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Rapid high-throughput detection of diethylstilbestrol by using the arrayed langasite crystal microbalance combined with gold nanoparticles through competitive immunoassay
Accepted Manuscript Title: Rapid high-throughput detection of diethylstilbestrol by using the arrayed langasite crystal microbalance combined with gold nanoparticles through competitive immunoassay Author: Xiaoyan Liu Yuanwei Hu Xuan Sheng Yuan Peng Jialei Bai Quanjun Lv Hong Jia Huicong Jiang Zhixian Gao PII: DOI: Reference:
S0925-4005(17)30422-7 http://dx.doi.org/doi:10.1016/j.snb.2017.03.014 SNB 21926
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-12-2016 21-2-2017 3-3-2017
Please cite this article as: X. Liu, Y. Hu, X. Sheng, Y. Peng, J. Bai, Q. Lv, H. Jia, H. Jiang, Z. Gao, Rapid high-throughput detection of diethylstilbestrol by using the arrayed langasite crystal microbalance combined with gold nanoparticles through competitive immunoassay, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.03.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid high-throughput detection of diethylstilbestrol by using the arrayed
langasite
crystal
microbalance
combined
with
gold
nanoparticles through competitive immunoassay
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Xiaoyan Liua, Yuanwei Hu b, Xuan Sheng b, Yuan Peng b, Jialei Bai b, Quanjun Lva, Hong Jia c, Huicong Jianga,b, Zhixian Gao a* a
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Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou 450001, China b Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environment Medicine, Tianjin 300050, China c Department of Public Health, Southwest Medical University, Luzhou 646000, China
Highlights 1: The signals of sensor were amplified by intruducing AuNPs. 2: The method is wider in the detection range and lower in the detection limit. 3: An arrayed LCM sensor was developed to realize the high-throughput detection of diethylstilbestrol.
Corresponding author. Tel.: +86 22 84655403; fax: +86 22 84655403. E-mail addresses: [email protected] (X. Liu), [email protected] (Z. Gao). 1
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Abstract
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In this paper, a method for the rapid detection of diethylstilbestrol (DES) using quartz crystal microbalance (QCM) with gold nanoparticles (AuNPs) amplification was developed and this method was used on the arrayed langasite crystal microbalance (LCM) to realize the high-throughput detection of DES. A sensitive membrane which can recognize DES specifically was prepared on the surface of gold electrode through the molecular self-assembly technique. Gold nanoparticles (AuNPs) were coupled with anti-DES antibodies to amplify the signal of QCM. DES was detected through competitive immunoassay. At the same time, five gold electrodes were plated on a single langasite crystal in order to prepare an arrayed LCM sensor. The reliability and efficiency of the arrayed sensor were studied. The QCM sensor exhibits a linear detection range from 16 to 500 ng/mL and a detection limit of 13 ng/mL. The recovery rate of DES in the milk samples was 98.0-104.8%. By using structural analogs, the QCM sensor platform was demonstrated to be specific for the detection of DES. The arrayed LCM sensor was developed successfully and it could detect DES with arrayed method. Keywords: Quartz crystal microbalance sensor; Diethylstilbestrol; Gold nanoparticles; Arrayed langasite crystal microbalance sensor; competitive immunoassay.
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1. Introduction
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Diethylstilbestrol (DES), which is the first estrogenic substance to be easily synthesized and administered, is demonstrated to be beneficial in preventing premature deliveries and miscarriages [1] . Because of its good properties to increase the weight of animals, DES is widely used in livestock feeding [2, 3]. However, numerous unfortunate effects have shown that DES is one of the most potential carcinogens [4]. DES can enter human body by the biological accumulation and greatly threaten human health. Therefore, the use of growth-promoting drugs for fattening livestock has been banned in USA and many European countries [5], yet DES is still illegally abuse in animal production in pursuit of economical interests. Thus, it is indispensable to establish a novel method with high sensitivity, high specificity, as well as being able to detect many kinds of contaminants in food simultaneously. Traditional methods for the detection of DES is mainly to large-scale equipment, including high performance liquid chromatography (HPLC)[6-9], gas chromatography-mass spectrometry(GC-MS)[10-12], HPLC-MS[13]. However, these techniques have a disadvantage in that they are either time-consuming or expensive. Therefore, they are not suitable for the rapid detection of DES. In recent years, quartz crystal microbalance (QCM) sensor has been a new technology for the rapid detection of DES because of its simplicity in concept, ease of use, low cost, online monitoring, shorter analysis time and suitability for label-free measurement[14]. An increase in mass bound to the crystal surface causes the oscillation frequency shift of the crystal. The relationship between the frequency change and mass loading is shown by Sauerbrey equation[15]
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Where q is the quartz density (2.65 g cm-3), q is crystal shear module (2.95 1011 dyn cm-2), f 0 is crystal fundamental frequency of piezoelectric quartz crystal, A is the electrode area, and f is the measured resonant frequency decrease (Hz), m is the elastic mass change (g) . Piezoelectric quartz crystal is always the predominating piezoelectric material since QCM was first explored by Sauerbrey[15]. Quartz crystal resonators are relatively inexpensive and readily available. About 40 billion pieces of quartz crystal resonators are yearly manufactured in the world [16] . Therefore, QCM is in fact the standard term for piezoelectric sensors. It was successfully used for thin film thickness monitoring and for sorption studies in the gas phase [17]. For many applications, however, liquid phase operation is required. The quartz crystal will cease the oscillation in liquids with high viscosity when the oscillator method is employed. Langasite (La3Ga5SiO14) single crystal is a new piezoelectric substrate and can be used in acoustoelectron
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and piezoelectric devices [18-22]. Compared with quartz crystal, Langasite has a higher coupling coefficient and lower acoustic losses, which make it possess the ability to oscillate in liquid. Many articles have discussed the applications of QCM immunosensors in the detection of endocrine disrupting chemicals (EDCs) as alternatives to the conventional method [23-25]. However, label-free QCM immunosensors for DES detection still cannot achieve the demand that DES mustn’t be found in food. For their applications in food safety, the detection sensitivity definitely needs to be improved because the exposure dosage of the DES in food can be much lower. AuNPs possess excellent compatibility with almost all types of chemically (organic and metallic) and biologically active molecules. This means that the functional activity of these molecules remain unaffected even after their immobilization on AuNPs [26-28]. So AuNPs were often regarded as excellent signal amplification material for the design and fabrication of biosensors. In this work we proposed a method for the rapid detection of DES using QCM with AuNPs amplification. Fig.1 gives the preparation procedures of the QCM sensor. The 11-mercaptoundecanoic acid (MUA), a long-chain carboxylic acid-terminating alkanethiol, was introduced on the Au surface of QCM, then activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS) to generate a suitable intermediate for the immobilization of DES-BSA. Gold nanoparticles (AuNPs) were coupled with anti-DES antibodies to amplify the signal of QCM. The DES-BSA immobilized on the electrode surface and DES bound with AuNPs-DES Ab competitively. The binding would decrease the sensors’s resonant frequency, and the frequency shift was negatively correlated to the DES concentration. Based on this, we also developed a novel monolithic array piezoelectric sensor to detect DES in an arrayed manner. The method was applied on the array piezoelectric sensor successfully to realize the preliminary arrayed detection for DES. This work will be a pregnant explore for the detection of DES or other environmental endocrine disruptors (EDCs) with a high throughput manner. 2. Experimental
