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Sensors and Actuators B 191 (2014) 326– 331

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locate /snb

apid and multiple detections of staphylococcal enterotoxins bywo-dimensional molecularly imprinted film-coated QCM sensor

an Liu, Xiaoli Li, Xinhua Ma, Guorong Ou, Zhixian Gao ∗

ianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Health and Environmental Medicine,ianjin 300050, PR China

r t i c l e i n f o

rticle history:eceived 21 April 2013eceived in revised form9 September 2013ccepted 20 September 2013vailable online 29 September 2013

eywords:

a b s t r a c t

A quartz crystal microbalance with dissipation biosensor based on two-dimensional (2D) molecularlyimprinted film-coated by organosilanes and the template protein staphylococcal enterotoxins (SEs) in situsynthesizing on the surface of the piezoelectric quartz crystal (PQC) as transducer was developed for thedetection of SEs in real samples. By incorporated with the sensor, frequency shifts (�fs) and dissipationshifts (�Ds) were recorded and monitored in real time for the evaluation of the property of the sol–gelimprinted films for the detection of targets. Both of the working ranges for the detection of SEA and SEBwere 0.1–1000 �g/mL with the lowest detection limits 7.97 and 2.25 ng/mL, respectively. Performances

taphylococcal enterotoxinwo-dimensionaluartz crystal microbalance withissipationolecularly imprinted polymer

ol–gel

and surface structures of the fabricated 2D sol–gel films were also observed and confirmed by atomicforce microscopy. Results demonstrated that the 2D molecularly imprinted film-coated QCM could giverepeated usage and selective recognition to the template SEs. The intelligentized biosensor was employedto SEA and SEB detection in milk samples after pre-treatment. The recoveries ranged from 97.00% to114.20% and it can be used for ten times indicating that the developed method was feasible and applicable

owest detection limitin the detection of SEs.

. Introduction

Staphylococcus aureus (S. aureus) is one of the most commonlyound pathogenic bacteria and opportunistic pathogen. Staphy-ococcal food poisoning is one of the most common forms ofoodborne illness, resulting from the ingestion of staphylococcalnterotoxins (SEs) formed in foods by enterotoxinogenic strainsf S. aureus [1]. SE are one of the major causes or factors of bac-erial food poisoning outbreaks, toxigenic syndromes and one ofhe hospital infections ranging from superficial skin lesions to life-hreatening septicaemia [2]. They are divided into a number oferotypes, such as A, B, C1, C2, D, E, etc. SE is also considered to be aotential biological weapon owing to its severe toxicity comparedith previous biowarfare programs [3].

Routine detection of S. aureus in food and environment is usu-lly carried out by traditional methods based on morphologicalnd biochemical characterization [4]. However, these methodsre time consuming and tedious. In the past years, some ana-ytical methods divided into methods based on immunological

r biological testing and chemical methods based on spectralechniques have been developed for SEs detection. Thus far, mod-rn methods integrated with immunological methods, sensors

∗ Corresponding author. Tel.: +86 22 84655191; fax: +86 22 84655191.E-mail address: Gaozhx@163.com (Z. Gao).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.09.086

© 2013 Elsevier B.V. All rights reserved.

and micro/nanomaterials have been designed including enzymaticassay [5], piezoelectric-excited millimeter-sized cantilever sensor[6], piezoelectric crystal immunosensor [7–9], surface plasmonresonance (SPR) sensor [10], carbon nanotubes with enhancedchemiluminescence immunoassay [11], gold nanoparticle-basedenhanced chemiluminescence immunosensor [12] and resonantacoustic profiling [13], etc. Although they have highly/ultra-highlysensitivity with a lowest detection limit of �g to pg level, espe-cially with pg level by Campbell [6] and Yang’s report [11,12], mostof these methods are still based on Ab-Ag reactions. SEs’ antibodies,especially the monoclonal antibodies, are still the most expensiveand fragile biomaterial in immunological determination and sensorapplication which being considered as the bottleneck.

