that even

exposure to very low concentrations of BPA can significantly affect

cell proliferation and health, with the maximal effect at approxi-

mately 1010–108 M   (Salian et al., 2011;   Sheng and Zhu, 2011).

Meanwhile, researches also demonstrate that BPA can cause effect in

animal models at doses in the range of human exposures, indicating

that it can act at lower doses than predicted from some  in vitro and

in vivo assays (Rechter et al., 2007; Vandenberg et al., 2007; Wetherill

et al., 2007). Thus, ultrasensitive and rapid detection of BPA in food

samples and food containers is of great importance for assuring

human health. In particular, for drinking water, BPA released from the

plastic packages or cups should be accurately monitored on-site for

food safety (Hengstler et al., 2011; Vandenberg et al., 2007).

Various conventional instrument-based methods have been

widely adopted for detection of BPA, including high pressure

liquid chromatography (HPLC) (Inoue et al., 2000; Watabe et al.,

2004), liquid chromatography (LC) (Zafra-Gomez et al., 2008), and

gas chromatography coupled with mass spectrometry (GC–MS)

(Kawaguchi et al., 2004). Samples must be pretreated adequately

to improve the evaporative ability of the analyte in GC-based

analytical methods, which is complicated and prone to pretreat-ment error. It has been reported that BPA can be detected by a

standard HPLC system with LOD of 200 ng/mL in European Union

and with a LOD of 0.04 ng/mL using a fluorescent detector

(Kuroda et al., 2003). However, all of these instruments are

expensive, have strict sample preparation requirements and

require trained operators. Immunoassay-based methods have also

attracted considerable attention for BPA detection due to the high

sensitivity and comparable low costs of these techniques

(Freymuth et al., 1986;   Kim et al., 2007;   Zhao et al., 2002;

Zheng et al., 2008). Determinations with immunoassay-based

methods, however, are strongly dependent on the quality of the

prepared antibody, while there have been some reports about

Contents lists available at  SciVerse ScienceDirect

journal homepage:  w ww.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$- see front matter Crown Copyright &   2012 Published by Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bios.2012.06.027

n Corresponding authors. Tel./fax:  þ86 551 2919396.

E-mail addresses:  chenweishnu@163.com,

chenweishnu@hfut.edu.cn (W. Chen),  lei.zheng@yahoo.cn (L. Zheng).1 These two authors contributed equally to this paper.

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nonspecific binding to the analogs, such as Bisphenol B (BPB) and

4,4-Bis-(4-hydroxyphenyl) valeric acid (Marchesini et al., 2005;

Ohkuma et al., 2002). Besides, according to the recent published

review about monitoring BPA in different biological samples

including urine, serum, saliva and tissue etc., even some of the

instrumental based methods and ELISA methods could not meet

the requirements of risk assessments of BPA at low levels

(Vandenberg et al., 2010). Thus, developing new, rapid, field-

deployable methods for BPA detection with high sensitivity andspecificity is of great importance for food safety and human

health.

Aptamers have received much attention since the first reports

on selection by both Ellington and Szostak (1990) and Tuerk and

Gold (1990). Aptamers screened by the classic systematic evolu-

tion of ligands by exponential enrichment (SELEX) technique have

the inherent characteristics of a high association constant, high

specificity, easy production and modification, cost-effectiveness

and stability (Xu et al., 2009). Aptamers, a potential replacement

probe for antibodies in the field of analytical science, have been

widely used for the development of novel detection methods.

Extensive research has been conducted using aptamer as recogni-

tion probes for different targets, including metal ions, organic

molecules, peptides, proteins and even whole cells (Bang et al.,

2005;  Ho and Leclerc, 2004; Huang et al., 2005; Lin et al., 2006;

Stojanovic and Landry, 2002; Wilson and Szostak, 1999). To the

best of our knowledge, however, there is still no report on the use

of an aptamer probe for rapid, in-field BPA detection.

Herein, we present a one-step, rapid, label-free aptasensor for

BPA detection. The colorimetric detection results were visible to

the naked eye without the assistance of any instrumentation.