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DES was purchased from J&K CHEMICA (Beijing, China). 11-mercaptoundecanoic acid (MUA), 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl), N-hydrosuccinimide (NHS), and ethanolamine were supplied from Sigma Chemical Co. (St.Louis, MO, USA). All other chemicals used were of analytical grade. DES-BSA and anti-DES antibody were prepared in our laboratory. Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). The DES-BSA and anti-DES antibody solution were diluted using phosphate buffer solution (PBS) (0.01 mol/L, pH 7.2). 2.2. Equipment and apparatus We applied a QCM200 system for this study, controlled by a personal computer and connected with 5 MHz QCM devices provided by Stanford Research Systems Company. The AT-cut 5 MHz quartz crystals (Jiaxing crystal controlled Electronic Co., Ltd) (1 inch diameter, 331μm thickness, Au and chromium covered electrode surface on both sides) were used to fabricate the immunosensor. Compared with the large diameter (1 inch), the relatively small vibrating area (0.4 cm2) can not only make the separation between the effective electrode area and erecting structure of the holder keep better but also shrink the coupling generated in other resonance model. 2.3. Preparation of gold nanoparticles-antibody conjugates (AuNPs-DES Ab) 4
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2.3.1 Synthesis of AuNPs modified with amino
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The AuNPs with average diameter of approximately 40 nm were synthesized by the dextran reduction method according to Jang et al [29] with slight modifications. The first step: the synthesis of dex-AuNPs. 7.5 wt% dextran (160 mL) was boiled with vigorous stirring in a flask. Then 4.32 mL of HAuCl4 (0.01g/mL) was added to the boiling solution immediately, resulting in a color change from faint yellow to reddish-violet. After cooling to room temperature the dex-AuNPs was rinsed with distilled water for 4 times using Amicon filter (cutoff: 100 kDa) and redispersed in distilled water (4 mL). The second step: the synthesis of cl-dex-AuNPs. 200 μL of 1 M NaOH solution was added to the synthesized 4 mL of dex-AuNPs. After the mixture was vortexed vigorously for 15 s, 60 μL of epichlorohydrin was edded. Mixed solution was incubated overnight while shaking at 600 rpm at room temperature. The purification was done as the former. The third step: the synthesis of N-cl-dex-AuNPs. 3% ammonium hydroxide was added to the cl-dex-AuNPs solution for its amination. The mixture was incubated for 24 h while shaking at 600 rpm at room temperature. The purification was done as the former. 2.3.2 Synthesis of AuNPs-DES Ab
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Initially, 300 μL of aqueous solution of AuNPs at 15 nmol/L was centrifuged and the precipitate was dissolved into 300 μL of pH 7.2 PBS. Thereafter, 400 μL of glutaraldehyde solution (2.5 wt %) was added to the dispersion, and the mixture was stirred 30 min at room temperature. After centrifugation at 12000 rpm for 10 min, the resulting mixture was redispersed 300 μL of pH 7.2 PBS. Then 300 μL of anti-DES antibody was added and the mixture was stirred 30 min at 37 ℃. After centrifugation at 8000 rpm for 10 min, the resulting mixture was redispersed 500 μL of 1% BSA and stirred 40 min at 37 ℃. After centrifugation at 8000 rpm for 10 min, the resulting mixture was redispersed 300 μL of pH 7.2 PBS solution and stored at 4 ℃ for further use. 2.4. The detection of DES using a QCM sensor with gold nanoparticles
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2.4.1 Fabrication of QCM sensor
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The bare QCM sensor crystal was pretreated with 1 mol/L NaOH for 20 min, 1 mol/L HCl for 5 min in ultrasonic bath and piranha etch solution (H2O2:H2SO4 = 1:3) for 1 min, in sequence, to obtain a clean Au surface. After each step, the crystal was rinsed with ethanol and then water and dried with pure nitrogen gas. The cleaned crystal was submerged immediately into a solution of MUA (5 mmol/L in pure ethanol) for 16 h to form self-assembled monolayer (SAM). After rinsing with ethanol and water, the MUA-modified crystal surface was activated with 1:1 mixture of 400 mmol/L EDC·HCl and 100 mmol/L NHS for 20 min in order to convert the terminal carboxylic groups to active NHS esters. After rinsing with water and drying under nitrogen gas, 20 μL of 2.2 mg/mL DES-BSA was added onto one side of the crystal and spread over the entire Au electrode for 2 h at 37 °C. The excess DES-BSA was removed by rinsing with water, and then the crystal was dried under nitrogen gas again. Non-reacted NHS esters were then capped with 1 mol/L ethanolamine solution (pH 8.5) for 30 min at 37 °C. After rinsing with water, the QCM sensor crystal was dried in nitrogen, and finally the desired sensor was fabricated and stored at 4 °C till use. 2.4.2 Detection of DES with QCM sensor
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DES determination was based on the principle that the DES-BSA immobilized on the surface of the QCM sensor and DES can bound with AuNPs-DES Ab competitively. The binding of AuNPs-DES Ab with the antigen will decrease the sensors’s resonant frequency, and the frequency shift was negatively correlated to the DES concentration. Different concentrations of the DES solution were prepared in PBS that contained 10% methanol (0.032, 0.16, 0.8, 2.5, 4, 20, 100 and 500 ng/mL). 10 μL of the DES solution was taken to mix with 10 μL of 28 μg/mL AuNPs-DES Ab. The mixture was dropped on the surface of the QCM sensor and then the sensor was placed in the water bath kettle for 1 h at 37 °C. The excess AuNPs-DES Ab was removed by rinsing with water. After drying under nitrogen gas, the frequency shift was recorded by the data system. All the concentrations of the DES solution were measured following the same procedure. The limit of detection (LOD) was calculated as the signal obtained from the DES concentration that is equivalent to the 3 times the standard deviation of the signals obtained from the blank spot array. All assays were repeated in triplicates. 2.4.3 Specificity of the QCM sensor to DES
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To verify the specificity of the QCM sensor to DES, current responses were tested in the presence of several possible interfering substances such as hexoestrol, dienestrol, estradiol, and βestradiol. 20 ng/mL of DES and its four analogues solutions were prepared in PBS that contained 10% methanol. 10 μL of the DES and its analogues solutions were taken to mix with 10 μL of 28 μg/mL AuNPs-DES Ab respectively. The detection procedure for the mixture was the same as the detection process of DES with QCM sensor. All assays were repeated in triplicates. Blank samples were prepared as described above without DES and its analogues. 2.4.4 Preparation and measurement of real samples
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Milk samples obtained from a local supermarket were used in this work. 900 μL of milk was spiked with 100 μL of increasing DES concentrations (0.05, 0.5, and 5μg/mL) in PBS containing 10% methanol. Blank samples were prepared following the previous procedure without DES. The samples were mixed with equal ethyl acetate and mixed with an oscillator for 10 min, and then equilibrated for at least 30 min. 100 μL of the supernates were collected in 900 μL PBS containing 10% methanol. Afterwards, the milk samples containing the increasing concentrations DES (0.5, 5, and 50 ng/mL) were prepared. The measurement for real samples was the same as the detection process of DES with QCM sensor. The samples were also analyzed via HPLC-MS/MS for validation. 50 mL of milk sample were centrifuged at 3500 rpm for 5 min. The substrate was collected. Different concentrations of the DES solution (0.5, 5, and 50 ng/mL) were prepared in methanol. A quantitative of DES-D8 was added to each DES solution to make its concentration 10 ng/mL in the all DES solutions. The liquid was dried via mild nitrogen gas, and 5 mL milk samples were subsequently added. After the mixture was shaken until the solid was completely dissolved, 10 mL of acetonitrile was added. Then the mixture were mixed with an oscillator for 3 min and centrifuged at 3500 rpm for 10 min. The supernates were collected and 5 mL of acetonitrile was added again. Repeated the procedure above, the extracting solution was combined. After the liquid was dried via nitrogen gas at 50 ℃, 10 mL water was added and the pH was adjusted to 11.0. Then the liquid was centrifuged at 9000 rpm for 5 min at 4 ℃. Blank samples were prepared following the previous procedure without DES. The liquids were filtered with a 0.22 μm PTFE filter, followed by HPLC-MS/MS detection. The analysis was performed in triplicate.