Molecular imprinting has been established as a techniquethat allows the creation of tailor-made binding sites for certainmolecules such as chiral molecules [14,15]. Molecularly imprintedpolymers (MIPs) are expected to be applied more widely as arti-ficial synthetic materials for use in molecular recognition becauseof their high affinity and selectivity. Compared with the imprint-ing of small molecules, such as maleic hydrazide [16], daminozide[17], profenofos [18], and malachite green [19] etc. which we havereported in our previous works, imprinting of biomacromolecules,

especially protein, is still a challenge [20]. Two-dimensional (2D)imprinting technology allows free access to binding sites for largermolecules and allows specific areas of proteins to be a target forimprinting [21]. In our another previous work, 2D molecularly

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mprinted sol–gel thin film coated quartz crystal microbalanceQCM) was reported for the rapid detection of staphylococcalnterotoxin B (SEB) by combining organosilanes and the templaterotein SEB on the surface of piezoelectric quartz crystal (PQC)u-electrode by in situ immobilization [20]. The sol–gel thin filmith the characteristics of mild polymerization conditions, such asH, ionic strength and water compatibility making it suitable for

mprinting proteins. In this study, we presented the universal, sim-le, rapid, sensitive and multiplex QCM sensor with dissipation foriverse SEs. Compared with QCM 200 which produced by Stanfordesearch Systems used in our previous report [20], QCM with dis-ipation (QCM-D) instrument employed here can provide multipleEs determination and better consistency. The LDL is lower thanhe former. Also, QCM-D system can be used to characterize theormation of thin films from proteins template on surfaces by theissipation measurement. To our knowledge, this is the first studyo develop a multiplex and universal QCM sensor with sol–gel thinlm imprinting technology for the multi-analysis of large biologicalolecules.

. Experimental

.1. Materials

SEB was self-prepared and purified in our own laboratory.EA, SEC1, SED and rabbit anti-SEB IgG were purchased fromhe Institute of Microbiology and Epidemiology, Beijing, China.etraethylorthosilicate (98%, TEOS), 3-aminopropyltriethoxysilane99%, APTES), and triethoxy(octyl)silane (97.5%, OTES) were pur-hased from Aldrich, USA. 11-Mercaptopropionic acid (MUA) wasbtained from J&K Chemical Co, Beijing, China. Bovine serumlbumin (BSA) and ovalbumin (OVA) were bought from Roche,witzerland. For background solution, 0.1 M phosphate bufferaline solution (PBS), pH 7.4 was used. Packages of milk (250 mL perackage with Tetra PakTM) were bought from local supermarket inianjin, China. All other reagents were of analytical grade. The ultra-urified water used was prepared by the Milli-Q system (Millipore,edford, MA), which had a minimum resistivity of 18.2 M� cm.

.2. In situ synthesis of the 2D self-assembled molecularly sol–gelmprinted film on the PQC electrodes

2D self-assembled molecularly sol–gel imprinted film was syn-hesized according to our previous studies [20]. PQC Au-electrodesere immersed in an ethanol solution of 0.1 M MUA for 24 h, rinsedith 0.1 M NaOH and water, and dried under nitrogen before use.riefly, initial sol–gel was synthesized by mixing of 8 mL of TEOS,00 �L of APTES, 400 �L of OTES, 1 mL of HCl (0.1 M), and 4 mL of2O and then sonicated for 20 min. The template molecules, 300 �L0 mg/mL SEA and SEB were respectively dissolved and vortexedvenly into 3 mL initial sol–gel tubes. 100 �L obtained imprintedol–gel was dispensed onto the PQC Au-electrodes for spin-coatingt 2000 rpm for 30 s in ambient atmosphere by a spin coater whichupplied by Jingshengweina Technology Co. Ltd., Beijing, China.hereafter, the films were dried in N2 gas. The PQC Au-electrodesere maintained for 10 min, rinsed with water to remove thehysically adsorbed sol, and then dried under nitrogen gas. Theserocedures were repeated for 3–5 times for sufficient adsorption.ll these prepared electrodes were dried for 24 h at room temper-ture (RT). Removals of the templates from the sol–gel imprinted

lm were achieved by repeatedly washing with hot water at around0◦C until no template could be detected under UV spectrometriceasurement at 280 nm. A non-imprinted polymers (NIPs) coating

lm was synthesized under identical conditions except for absencef the template molecule.