More importantly, the sensitivity of this one-step, label-free

aptasensor is comparable to that of the traditional ELISA and

some instrument-based methods, which is of great importance

for the current BPA risk assessments through the toxicokinetic

models. This label free BPA aptasensor is inexpensive, less time-

consuming, easy to operate and will likely facilitate sensitive, on-

site detection of BPA.

2. Materials and methods

 2.1. Chemicals

Bisphenol A (BPA) was purchased from J&K Chemical Com-

pany, Shanghai. Chloroauric acid (HAuCl4) and sodium citrate

were obtained from Sigma-Aldrich, USA. Other normal chemicals,

including sodium chloride (NaCl), disodium hydrogen phosphate

(Na2HPO4) and monosodium phosphate (NaH2PO4), were all

purchased from Shanghai Chemical Reagents Company (Shanghai,

China). Anti-BPA aptamer was synthesized by Shengon Biotech-

nology Co. Ltd. (Shanghai, China). The sequence of the BPA was

designed as 5–CCG GTG GGT GGT CAG GTG GGA TAG CGT TCCGCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA–3 according

to reported studies ( Jo et al., 2011). Millipore Milli-Q ultrapure

(418 MO) water was used throughout the whole research.

Actual water samples were taken from the tap water in our

laboratory and Caohu Lake in Anhui province, China. The UV–vis

spectra were measured with a UV 5100 spectrophotometer

(Wanyi Science & Technology Co. Ltd., Hefei, China).

 2.2. Methods

Gold nanoparticles were prepared by the traditional chemical

reduction methods. Briefly, 0.1% chloroauric acid solution was

added to a flask and heated until boiling. Sodium citrate was

subsequently added quickly to the boiling solution. An obvious

color change of the reaction mixture was observed from trans-

parent to dark blue and finally wine red. The mixture was further

boiled for another 5 min and cooled to room temperatures.

Different amounts of sodium citrate solutions were added in the

process of preparation to obtain GNPs with various diameters.

The GNPs were subsequently cultured with anti-BPA aptamer

for 15 min and purified by centrifugation at the speed of 

10,000 rpm for 5 min to obtain the aptamer-protected GNPs. To

test the salt tolerance of aptamer-protected GNPs, sodium chlor-ide (NaCl) solutions at different concentrations were added to the

aptamer–GNP solutions. The optimal concentration of NaCl was

defined as the highest concentration of NaCl which did not induce

the aggregation of aptamer–GNPs.

For BPA detection, anti-BPA aptamer was initially added to

form the GNP–aptamer complex, followed by addition of a NaCl

solution at a certain concentration. Subsequently, BPA standard

solutions at different concentrations were added to different

aliquots of aptamer–GNPs/NaCl solutions and cultured for

another 15 min. The qualitative results were visually determined

by naked-eye observation while the quantitative results were

obtained by measurements of UV–vis spectra.

3. Results and discussions

A schematic of the sensing principle is shown in   Fig. 1. It is

easy to understand that salt solutions at high concentrations

could induce GNP aggregation by the electrostatic screening

effect. Visually, the color of the GNP solution changes from wine

red to blue during the aggregation. Moreover, the characteristic

plasmon peak of GNPs, which is very sensitive to the diameter of 

GNPs in the solution, changes accordingly. Following this rule,

considering the sensitivity of the method, GNPs with the strong

surface plasmon signal, which is highly related to the size of 

prepared gold nanoparticles, and the color variation under the

condition of BPA induced aggregations should be chosen as the

optimal indicator (See size optimization result in  Fig. S2). In thisresearch, GNPs with the diameter of about 18 nm were adopted

(TEM image shown in Fig. S1). The intensity of GNP aggregation

can be analyzed from the variations in the plasmon peak;

a principle upon which many methods are based (Li et al., 2010;

Liu et al., 2011). We have previously reported on a colorimetric

method for melamine detection based on plasmon peak varia-

tions (Kuang et al., 2011). Herein, we first used an anti-BPA

aptamer to protect GNPs and enhance their salt tolerance. Then,

BPA was added to the protected-GNPs system to competitively

Fig. 1.   Schematic diagram of rapid label-free BPA detection.