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2.5. The detection of DES using LCM sensor 2.5.1 Development and screening test of arrayed LCM sensor
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Five gold electrodes (recording element 1 to 5 respectively) were plated on a single langasite crystal to prepare an arrayed LCM sensor. In order to evaluate the vibration characteristics of the five array elements, the network analyzer HP8752A was used to test their vibration performance. The LCM sensor was fabricated according to the procedure described in the fabrication of QCM sensor (2.4.1). Different electrodes were modified at the same time. 2.5.2. Control experiment
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The frequencies of three DES-BSA-modified crystals were detected by arrayed LCM sensor and the frequency values were recorded F1. 20 μL of 14 μg/mL anti-DES antibody was dropped on the element 4 and element 5 respectively, and 20 μL of PBS solution was dropped on the element 3 as control group. After the reaction cell was placed in an incubator chamber at 37 ℃ for 1 h, the frequencies were detected by arrayed LCM sensor and the frequency values were recorded F2. Then the frequency shifts (ΔF=F2-F1) represent the changes caused by environment factors or both environment factors and anti-DES antibody. 2.5.3. The detection for different concentrations of anti-DES antibody
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The frequencies of three DES-BSA-modified crystals were detected by arrayed LCM sensor and the frequency values were recorded F3. 20 μL of different concentrations of anti-DES antibody (3.5 μg/mL, 7 μg/mL and 14 μg/mL) were respectively dropped on the element 3, element 4 and element 5. After the reaction cell was placed in an incubator chamber at 37 ℃ for 2 h, the frequencies were detected by arrayed LCM sensor and the frequency values were recorded F4. Then the frequency shifts (ΔF=F4-F3) represent the changes caused by both environment factors and different concentrations of anti-DES antibody. 3. Results and discussion
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Topographic images were taken by AFM in the non-contact mode (5 μm×5 μm surface). In order to compare the topologies of each surface, surface roughness (Ra) and root mean square roughness (Rq) were estimated from the AFM image [30]. Fig.2 represent surface morphology of the bare Au (Fig.2A), DES-BSA immobilization (Fig.2B), anti-DES antibody binding (Fig.2C), AuNPs-DES Ab binding (Fig.2D) based QCM sensor. Because a highly ordered and densely packed self-assembled layer of MUA can appear on the gold surface [31] and the Ra and Rq values for the bare Au surface and MUA electrode surface will be similar, the AFM characterization for the MUA electrode surface was not been conducted. The increase of surface roughness of DES-BSA electrode (Fig.2B) as indicated by the values of Ra (2.31 nm from 2.40 nm) and Rq (1.83 nm from 1.87 nm) demonstrates immobilization of DES-BSA on to electrode surface. The Ra and Rq have been found to be increase to 2.79 nm and 2.22 nm of anti-DES antibody electrode (Fig.2C) after the interaction of antigen with the antibody. Compared with anti-DES antibody, the Ra (from 2.40 to 14.7) and Rq (from 1.87 to 9.99) had drastic increase after the binding of AuNPs-DES Ab (Fig.2D). This may result from the gold nanoparticels amplification. The results shown by AFM images after each treated step indicated that DES-BSA and AuNPs-DES Ab have been successfully performed on a QCM device.
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3.2. Optimization of DES-BSA immobilization Efficient antigen immobilization is important for enabling sensitive antibody detection, hence different concentrations of DES-BSA were examined to optimize the assay. Six different concentrations of DES-BSA were immobilized (0.14 to 2.20 mg/mL) on the MUA immobilized surface. Fig.3 shows the frequency changes achieved with the increasing DES-BSA concentrations. Shown from the figure, DES-BSA concentration of 2.20 mg/mL gave the optimal response change (31 Hz) and therefore, this concentration was chosen for the following assay.