rs B 191 (2014) 326– 331 327

2.3. Fabrication of 2D molecularly imprinted film-coated QCMsensor for the rapid and multiple detection of SEs

The QCM-D system, i.e., Q-Sense E4 instrument (Q-Sense AB,Västra Frölunda, Sweden) was employed for on-line measurementwith gold-coated quartz crystals (Ø 14 mm, AT-cut) with a reso-nance frequency of 5 MHz (HRbio Biotechnology Co., LTD, Beijing,China) as the sensing elements, support and transducer for the thinsol–gel film with circular electrodes. All the Au electrodes usedwere immersed in a 1:1:5 solution of 30% H2O2, 28% ammonia, anddeionized water for 10 min at 80 ◦C. After rinsing of the surfacesrepeatedly with water, they were dried under N2 flow prior to mod-ification. Prior to the measurements, all the four QCM-D chamberswere sonicated for 5 min in a 10% alkaline cleaning solution andrinsed twice in water followed by N2 blow. The Au electrodes withthe self-assembled molecularly imprinted thin film in situ immobi-lization were carefully placed in the flow modules. After mountingthe crystal in the cell, 0.1 M PBS as the background solution wasintroduced into the cell by a peristaltic pump that produced stableflows with a flow rate of 20 �L/min. All measurements were per-formed at RT. The baseline was not recorded until the frequencystabilized for about 10 min. Afterwards, time-dependent change inthe frequency shift (�f) and dissipation shift (�D) were measuredand recorded by injection into the cell of different concentrations of0.1, 1, 10, 100, and 1000 �g/mL SEA and SEB which solved in 0.1 MPBS. After one time test, the template was washed out for anotherbinding. The Au electrode with non-imprinted film was treated asmentioned above.

2.4. Characterization of PQC Au-electrodes

The prepared activated 2D thin film coating with/without tem-plates (SEA) and template-eluted PQC Au-electrodes were washedwith water and dried under N2 flow in 50% relative humidity. Thedetailed surface topographies of these electrodes were character-ized using Nanoscope III � multimode atomic force microscopy(AFM, Digital Instrument Inc., USA) at RT, with the parameters usedas follows:

Mode: Tipping ModeTM; resonance frequency: 300 kHz,K = 40 N/m, L = 120 �m, W = 40 �m, H = 4 �m.

The Si cantilever (NSG 20, NT-MDT Inc., USA) was used for AFMimages. All the images were automatically recorded by NanoScope(R) III software (version: 5.30r3.sr3). To avoid electrostatic interac-tions, all the QCM wafers were grounded during imaging.

2.5. Specificity evaluation

For specificity evaluation, 100 �g/mL SEB, SEC1, BSA, OVA and0.1 M PBS were used in the chamber of SEA-MIP coated QCM-Dsensor. 100 �g/mL SEA, SEC1, BSA, OVA and 0.1 M PBS were used inthe chamber of SEB-MIP coated QCM-D sensor correspondingly.