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bind with the aptamer due to the stronger affinity effects includ-

ing the electrostatic effect, hydrogen bond effect, and spatial

matching effect etc. ( Jo et al., 2011), which decreases salt

tolerance. In the presence of high salt concentration, the GNPs

re-aggregate and cause a color change in the detection system.

Finally, the relationship between BPA concentrations and varia-

tions of the plasmon peak can be developed, making both

qualitative and quantitative detection of BPA possible.

We first tested the salt tolerance of the prepared pure GNPs asa control. The images of the GNPs solution demonstrate what

happens in the presence of increased NaCl concentrations (see

details in Fig. S3). Only 10 mM NaCl induced GNP aggregation (Fig.

S3). After culturing with the aptamer solution, the salt tolerance

was greatly enhanced and GNP aggregation was not observed until

the NaCl concentration reached 40 mM (Fig. S4). From this result,

we can confirm that the ssDNA aptamer is indeed adsorbed onto

the surface of GNPs and protects the stability of GNPs under

conditions of high salt concentrations. It should be pointed out

that the concentration of the salt solution also needs to be

optimized for improvement of sensitivity. For one thing, the

higher concentration of used salt solution is good for the improve-

ment of sensitivity. For the other, higher concentration of used salt

solution would decrease the stability of intra-assay and reprodu-

cibility of the sensing system. Of note, due to the pretreatment of 

aptamer protected gold nanoparticles with centrifugation, residue

citrate was not considered for the optimization of the detection.

Thus, 15 mM NaCl was adopted as the suitable concentration for

further experiments in this research. Besides, the ratio between

gold nanoparticles and aptamer could affect the final sensitivity.

Too many aptamers in the sensing system would sacrifice the

sensitivity while too few would decrease the stability of the

sensing system (see details of the gold nanoparticle/aptamer ratio

results in   Fig. S5). 0.05 mM aptamer (final concentration) in the

100 mL 40 nM gold nanoparticle was adopted for following BPA

detection. Meanwhile, we also confirmed the effect of BPA to the

aggregation of gold nanoparticles as the control (Fig. S6). BPA at

different concentrations was added into the pure gold nanoparti-

cle solution and cultured for 15 min. From the results shown inFig. S6, it is found that the color of the gold nanoparticles is not

changed under the condition of added BPA at the concentration

from 0.01 to 10,000 ppb when compared with blank sample.

Therefore, we could confirm that the GNPs aggregation is not

induced by the existence of BPA.

Then, under optimized conditions, BAP standard stock solu-

tions at different concentrations were added to the aptamer-GNPs

solutions. After culturing for approximately 5 min, the sensing

results were first determined by naked-eye observation and then

further quantified by measurements of UV–vis spectra. As shown

in Fig. 2, there was an obvious color change due to the addition of 

BPA. More importantly, we found that only 0.01 ng/mL (ppb) BPA

in the detection system could induce a distinguishable color

change from red to slightly purple compared with a blank sample.

With an increase in the concentration of added BPA in the

detection system, the color changed to purple at 1 ng/mL, a dark

purple at 50 ng/mL and to blue at concentrations higher than

100 ng/mL. The following two aspects can be surmised from these

results. First, the addition of BPA induces the aggregation by

competitive binding with the aptamer. Second, 0.1 ng/mL is the

visual limit of detection (visual LOD) of this label-free aptasensorby naked-eye observation, which is much lower than the Chinese

Standard requirement of 50 ng/mL (the red dashed rectangle in

Fig. 2). The reason for the color change could be explained as

previously described. Namely, the added BPA could competitively

bind with the aptamer and induce the release of aptamer from the

surface of GNPs, which decreases the salt tolerance of GNPs and,

in turn, induces GNPs aggregation.