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3.3. Optimisation of AuNPs-DES Ab binding assay
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The concentration of AuNPs-DES Ab was optimized in order to achieve the widest detection range and the optimal detection limit for the sensor. Six different concentrations of AuNPs-DES Ab were immobilized (1.75 to 56.00 μg/mL) on the DES-BSA immobilized surface. Fig.4 shows the frequency changes response with the increasing concentrations of the AuNPs-DES Ab. Shown from the figure, AuNPs-DES Ab concentration of 14 μg/mL gave the optimal response change (101 Hz) and then the frequency shift levels off with the concentration increasing, therefore, this concentration was chosen for the following assay. 3.4. Analytical performance of the QCM sensor and calibration curve
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Typical temporal response curves of the QCM sensor at different concentration of DES are demonstrated in Fig.5A. It is shown that in 0.16~500 ng/mL of DES solution, the sensor ’s resonant frequency decreased over time due to the binding of AuNPs-DES Ab onto the immobilized DES-BSA, and the time to reach a plateau at ~15 min. When the DES concentration was 0.032 ng/mL, the temporal response curves could not be distinguished from the baseline of negative control. The calibration graph of frequency decrease vs. logarithm of DES concentration was displayed in Fig.5B. It can be seen that in the entire working range of 0.16~500 ng/mL, the higher the concentration, the smaller the sensor response. The calibration curve formula is y=﹣ 24.17 logx+69.72, with a correlation coefficient of 0.998. The limit of detection (LOD) was determined at 0.13 ng/mL based on the 3σ values of the blank signals. 3.5. Cross-reactivity of the QCM sensor with the analogues
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Cross-reactivity describes the specificity of the antibody applied in the assay and is an important analytical parameter regarding specificity and reliability of immunosensor devices[32]. To study the specificity of the QCM sensor, cross-reactivity with DES and structural analogs were compared. Fig.6 shows the frequency shifts caused by DES and the structural analogs at the same concentration (20 ng/mL) in the detection solution. The results reveal that the detection of DES could be distinguished from the other structural analogs, so the sensor exhibited higher selective recognition to DES. 3.6. Real samples analysis Immunoassays are known to suffer from matrix effects because of nonspecific binding and denaturation of the Ab and the enzyme, which results in false-positive responses [33]. To investigate the matrix effect and the applicability of the proposed sensor, the content of DES in milk samples was detected using spiked recovery method. Prepared samples were analyzed using the optimized QCM sensor at three levels. The determination results were shown in Table 1. Good recovery rates ranged from 98.00% to 104.80% were obtained with the milk samples. Thus, the proposed sensor allows direct DES analysis in milk samples. The samples were also analyzed by HPLC-MS/MS 8
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for confirmation. No significant differences were obtained with the recovery rates ranged from 102.00% to 104.90%, which demonstrated the good performance of the optimized QCM sensor. Besides, compared with the HPLC-MS/MS method, the QCM sensor could detect DES in real milk samples without any further treatment. The method is efficient and environmental friendly because it’s not necessary for the complex pretreatment with much organic solvent. 3.7. Stability and repeatability
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Stability and reusability are major concerns in evaluating a QCM immunosensor. It was found that the crystal modified with DES-BSA could retain its activity for at least 1 week at 4 °C before its first use. In our work, all the results were obtained on a QCM sensor surface with freshly immobilized DES-BSA and limited experiments were carried out to inspect the repeatability of the sensor. For regeneration, we could release the antibody from the sensor by rinsing the crystal in the buffer with low pH (commonly to pH 1) whereas minor DES-BSA immobilized would also be released from the sensor. This would decrease its ability to detect the analyte inevitably. So an investigation should be implemented to reduce the loss of activity and improve the regeneration of sensors.
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The arrayed langasite crystal device was designed (Fig.7). Five gold electrodes were plated on a single gallium silicate crystal by the vacuum coating way, and each array element oscillate without disturbing each other by time sharing gate model. Besides, only one side of the sensor can be used in liquid, which can make five electrodes work in the same liquid and this environment is free from outside distraction. The sensitive devices were connected to the testing instruments to complete the assembling of the arrayed LCM sensor (Fig.8). Possessing the function of time sharing gate make the five electrodes on a single crystal can work well but without interfering with each so that the device can detect DES in arrayed methods.
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3.9. The evaluation and screening for the array elements
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The piezoelectric crystal may cease the oscillation in liquids of high viscosity. Because of the difference in the production process, it is difficult to ensure that every array element can possess good vibration performance. Fig.9 was the result of screening test for the five array elements. The curve reflected the impedance of analytical device. If the peak of the curve was more incisive, the impedance of the device was smaller and it will have good vibration characteristics. As can be seen from figure 8, the element 1 and 2 were found the obvious spurious resonance, which may be caused in the production process and interfere the measurement. In order to avoid the interference for the detection, the element 3, 4 and 5 were chosen for the subsequent experiments. 3.10. The results of control experiment Compared with element 3 (control group), element 4 and element 5 (experiment group) performed significant different in the frequency shifts, and at the same time, the frequency shifts between element 4 and element 5 had a few minor differences (Fig.10), which indicated that the arrayed LCM sensor could detect DES with arrayed methods. Setting up a control group during the experiment can compensate the interference of environment factors by reference method, which boosts this sensor’s resistance to interference. Anti-DES antibodies with the same concentration were detected on element 4 and element 5 and the frequency shifts caused on the
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two elements were similar, which revealed that the sensor had high accuracy and reliability. 3.11. The detection results for different concentrations of anti-DES antibody
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A good arrayed sensor can not only conduct reference experiment but also detect different samples at the same time. In order to evaluate the efficiency of high-through detection of the arrayed sensor, different concentrations of anti-DES antibody were used on the arrayed LCM sensor. The results were shown in Fig.11. With the increasing of anti-DES antibody concentration, the frequency shifts show a rising trend. The detection efficiency was improved by detect several concentrations of anti-DES antibody at the same time. 4. Conclusion
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This study successfully developed a method based QCM sensor for the detection of DES in milk with AuNPs amplification. Compared with the large-scale equipment, this method was more simply during the operation and more sensitive with the lower detection limit of 13 ng/mL. In addition, this QCM sensor has high specificity to DES. Therefore, this method can be further applied to detect other environmental pollutants. At the same time, an arrayed LCM sensor by plating five gold electrodes on a single gallium silicate crystal was also developed in this study for the arrayed detection of DES. It was found that on the different electrodes of the same crystal, the frequency shifts caused by the same concentrations of anti-DES antibody were similar; and with the increasing of concentrations of anti-DES antibody, the frequency shifts were rising. Therefore, the arrayed LCM sensor can preliminary achieve the detection of DES with high throughput. Acknowledgements
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The authors gratefully acknowledge financial support from the National Nature Science Foundation of China (No.21177159).
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3-34. DOI: 10.1016/S0003-2670(97)00608-9
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Figure Caption
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Fig.1. Schematic illustration of the fabrication procedures for the QCM sensor
Fig.2. AFM of the surface of QCM sensor crystal. A: bare surface and B: DES-BSA immobilization
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and C: anti-DES antibody binding and D: AuNPs-DES Ab binding
Fig.3. Frequency changes for the different concentrations of DES-BSA
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Fig.4. Frequency changes for the different concentrations of AuNPs-DES Ab
Fig.5.（A）Frequency change of the QCM sensor as a function of time upon interaction with different
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concentration of DES; and (B) Calibration curves for DES determination based on the QCM sensor.
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DES concentration was between 0.16 and 500 ng/mL.
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Fig.6. Frequency shifts of DES and its structural analogs on the QCM sensor. The concentration of
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DES and its structural analogs is 20 ng/mL.