2.6. Analysis of standard spiked milk sample

Pasteurized milk was bought from local supermarket. The pre-treatment procedures were followed by Zhou’s method [22]. Inthis assay, SEA and SEB were spiked at the concentrations of 5, 50,and 100 �g/mL in the treated milk samples. Briefly, 0.3 mL 300 g/Ltrichloroacetic acid was added into the microtube containing of4.0 mL of milk; and the tube was shaken several times by hand tothoroughly mix the solution. After being left to stand for 1–2 min,

the mixture was filtrated using filter paper, and a drop of 3 M NaOHwas added to adjust the pH to 7 (measured using a pH testing strip).Finally, the filtrate was refiltered using a syringe and 0.22 �m fil-ters. The extracts were used for the SEA and SEB detection thrice

328 N. Liu et al. / Sensors and Actuators B 191 (2014) 326– 331

onses of the 2D MIP/NIP imprinted film-coated QCM sensor.

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y the QCM sensor. For reproducibility test, we repeatedly used itor ten times, �fs have been recorded and collected.

. Results and discussion

.1. Recognition of the 2D molecularly imprinted film-coatedCM sensor

E4 QCM-D was used for monitoring �f of the 2D molecularlymprinted films for SEA and SEB. To evaluate the molecular rebind-ng properties of the MIP film, NIPs film was also analyzed aseference under the same conditions. After binding, the templatehould be washed out for another binding. As was shown in Fig. 1,f and �D could be remained relatively stable about 10 min for

he imprinted and non-imprinted thin films. When 100 �g/mL SEAnd SEB mixed solution was added in the flow cells, �f of SEA-MIPlm coated QCM was sharply decreased to about 346 Hz and slowlyeached to 337 Hz; similarly �f of SEB-MIP film coated QCM washarply decreased to about 650 Hz and slowly reached to 540 Hz. Itndicated decreases of �f when the specific recognition and bind-ng of the imprinted film coated on the electrode of QCM to thearget protein in the injected sample giving a greater adsorptionnd a smaller diffusion resistance in this work which are similaro our previous studies [20]. Meanwhile, no obvious �f has beenbserved with the non-imprinted thin films. The whole detectioneriod was within 0.5 h. The response time was shorter than 2 h11,23], 1 h [24] and 37 min [25].

When mentioned dissipation response (�D), it providesnformation on amount of mass bound and assessment of con-ormational changes of the adsorbed layer [26]. Similar significanthifts of SEA/SEB-MIP films were observed compared with NIP film,s shown in Fig. 1(B). The largely amplified �Ds were approxi-ately 5 × 10−6 and 7 × 10−6 indicating the targets-SEA and SEBere captured by the 2D molecularly imprinted sol–gel thin films.

he degree of �DSEB was greater than that of �DSEA which wasoincident with that of �fs. We speculated that SEB-MIP film couldrap more SEB molecules than that of SEA-MIP film.

A series of 10× gradient concentrations (0.1–1000 �g/mL) ofEA solved in 0.1 M PBS were employed on the flow cell of SEA-IP thin film coated PQC, simultaneously, 0.1–1000 �g/mL of SEB

olved in 0.1 M PBS with 10× gradient solved in 0.1 M PBS wasespectively added into the flow cells of NIPs film and SEB-MIPhin film coated PQC. Fig. 2 shows the responses of SEA-MIP andEB-MIP thin film coated PQC to different concentrations of SEs.he response of the �fs increased significantly with the incrementf SEs concentration. We also found that, a certain non-specific

dsorption of the added protein could cause the increase of �f ofhe NIP thin film coated PQC (data were not shown). Nevertheless,he response was much lower than that of the SEA/SEB-MIP thinlm coated PQC under the same concentration. The fitting curves

Fig. 2. �f of the imprinted thin film coated PQC to different concentration of SEAand SEB in PBS (n = 3).

between �f (non-specific signal has been subtracted from the mea-surements) and the concentration (Ci, ng/mL) of SEA and SEB in therange of 0.1–1000 �g/mL can be described as follows:

�fSEA = 73.23 C0.26SEA (R2 = 0.9841) (1)

�fSEB = 121.61 C0.28SEB (R2 = 0.9722). (2)

Since the 2D molecularly imprinted sol–gel thin film is not con-sidered as rigid film, the equations were not in accordance withSauerbrey equation [27]. We have obtained the similar results asour previous study [20]. Due to the injection of PBS buffer, �fs ofSEA-MIP and SEB-MIP film coated PQC were about 12 and 60 Hz, theLDLs were 7.97 and 2.25 ng/mL (S/N = 3), respectively. Comparedwith the former study, the sensitivity was improved and we alsoobtained the dissipation information.