To further demonstrate the competitive binding of aptamer with

BPA in the detection system, some additional characterization was

carried out. It is well known that the conjugation constant of 

aptamer is stronger than that of antibody and nonspecific adsorption

between aptamer and GNPs. The interaction between the aptamer

and target analyte BPA includes electrostatic force, van der Waals

force and spatial configurations and measurements of circular

dichroism (CD) spectra were conducted to characterize the aptamer

conformation after binding to the target BPA. The CD spectra of the

pure aptamer before and after the binding of BPA (with the removing

of gold nanoparticles and aggregations) were compared (see Sup-

porting Information Fig. S7). It is clear that the CD spectra of aptamer

after binding are stronger than that of the unbound aptamer. We also

found a new shoulder peak of the aptamer after binding, which

indicates the new structural conformation of the aptamers in the

solution. Collectively, all of these results confirm the binding of 

aptamer and target BPA, which induces the release of aptamer from

GNPs and, in turn, induces GNP aggregation.

UV–vis spectroscopy is often used to assess the stability of 

GNPs in solution as the intensity and peak position are both

sensitive to any aggregation that occurs. Therefore, we also

further interrogated the sensing system for quantitative BPAdetection by the UV–vis results. As shown by the UV–vis results

in Fig. 3, with an increase in the concentration of added BPA in the

label-free aptasensor system, the characteristic plasmon peak of 

GNPs red shifted from the original 521 nm to 680 nm. It should be

noted that in the presence of 0.01 ng/mL BPA, the peak intensity

at 521 nm decreased slightly and an observable red shift occurred

compared with that of when no BPA was in the system. With an

increase of BPA in the detection system, the characteristic peak

intensity decreased further while the new peak red shifted much

more with the increased intensity. Apparently, at a concentration

of 1000 ng/mL, the peak at 521 nm almost completely disap-

peared and a new integrated peak formed at 680 nm. The UV–vis

spectra did not change with any further increase of BPA. The

quantitative detection results were achieved by determining therelationship between the absorption ratio ( A680/ A521) and BPA

concentrations. A good linear correlation was observed ranging

from 0.01 to 100 ng/mL (Fig. 3, with the   R2 of 0.9904). Each

experimental point in   Fig. 3   is the mean of five experiments

carried out under identical conditions. It should be noted that the

red, vertical dashed line in Fig. 3 represents the requirements of 

the Chinese Standards and the standards of the United States

Federal Drug Administration (FDA) (50 ng/mL), which is lower

than those of Japan (2.5 mg/g) and EU (3 mg/g kg). The black,

vertical dashed line in Fig. 3   indicates the calculated LOD of the

developed aptasensor-based BPA detection method, 0.049 ng/mL 

(3 standard deviations above the blank). From these comparisons,

it can be concluded that the LOD of the developed aptasensor-

based method is much lower than the requirements of other

G BLOD

Fig. 2.  Photograph of label-free BPA detection results. (For interpretation of the

references to color in this figure, the reader is referred to the web version of this

article.)

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countries and comparable to that of the ELISA and instrument-

based methods. More importantly, no more than 30 min is

required for BPA detection by this one-step, label-free aptasensor

method, which is the most obvious advantage compared with

other BPA detection methods, without considering the sample

pretreatments, operation procedures, and cost of the detections.

The specificity of the developed method was also tested using

different analogs in the BPA label-free aptasensor system. Three

kinds of analogs, including bisphenol B (BPB), 4, 4 0-biphenol (BP)

and 6F bisphenol A (6F-BPA) were added to the BPA detection

system at different concentrations (see   Fig. S8   for chemical

structures of the analogs). As shown in  Fig. 4, addition of 0.1 ng/mL 

BPA induced a distinguishable color change compared with the

blank. However, 10 or 100 ng/mL BPB did not induce any obvious

color change in the detection system. Similarly, 100 ng/mL BP and6F-BPA in the detection system did not induce any color change.

Furthermore, the corresponding UV–vis spectra of the sensing

system were recorded and a red shift was only observed under

the condition of 0.1 ng/mL BPA, which confirmed the excellent

specificity of the developed aptasensor-based BPA detection

method. This high specificity of the method is attributed to the

high specificity and binding coefficient of the selected aptamer,

which is in accordance with previous selection researches.

Finally, the application of this one-step, label-free aptasensor

BPA detection method was evaluated by testing spiked samples of 

tap water and water from Caohu Lake. A known quantity of BPA

was added into the two different water samples and measured

using the developed detection method. Meanwhile, the spiked

water samples were also analyzed by HPLC.