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Fig.7. Arrayed langasite crystal device
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Fig.8. Arrayed langasite crystal microbalance
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Fig.9. The vibration characteristics of five elements
Fig.10. The results of reference experiment
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Fig.11. The detection results of different concentrations of anti-DES antibody
Table 1 Results of the determination of DES in spiked milk samples (n=3) QCM sensor (mean ± SD)(ng/mL)
Recovery ratio （%）
HPLC-MS/MS (mean ± SD)(ng/mL)
Recovery ratio （%）
0.50 5.00 50.00
0.49±0.01 5.24±0.35 51.65±1.00
98.00 104.80 103.30
0.50±0.01 5.16±0.30 52.45±1.30
102.00 103.30 104.90
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Added (ng/mL)
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S0925-4005(17)30422-7 http://dx.doi.org/doi:10.1016/j.snb.2017.03.014 SNB 21926
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-12-2016 21-2-2017 3-3-2017
Please cite this article as: X. Liu, Y. Hu, X. Sheng, Y. Peng, J. Bai, Q. Lv, H. Jia, H. Jiang, Z. Gao, Rapid high-throughput detection of diethylstilbestrol by using the arrayed langasite crystal microbalance combined with gold nanoparticles through competitive immunoassay, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.03.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid high-throughput detection of diethylstilbestrol by using the arrayed
langasite
crystal
microbalance
combined
with
gold
nanoparticles through competitive immunoassay
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Xiaoyan Liua, Yuanwei Hu b, Xuan Sheng b, Yuan Peng b, Jialei Bai b, Quanjun Lva, Hong Jia c, Huicong Jianga,b, Zhixian Gao a* a
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Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou 450001, China b Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environment Medicine, Tianjin 300050, China c Department of Public Health, Southwest Medical University, Luzhou 646000, China
Highlights 1: The signals of sensor were amplified by intruducing AuNPs. 2: The method is wider in the detection range and lower in the detection limit. 3: An arrayed LCM sensor was developed to realize the high-throughput detection of diethylstilbestrol.
Corresponding author. Tel.: +86 22 84655403; fax: +86 22 84655403. E-mail addresses: [email protected] (X. Liu), [email protected] (Z. Gao). 1
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Abstract
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In this paper, a method for the rapid detection of diethylstilbestrol (DES) using quartz crystal microbalance (QCM) with gold nanoparticles (AuNPs) amplification was developed and this method was used on the arrayed langasite crystal microbalance (LCM) to realize the high-throughput detection of DES. A sensitive membrane which can recognize DES specifically was prepared on the surface of gold electrode through the molecular self-assembly technique. Gold nanoparticles (AuNPs) were coupled with anti-DES antibodies to amplify the signal of QCM. DES was detected through competitive immunoassay. At the same time, five gold electrodes were plated on a single langasite crystal in order to prepare an arrayed LCM sensor. The reliability and efficiency of the arrayed sensor were studied. The QCM sensor exhibits a linear detection range from 16 to 500 ng/mL and a detection limit of 13 ng/mL. The recovery rate of DES in the milk samples was 98.0-104.8%. By using structural analogs, the QCM sensor platform was demonstrated to be specific for the detection of DES. The arrayed LCM sensor was developed successfully and it could detect DES with arrayed method. Keywords: Quartz crystal microbalance sensor; Diethylstilbestrol; Gold nanoparticles; Arrayed langasite crystal microbalance sensor; competitive immunoassay.
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1. Introduction
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Diethylstilbestrol (DES), which is the first estrogenic substance to be easily synthesized and administered, is demonstrated to be beneficial in preventing premature deliveries and miscarriages [1] . Because of its good properties to increase the weight of animals, DES is widely used in livestock feeding [2, 3]. However, numerous unfortunate effects have shown that DES is one of the most potential carcinogens [4]. DES can enter human body by the biological accumulation and greatly threaten human health. Therefore, the use of growth-promoting drugs for fattening livestock has been banned in USA and many European countries [5], yet DES is still illegally abuse in animal production in pursuit of economical interests. Thus, it is indispensable to establish a novel method with high sensitivity, high specificity, as well as being able to detect many kinds of contaminants in food simultaneously. Traditional methods for the detection of DES is mainly to large-scale equipment, including high performance liquid chromatography (HPLC)[6-9], gas chromatography-mass spectrometry(GC-MS)[10-12], HPLC-MS[13]. However, these techniques have a disadvantage in that they are either time-consuming or expensive. Therefore, they are not suitable for the rapid detection of DES. In recent years, quartz crystal microbalance (QCM) sensor has been a new technology for the rapid detection of DES because of its simplicity in concept, ease of use, low cost, online monitoring, shorter analysis time and suitability for label-free measurement[14]. An increase in mass bound to the crystal surface causes the oscillation frequency shift of the crystal. The relationship between the frequency change and mass loading is shown by Sauerbrey equation[15]
ce pt
2mf0 f , A q q 2
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Where q is the quartz density (2.65 g cm-3), q is crystal shear module (2.95 1011 dyn cm-2), f 0 is crystal fundamental frequency of piezoelectric quartz crystal, A is the electrode area, and f is the measured resonant frequency decrease (Hz), m is the elastic mass change (g) . Piezoelectric quartz crystal is always the predominating piezoelectric material since QCM was first explored by Sauerbrey[15]. Quartz crystal resonators are relatively inexpensive and readily available. About 40 billion pieces of quartz crystal resonators are yearly manufactured in the world [16] . Therefore, QCM is in fact the standard term for piezoelectric sensors. It was successfully used for thin film thickness monitoring and for sorption studies in the gas phase [17]. For many applications, however, liquid phase operation is required. The quartz crystal will cease the oscillation in liquids with high viscosity when the oscillator method is employed. Langasite (La3Ga5SiO14) single crystal is a new piezoelectric substrate and can be used in acoustoelectron
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2.1. Reagents and materials
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and piezoelectric devices [18-22]. Compared with quartz crystal, Langasite has a higher coupling coefficient and lower acoustic losses, which make it possess the ability to oscillate in liquid. Many articles have discussed the applications of QCM immunosensors in the detection of endocrine disrupting chemicals (EDCs) as alternatives to the conventional method [23-25]. However, label-free QCM immunosensors for DES detection still cannot achieve the demand that DES mustn’t be found in food. For their applications in food safety, the detection sensitivity definitely needs to be improved because the exposure dosage of the DES in food can be much lower. AuNPs possess excellent compatibility with almost all types of chemically (organic and metallic) and biologically active molecules. This means that the functional activity of these molecules remain unaffected even after their immobilization on AuNPs [26-28]. So AuNPs were often regarded as excellent signal amplification material for the design and fabrication of biosensors. In this work we proposed a method for the rapid detection of DES using QCM with AuNPs amplification. Fig.1 gives the preparation procedures of the QCM sensor. The 11-mercaptoundecanoic acid (MUA), a long-chain carboxylic acid-terminating alkanethiol, was introduced on the Au surface of QCM, then activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS) to generate a suitable intermediate for the immobilization of DES-BSA. Gold nanoparticles (AuNPs) were coupled with anti-DES antibodies to amplify the signal of QCM. The DES-BSA immobilized on the electrode surface and DES bound with AuNPs-DES Ab competitively. The binding would decrease the sensors’s resonant frequency, and the frequency shift was negatively correlated to the DES concentration. Based on this, we also developed a novel monolithic array piezoelectric sensor to detect DES in an arrayed manner. The method was applied on the array piezoelectric sensor successfully to realize the preliminary arrayed detection for DES. This work will be a pregnant explore for the detection of DES or other environmental endocrine disruptors (EDCs) with a high throughput manner. 2. Experimental