3.2. Characterization of the 2D MIP/NIP film coated PQC surfacestructure by AFM

Since AFM is a powerful analytical tool that has been used tostudy a variety of biomolecular interactions at the molecular level[28], in order to visualize the surface of PQC Au-electrodes for QCMsensors including the mercapto-activated, sol–gel with/withouttemplates coated and after elution of the template, AFM wasemployed and the topographies were shown in Fig. 3. The param-

eters of mean roughness (Ra) and maximum height (Rmax) of theseAFM topographies were listed in Table 1.

The surface of the mercapto-activated unmodified PQC chip wasrelatively smooth (Fig. 3A), while the surfaces were displayed as

N. Liu et al. / Sensors and Actuators B 191 (2014) 326– 331 329

Fig. 3. AFM topographic images of the mercapto-activated chip (A), the sol–gel without

with elution of the template (D).

Table 1The parameters of AFM topographies in Fig. 3A–D.

Parameter Fig. 3A Fig. 3B Fig. 3C Fig. 3D

Ra (nm) 0.173 0.911 3.457 1.457

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with the inter-batch; whereas, there were no significance between

Rmax (nm) 1.143 6.422 23.989 10.062

rderly spiky peaks (Fig. 3B and C) which were much differentrom Fig. 3A. The overlaid layers of sol–gel thin film with an aver-ge roughness (Ra) of 0.911 nm were observed as elongated bumpsompared with Fig. 3A (0.173 nm). It suggests that the thicknessf the 2D molecularly imprinted sol–gel thin film is approximately.738 nm which is much more thinner than our previous studiesy using surface-initiated radical polymerization method coatedn the surface of gold chip (300, 40 and 20 nm) [18,19,28]. The sur-ace of sol–gel with templates coated chip was displayed as orderlypiky peaks with Ra = 3.457 nm which was the highest among theour topographies. The thickness of SEA template can be calculateds approximately 2.719–3.457 nm which implying the templateeing embedded in the sol–gel film or adsorbed on the surface of theol–gel layer. In Schad’s report [29], the size of SEA is 50 × 45 × 30 A3

nd forms a well-defined core with a few regions of high mobility.he calculated size in our research is very close to the former one.nd also it can be learned that only monolayer of SEA template areaptured by 2D sol–gel film. After elution of the template moleculerom the chip, the Ra (Fig. 3D) received was less than half of thatf Fig. 3C and relatively coincided with Fig. 3B. The concavitiesould be attributed to the extraction of embedded templates in the

rocess of molecularly imprinted SEs sol–gel film formation. It sug-ested that the template has been washed out from the molecularlymprinted film and the cavities have been remained. Being absent

templates coated chip (B), the sol–gel with templates coated chip (C) and the chip

of supporting of the template, the cavities fabricated with sol–gelturned to be collapsed, deflated and plump compared with theformer one as sol–gel is the soft and flexible material. Ra reflectedthe thickness of the films with the order of Fig. 3C > D > B > A. Fromthe AFM topographies and based on the reckoning thickness of the2D sol–gel film of PQC Au-electrodes for QCM sensors; and givenone layer of 2D sol–gel film can imprint one layer of template pro-tein, we presume that the film is one kind of monolayer and directlyproved the formation of 2D molecularly imprinted film-coated Auelectrode on chip surface.