The actual spiked tap water samples were directly detected with

naked-eye observation with the developed label-free aptasensor

method without any further sample pretreatments. The results

shown in   Fig. 5   indicate that direct detection of BPA in actual

Fig. 3.   UV–vis spectrum of aptasensor at different BPA concentrations (a) and

(b) calibration curve for BPA aptasensor. (For interpretation of the references to

color in this figure, the reader is referred to the web version of this article.)

Fig. 4.   The specificity testing of the developed aptasensor based BPA detection method.

Fig. 5.   Detections of BPA spiked tap water samples.

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samples of tap water samples is totally possible without the use of 

any instrumentation and without the need for sample pretreat-

ments. The actual spiked samples of water from Caohu Lake were

filtrated and detected by the label-free aptasensor method and HPLC

method with the identical sample pretreatments procedures. The

data presented in Table 1 indicate that results from the developed

aptasensor method correlate well with those from HPLC. However, it

also should be noted that the SD of the recovery detection of the

spiked samples was a little higher than the conventional instru-

mental based methods at some specific concentrations. This may be

induced by the intrinsic properties of this kind of colorimetric

methods. The quantitative analysis is based on the intensity ration

of two characteristic peaks, which is not much greater than the

noise of the technique itself and it normally creates obvious

deviation in quantitative measurement.

4. Conclusion

In the present study, we demonstrated the use of a novel, one-

step, label-free aptasensor-based colorimetric method for BPA detec-

tion. An anti-BPA aptamer was used to protect GNPs under conditions

of high salt concentrations. Added BPA competitively bound with theaptamer and induced the release of aptamer from the surface of 

GNPs, which, in turn, induced GNP aggregation. The color change and

corresponding plasmon peak shift exhibited a linear relationship with

the concentration of added BPA. Qualitative and quantitative detec-

tion of BPA were successfully realized with LODs of 0.1 and 0.049 ng/

mL by naked-eye observation and measurements of UV–vis spectra,

respectively. All detection results meet the Chinese Standards as well

as the requirements of other countries. Results indicate that this

label-free, aptasensor-based, one-step BPA detection method is com-

parable to the traditional ELISA and instrument-based methods, and

detection of actual spiked samples confirms a good correlation with

traditional detection methods. More importantly, this method could

be extended to the detection of other analytes by simply replacing the

aptamer sequence. Work in our laboratory on optimization of thequantitative detections and signal enhancement for BPA detection

and general applications are ongoing.

 Acknowledgments

This work is financially supported by the Huangshan Young

Scholar Fund of Hefei University of Technology (407-037025), the

National Basic Research Program of China (2012CB525003), the

Science and Technology Research Project of General Administra-

tion of Quality Supervision, Inspection and Quarantine of PR China

(201210127), the 12th Five Years Key Programs (2012BAK08B01,

2012BAK17B10, SS2012AA101001) and the National Science

Foundation of China (no. 20906039).

 Appendix A. Supporting information

Supplementary data associated with this article can be found

in the online version at   http://dx.doi.org/10.1016/j.bios.2012.06.027.

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 Table 1

Detection results of spiked samples by label-free aptasensor and HPLC.

Caohu lake

samples

Spiked

(ng/mL)

Measured (ng/mL)a Recovery (%), (%)7SD,  n¼5

Apt asensor H PLC Apt asensor H PLC

1 4.5 4.370.8 4.570.3 95.6717.4b 10076.7

2 11.3 10.971 .4 1 1.170.4 96.5712.4b 98.273.5

3 0.50 0.4870.07 0.4970.04 96.071 4.0 9 8.078.0

4 105 10972.9 10475.1 103.872.76 99.0574.95 98 9976.3 9870.4 101.076.43 101.776.0

a Each value is the average of five samples detected at the same conditions.b The obvious SD of the detection was determined by the intrinsic character-

istic of the developed method and explained in the main text.

 Z. Mei et al. / Biosensors and Bioelectronics 39 (2013) 26 –3030