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DES was purchased from J&K CHEMICA (Beijing, China). 11-mercaptoundecanoic acid (MUA), 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl), N-hydrosuccinimide (NHS), and ethanolamine were supplied from Sigma Chemical Co. (St.Louis, MO, USA). All other chemicals used were of analytical grade. DES-BSA and anti-DES antibody were prepared in our laboratory. Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). The DES-BSA and anti-DES antibody solution were diluted using phosphate buffer solution (PBS) (0.01 mol/L, pH 7.2). 2.2. Equipment and apparatus We applied a QCM200 system for this study, controlled by a personal computer and connected with 5 MHz QCM devices provided by Stanford Research Systems Company. The AT-cut 5 MHz quartz crystals (Jiaxing crystal controlled Electronic Co., Ltd) (1 inch diameter, 331μm thickness, Au and chromium covered electrode surface on both sides) were used to fabricate the immunosensor. Compared with the large diameter (1 inch), the relatively small vibrating area (0.4 cm2) can not only make the separation between the effective electrode area and erecting structure of the holder keep better but also shrink the coupling generated in other resonance model. 2.3. Preparation of gold nanoparticles-antibody conjugates (AuNPs-DES Ab) 4
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2.3.1 Synthesis of AuNPs modified with amino
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The AuNPs with average diameter of approximately 40 nm were synthesized by the dextran reduction method according to Jang et al [29] with slight modifications. The first step: the synthesis of dex-AuNPs. 7.5 wt% dextran (160 mL) was boiled with vigorous stirring in a flask. Then 4.32 mL of HAuCl4 (0.01g/mL) was added to the boiling solution immediately, resulting in a color change from faint yellow to reddish-violet. After cooling to room temperature the dex-AuNPs was rinsed with distilled water for 4 times using Amicon filter (cutoff: 100 kDa) and redispersed in distilled water (4 mL). The second step: the synthesis of cl-dex-AuNPs. 200 μL of 1 M NaOH solution was added to the synthesized 4 mL of dex-AuNPs. After the mixture was vortexed vigorously for 15 s, 60 μL of epichlorohydrin was edded. Mixed solution was incubated overnight while shaking at 600 rpm at room temperature. The purification was done as the former. The third step: the synthesis of N-cl-dex-AuNPs. 3% ammonium hydroxide was added to the cl-dex-AuNPs solution for its amination. The mixture was incubated for 24 h while shaking at 600 rpm at room temperature. The purification was done as the former. 2.3.2 Synthesis of AuNPs-DES Ab
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Initially, 300 μL of aqueous solution of AuNPs at 15 nmol/L was centrifuged and the precipitate was dissolved into 300 μL of pH 7.2 PBS. Thereafter, 400 μL of glutaraldehyde solution (2.5 wt %) was added to the dispersion, and the mixture was stirred 30 min at room temperature. After centrifugation at 12000 rpm for 10 min, the resulting mixture was redispersed 300 μL of pH 7.2 PBS. Then 300 μL of anti-DES antibody was added and the mixture was stirred 30 min at 37 ℃. After centrifugation at 8000 rpm for 10 min, the resulting mixture was redispersed 500 μL of 1% BSA and stirred 40 min at 37 ℃. After centrifugation at 8000 rpm for 10 min, the resulting mixture was redispersed 300 μL of pH 7.2 PBS solution and stored at 4 ℃ for further use. 2.4. The detection of DES using a QCM sensor with gold nanoparticles
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amplification
2.4.1 Fabrication of QCM sensor
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The bare QCM sensor crystal was pretreated with 1 mol/L NaOH for 20 min, 1 mol/L HCl for 5 min in ultrasonic bath and piranha etch solution (H2O2:H2SO4 = 1:3) for 1 min, in sequence, to obtain a clean Au surface. After each step, the crystal was rinsed with ethanol and then water and dried with pure nitrogen gas. The cleaned crystal was submerged immediately into a solution of MUA (5 mmol/L in pure ethanol) for 16 h to form self-assembled monolayer (SAM). After rinsing with ethanol and water, the MUA-modified crystal surface was activated with 1:1 mixture of 400 mmol/L EDC·HCl and 100 mmol/L NHS for 20 min in order to convert the terminal carboxylic groups to active NHS esters. After rinsing with water and drying under nitrogen gas, 20 μL of 2.2 mg/mL DES-BSA was added onto one side of the crystal and spread over the entire Au electrode for 2 h at 37 °C. The excess DES-BSA was removed by rinsing with water, and then the crystal was dried under nitrogen gas again. Non-reacted NHS esters were then capped with 1 mol/L ethanolamine solution (pH 8.5) for 30 min at 37 °C. After rinsing with water, the QCM sensor crystal was dried in nitrogen, and finally the desired sensor was fabricated and stored at 4 °C till use. 2.4.2 Detection of DES with QCM sensor
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DES determination was based on the principle that the DES-BSA immobilized on the surface of the QCM sensor and DES can bound with AuNPs-DES Ab competitively. The binding of AuNPs-DES Ab with the antigen will decrease the sensors’s resonant frequency, and the frequency shift was negatively correlated to the DES concentration. Different concentrations of the DES solution were prepared in PBS that contained 10% methanol (0.032, 0.16, 0.8, 2.5, 4, 20, 100 and 500 ng/mL). 10 μL of the DES solution was taken to mix with 10 μL of 28 μg/mL AuNPs-DES Ab. The mixture was dropped on the surface of the QCM sensor and then the sensor was placed in the water bath kettle for 1 h at 37 °C. The excess AuNPs-DES Ab was removed by rinsing with water. After drying under nitrogen gas, the frequency shift was recorded by the data system. All the concentrations of the DES solution were measured following the same procedure. The limit of detection (LOD) was calculated as the signal obtained from the DES concentration that is equivalent to the 3 times the standard deviation of the signals obtained from the blank spot array. All assays were repeated in triplicates. 2.4.3 Specificity of the QCM sensor to DES
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To verify the specificity of the QCM sensor to DES, current responses were tested in the presence of several possible interfering substances such as hexoestrol, dienestrol, estradiol, and βestradiol. 20 ng/mL of DES and its four analogues solutions were prepared in PBS that contained 10% methanol. 10 μL of the DES and its analogues solutions were taken to mix with 10 μL of 28 μg/mL AuNPs-DES Ab respectively. The detection procedure for the mixture was the same as the detection process of DES with QCM sensor. All assays were repeated in triplicates. Blank samples were prepared as described above without DES and its analogues. 2.4.4 Preparation and measurement of real samples
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Milk samples obtained from a local supermarket were used in this work. 900 μL of milk was spiked with 100 μL of increasing DES concentrations (0.05, 0.5, and 5μg/mL) in PBS containing 10% methanol. Blank samples were prepared following the previous procedure without DES. The samples were mixed with equal ethyl acetate and mixed with an oscillator for 10 min, and then equilibrated for at least 30 min. 100 μL of the supernates were collected in 900 μL PBS containing 10% methanol. Afterwards, the milk samples containing the increasing concentrations DES (0.5, 5, and 50 ng/mL) were prepared. The measurement for real samples was the same as the detection process of DES with QCM sensor. The samples were also analyzed via HPLC-MS/MS for validation. 50 mL of milk sample were centrifuged at 3500 rpm for 5 min. The substrate was collected. Different concentrations of the DES solution (0.5, 5, and 50 ng/mL) were prepared in methanol. A quantitative of DES-D8 was added to each DES solution to make its concentration 10 ng/mL in the all DES solutions. The liquid was dried via mild nitrogen gas, and 5 mL milk samples were subsequently added. After the mixture was shaken until the solid was completely dissolved, 10 mL of acetonitrile was added. Then the mixture were mixed with an oscillator for 3 min and centrifuged at 3500 rpm for 10 min. The supernates were collected and 5 mL of acetonitrile was added again. Repeated the procedure above, the extracting solution was combined. After the liquid was dried via nitrogen gas at 50 ℃, 10 mL water was added and the pH was adjusted to 11.0. Then the liquid was centrifuged at 9000 rpm for 5 min at 4 ℃. Blank samples were prepared following the previous procedure without DES. The liquids were filtered with a 0.22 μm PTFE filter, followed by HPLC-MS/MS detection. The analysis was performed in triplicate.