3.3. Repeatability test of the 2D molecularly imprintedfilm-coated QCM sensor

200 �L 10 �g/mL SEA and SEB solution was respectively injectedinto the corresponding flow cells in situ coated with PQC electrodescoated with SEA-MIP and SEB-MIP after the stability of �fs andbeing recorded in the same conditions for the intra–batch test (testfor 3 times with the same PQC electrode) and inter-batch (test for 3times with different PQC electrode) test. The process was repeatedthrice. Next, we used different PQC electrode for the inter-batchtest under the same conditions. The determined values of intra-and inter-batch were 12.35 and 12.86 �g/mL for SEA, 12.76 and13.95 �g/mL for SEB. The recoveries were 123.5% and 128.6% forSEA, 127.6% and 139.5% for SEB respectively, as presented in Table 2.The performance of the intra-batch was slightly better compared

the two groups. These results indicated that the developed QCMsensor based on 2D molecularly imprinted film was satisfactory forthe detection of SEs.

330 N. Liu et al. / Sensors and Actuators B 191 (2014) 326– 331

Table 2Comparison of the detection result of the Intra- and inter-batch 2D molecularly imprinted film coated QCM.

Target Batch �f (x ± s, n = 3, Hz) CV (%) Amount added (�g/mL) Found (�g/mL) Recovery (%)

SEA Intra 39.99 ± 1.14* 2.85 10 12.35 125.5Inter 39.64 ± 1.71� 4.32 10 12.86 128.6

SEB Intra 287.45 ± 21.19* 7.37 10 12.76 127.6Inter 294.46 ± 25.35� 8.61 10 13.95 139.5

Compared * to � , P > 0.05.

Table 3Detection results of spiked samples by 2D molecularly imprinted film coated QCM (n = 3).

Samples (ng/mL) SEA-MIP coated QCM SEB-MIP coated QCM

Found (x ± s, �g/mL) Recovery (%) Found (x ± s, �g/mL) Recovery (%)

Blank (0) –a – –a –5 4.85 ± 0.92 97.00 5.71 ± 0.35 114.2050 52.06 ± 3.66 104.12

100 97.02 ± 1.46 97.02

a Undetectable concentrations or no results.

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ig. 4. Selectivity of the sol–gel film by addition of SEA, SEB, SEC1, SED, BSA and OVAn 0.1 and 10 �g/mL.

.4. Selectivity of the 2D molecularly imprinted film-coated QCM

A key component of this study involves proof of the specificitynd selectivity of the 2D molecularly imprinted film-coated QCMased on �f of different adsorbates injected into the QCM sen-or. When a QCM sensor is used to detect targets in samples, its inevitable that some structurally related compounds and othernterferents would affect the detection of targets [18]. So in thistudy, analogs of SEA and SEB such as SEC1 and SED and interferen-ial proteins of OVA and BSA with the concentration of 100 �g/mLere selected to evaluate the selectivity of the 2D molecularly

mprinted film-coated QCM. The relationship between �fs and theeterminands were illustrated in Fig. 4.

The �f values of the sensor for targets are much higher thanhose of the other analogs and irrelevant proteins with relativereat concentration, i.e., 100 �g/mL. Compared with irrelevant pro-

eins and the blank, the �fs of analogs are greater than those ones.he results are in accordance with those in previous reports wherehe shape effect seems to play a crucial role in the selective bindingf the analyte [24].

46.71 ± 2.13 93.42109.02 ± 3.25 109.02

SEA and SEB have a highly similar topology with �-barrel and �-grasp motifs present in both structures [30], and SEB and SEC1 havesignificant serological cross-reactivity and sequence homology ofabout 65% [31]. However, the structure of SEA differs significantlyfrom that of SEB or SEC1, and extensive homology is found partic-ularly in the carboxyl-terminal region between SEB and SEC1 [31].SEA and SEC1 are both analogs of SEB; thus, adsorption is hard toavoid by structural similarity, especially SEC1. The sensor could stilldiscriminate the template molecule from its analogs in the rapiddetection. This finding proves the acceptable selectivity in accor-dance with the previous results of the specific adsorption bindingof the sensor.