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2.5. The detection of DES using LCM sensor 2.5.1 Development and screening test of arrayed LCM sensor
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Five gold electrodes (recording element 1 to 5 respectively) were plated on a single langasite crystal to prepare an arrayed LCM sensor. In order to evaluate the vibration characteristics of the five array elements, the network analyzer HP8752A was used to test their vibration performance. The LCM sensor was fabricated according to the procedure described in the fabrication of QCM sensor (2.4.1). Different electrodes were modified at the same time. 2.5.2. Control experiment
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The frequencies of three DES-BSA-modified crystals were detected by arrayed LCM sensor and the frequency values were recorded F1. 20 μL of 14 μg/mL anti-DES antibody was dropped on the element 4 and element 5 respectively, and 20 μL of PBS solution was dropped on the element 3 as control group. After the reaction cell was placed in an incubator chamber at 37 ℃ for 1 h, the frequencies were detected by arrayed LCM sensor and the frequency values were recorded F2. Then the frequency shifts (ΔF=F2-F1) represent the changes caused by environment factors or both environment factors and anti-DES antibody. 2.5.3. The detection for different concentrations of anti-DES antibody
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The frequencies of three DES-BSA-modified crystals were detected by arrayed LCM sensor and the frequency values were recorded F3. 20 μL of different concentrations of anti-DES antibody (3.5 μg/mL, 7 μg/mL and 14 μg/mL) were respectively dropped on the element 3, element 4 and element 5. After the reaction cell was placed in an incubator chamber at 37 ℃ for 2 h, the frequencies were detected by arrayed LCM sensor and the frequency values were recorded F4. Then the frequency shifts (ΔF=F4-F3) represent the changes caused by both environment factors and different concentrations of anti-DES antibody. 3. Results and discussion
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3.1. Atomic force microscope (AFM) characterization of the QCM sensor
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Topographic images were taken by AFM in the non-contact mode (5 μm×5 μm surface). In order to compare the topologies of each surface, surface roughness (Ra) and root mean square roughness (Rq) were estimated from the AFM image [30]. Fig.2 represent surface morphology of the bare Au (Fig.2A), DES-BSA immobilization (Fig.2B), anti-DES antibody binding (Fig.2C), AuNPs-DES Ab binding (Fig.2D) based QCM sensor. Because a highly ordered and densely packed self-assembled layer of MUA can appear on the gold surface [31] and the Ra and Rq values for the bare Au surface and MUA electrode surface will be similar, the AFM characterization for the MUA electrode surface was not been conducted. The increase of surface roughness of DES-BSA electrode (Fig.2B) as indicated by the values of Ra (2.31 nm from 2.40 nm) and Rq (1.83 nm from 1.87 nm) demonstrates immobilization of DES-BSA on to electrode surface. The Ra and Rq have been found to be increase to 2.79 nm and 2.22 nm of anti-DES antibody electrode (Fig.2C) after the interaction of antigen with the antibody. Compared with anti-DES antibody, the Ra (from 2.40 to 14.7) and Rq (from 1.87 to 9.99) had drastic increase after the binding of AuNPs-DES Ab (Fig.2D). This may result from the gold nanoparticels amplification. The results shown by AFM images after each treated step indicated that DES-BSA and AuNPs-DES Ab have been successfully performed on a QCM device.
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3.2. Optimization of DES-BSA immobilization Efficient antigen immobilization is important for enabling sensitive antibody detection, hence different concentrations of DES-BSA were examined to optimize the assay. Six different concentrations of DES-BSA were immobilized (0.14 to 2.20 mg/mL) on the MUA immobilized surface. Fig.3 shows the frequency changes achieved with the increasing DES-BSA concentrations. Shown from the figure, DES-BSA concentration of 2.20 mg/mL gave the optimal response change (31 Hz) and therefore, this concentration was chosen for the following assay.
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3.3. Optimisation of AuNPs-DES Ab binding assay
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The concentration of AuNPs-DES Ab was optimized in order to achieve the widest detection range and the optimal detection limit for the sensor. Six different concentrations of AuNPs-DES Ab were immobilized (1.75 to 56.00 μg/mL) on the DES-BSA immobilized surface. Fig.4 shows the frequency changes response with the increasing concentrations of the AuNPs-DES Ab. Shown from the figure, AuNPs-DES Ab concentration of 14 μg/mL gave the optimal response change (101 Hz) and then the frequency shift levels off with the concentration increasing, therefore, this concentration was chosen for the following assay. 3.4. Analytical performance of the QCM sensor and calibration curve
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Typical temporal response curves of the QCM sensor at different concentration of DES are demonstrated in Fig.5A. It is shown that in 0.16~500 ng/mL of DES solution, the sensor ’s resonant frequency decreased over time due to the binding of AuNPs-DES Ab onto the immobilized DES-BSA, and the time to reach a plateau at ~15 min. When the DES concentration was 0.032 ng/mL, the temporal response curves could not be distinguished from the baseline of negative control. The calibration graph of frequency decrease vs. logarithm of DES concentration was displayed in Fig.5B. It can be seen that in the entire working range of 0.16~500 ng/mL, the higher the concentration, the smaller the sensor response. The calibration curve formula is y=﹣ 24.17 logx+69.72, with a correlation coefficient of 0.998. The limit of detection (LOD) was determined at 0.13 ng/mL based on the 3σ values of the blank signals. 3.5. Cross-reactivity of the QCM sensor with the analogues
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Cross-reactivity describes the specificity of the antibody applied in the assay and is an important analytical parameter regarding specificity and reliability of immunosensor devices[32]. To study the specificity of the QCM sensor, cross-reactivity with DES and structural analogs were compared. Fig.6 shows the frequency shifts caused by DES and the structural analogs at the same concentration (20 ng/mL) in the detection solution. The results reveal that the detection of DES could be distinguished from the other structural analogs, so the sensor exhibited higher selective recognition to DES. 3.6. Real samples analysis Immunoassays are known to suffer from matrix effects because of nonspecific binding and denaturation of the Ab and the enzyme, which results in false-positive responses [33]. To investigate the matrix effect and the applicability of the proposed sensor, the content of DES in milk samples was detected using spiked recovery method. Prepared samples were analyzed using the optimized QCM sensor at three levels. The determination results were shown in Table 1. Good recovery rates ranged from 98.00% to 104.80% were obtained with the milk samples. Thus, the proposed sensor allows direct DES analysis in milk samples. The samples were also analyzed by HPLC-MS/MS 8
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for confirmation. No significant differences were obtained with the recovery rates ranged from 102.00% to 104.90%, which demonstrated the good performance of the optimized QCM sensor. Besides, compared with the HPLC-MS/MS method, the QCM sensor could detect DES in real milk samples without any further treatment. The method is efficient and environmental friendly because it’s not necessary for the complex pretreatment with much organic solvent. 3.7. Stability and repeatability
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Stability and reusability are major concerns in evaluating a QCM immunosensor. It was found that the crystal modified with DES-BSA could retain its activity for at least 1 week at 4 °C before its first use. In our work, all the results were obtained on a QCM sensor surface with freshly immobilized DES-BSA and limited experiments were carried out to inspect the repeatability of the sensor. For regeneration, we could release the antibody from the sensor by rinsing the crystal in the buffer with low pH (commonly to pH 1) whereas minor DES-BSA immobilized would also be released from the sensor. This would decrease its ability to detect the analyte inevitably. So an investigation should be implemented to reduce the loss of activity and improve the regeneration of sensors.
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3.8. Development of arrayed LCM sensor
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The arrayed langasite crystal device was designed (Fig.7). Five gold electrodes were plated on a single gallium silicate crystal by the vacuum coating way, and each array element oscillate without disturbing each other by time sharing gate model. Besides, only one side of the sensor can be used in liquid, which can make five electrodes work in the same liquid and this environment is free from outside distraction. The sensitive devices were connected to the testing instruments to complete the assembling of the arrayed LCM sensor (Fig.8). Possessing the function of time sharing gate make the five electrodes on a single crystal can work well but without interfering with each so that the device can detect DES in arrayed methods.
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3.9. The evaluation and screening for the array elements
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The piezoelectric crystal may cease the oscillation in liquids of high viscosity. Because of the difference in the production process, it is difficult to ensure that every array element can possess good vibration performance. Fig.9 was the result of screening test for the five array elements. The curve reflected the impedance of analytical device. If the peak of the curve was more incisive, the impedance of the device was smaller and it will have good vibration characteristics. As can be seen from figure 8, the element 1 and 2 were found the obvious spurious resonance, which may be caused in the production process and interfere the measurement. In order to avoid the interference for the detection, the element 3, 4 and 5 were chosen for the subsequent experiments. 3.10. The results of control experiment Compared with element 3 (control group), element 4 and element 5 (experiment group) performed significant different in the frequency shifts, and at the same time, the frequency shifts between element 4 and element 5 had a few minor differences (Fig.10), which indicated that the arrayed LCM sensor could detect DES with arrayed methods. Setting up a control group during the experiment can compensate the interference of environment factors by reference method, which boosts this sensor’s resistance to interference. Anti-DES antibodies with the same concentration were detected on element 4 and element 5 and the frequency shifts caused on the
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two elements were similar, which revealed that the sensor had high accuracy and reliability. 3.11. The detection results for different concentrations of anti-DES antibody
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A good arrayed sensor can not only conduct reference experiment but also detect different samples at the same time. In order to evaluate the efficiency of high-through detection of the arrayed sensor, different concentrations of anti-DES antibody were used on the arrayed LCM sensor. The results were shown in Fig.11. With the increasing of anti-DES antibody concentration, the frequency shifts show a rising trend. The detection efficiency was improved by detect several concentrations of anti-DES antibody at the same time. 4. Conclusion
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This study successfully developed a method based QCM sensor for the detection of DES in milk with AuNPs amplification. Compared with the large-scale equipment, this method was more simply during the operation and more sensitive with the lower detection limit of 13 ng/mL. In addition, this QCM sensor has high specificity to DES. Therefore, this method can be further applied to detect other environmental pollutants. At the same time, an arrayed LCM sensor by plating five gold electrodes on a single gallium silicate crystal was also developed in this study for the arrayed detection of DES. It was found that on the different electrodes of the same crystal, the frequency shifts caused by the same concentrations of anti-DES antibody were similar; and with the increasing of concentrations of anti-DES antibody, the frequency shifts were rising. Therefore, the arrayed LCM sensor can preliminary achieve the detection of DES with high throughput. Acknowledgements
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The authors gratefully acknowledge financial support from the National Nature Science Foundation of China (No.21177159).
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Figure Caption
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Fig.1. Schematic illustration of the fabrication procedures for the QCM sensor
Fig.2. AFM of the surface of QCM sensor crystal. A: bare surface and B: DES-BSA immobilization
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and C: anti-DES antibody binding and D: AuNPs-DES Ab binding
Fig.3. Frequency changes for the different concentrations of DES-BSA
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Fig.4. Frequency changes for the different concentrations of AuNPs-DES Ab
Fig.5.（A）Frequency change of the QCM sensor as a function of time upon interaction with different
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concentration of DES; and (B) Calibration curves for DES determination based on the QCM sensor.
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DES concentration was between 0.16 and 500 ng/mL.
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Fig.6. Frequency shifts of DES and its structural analogs on the QCM sensor. The concentration of
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DES and its structural analogs is 20 ng/mL.
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Fig.7. Arrayed langasite crystal device
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Fig.8. Arrayed langasite crystal microbalance
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Fig.9. The vibration characteristics of five elements
Fig.10. The results of reference experiment
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Fig.11. The detection results of different concentrations of anti-DES antibody
Table 1 Results of the determination of DES in spiked milk samples (n=3) QCM sensor (mean ± SD)(ng/mL)
Recovery ratio （%）
HPLC-MS/MS (mean ± SD)(ng/mL)
Recovery ratio （%）
0.50 5.00 50.00
0.49±0.01 5.24±0.35 51.65±1.00
98.00 104.80 103.30
0.50±0.01 5.16±0.30 52.45±1.30
102.00 103.30 104.90
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