3.5. Analysis of SEA and SEB standard spiked in milk samples

To demonstrate the applicability of the sensor, the 2D molec-ularly imprinted film-coated QCM sensor was employed to SEAand SEB detection in milk samples after pre-treatment. The resultswere shown in Table 3. The recoveries ranged from 97.00% to114.20%. We also test the reproducibility of this sensor. Afterten times repeatedly usage with moderate hot water rinsing, �fsslightly decreases about 5–10% loss (detailed data were not shown)which possibly due to the incomplete elution of SEA/SEB templateadsorbed from the 2D molecularly imprinted-coated QCM film sen-sor chip. It is relatively stable compared to traditional piezoelectriccrystal immunosensor [9]. These results indicated that the devel-oped method was feasible for the detection of ESs in milk. The entireprocess could be completed in less than 30 min.

4. Conclusions

In this study, we have developed a 2D molecularly imprintedfilm-coated QCM by in situ immobilization using sol–gel imprint-ing for the rapid detection of SEs, the important pathogenic factorfor food poisoning. The surface of gold-coated quartz crystal wasmodified by thin sol–gel film through the in situ self-assembly oforganosilane sol–gel synthesis which is inclined to replace naturalantibodies. By using this film as the recognition material, a novelQCM sensor with short response time, wide linear range, low costand relatively high selectivity has been developed for the detec-tion of SEs. The dissipation shifts were also measured. The LDL is

much lower than Harteveld’s report by using of piezoelectric crystalimmunosensor (0.1 �g/mL) [8] and our previous study (6.1 ng/mL)[20]. The sensitivity is lower than Yang’s reports [11,12] becausethey have employed cooled charge-coupled device detector

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N. Liu et al. / Sensors and A

ombined with carbon nanotubes or gold nanoparticle-basednhanced chemiluminescence technologies for greatly increas-ng the signals of the immunological reaction. In further studies,ensitivity could be potentially improved by coupling with sig-al amplifying technologies, such as chemiluminescence, quantumots, and/or gold nanoparticles, etc.; or other types of transduc-rs (SPR, cantilever, and electrochemical detectors). The resultslso demonstrated the feasibility of SEs determination in milk. Thisork will be promisingly applied in the areas of food safety, envi-

onmental monitoring and biological engineering, etc.

cknowledgments

The authors gratefully acknowledge the financial support by theational Nature Science Foundation of China (Grant No. 30800915),

he National High Technology R&D Program of China (Grant No.010AA06Z302) and National Science and Technology Supportingrogram of China (No. 2011BAK10B02).

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Biographies

Nan Liu received M.D. in Biosensor from Academy Military Medical Science in 2009,Beijing, China. Now he is serving as an associate professor in Institute of Health andEnvironmental Medicine. His research is focused on rapid and multiple detectionsof pollutants in environment and food by biosensors and biochips.

Xiaoli Li received master’s degree from Academy Military Medical Science in 2009.Now, she has been an assistant professor at Institute of Health and EnvironmentMedicine. Her current interests are in the fields of rapid detection of pesticidesand veterinary drugs based on molecular imprinting technology and quartz crystalmicrobalance sensor.

Xinhua Ma received master’s degree from Academy Military Medical Science in2012, China. His research interests includes fabrication of include microfluidic chipand synthesis of ultrathin molecularly imprinted polymer film on gold surfaces andtheir applications for the detection of pesticides and veterinary drugs.

Guorong Ou serves as the senior professor in Institute of Health and EnvironmentalMedicine from 1998. His current research fields are focused on the rapid detectiontechnologies of hazard factors in water and food.

Zhixian Gao holds a position of professor at Institute of Health and EnvironmentMedicine, China, since 2006. He received PhD degree from Institute of Health andEnvironment Medicine in 2000. His current interests are nanobiotechnology, foodsafety and biosensors.