- Email: [email protected]
A bovine serum albumin-coated magnetoelastic biosensor for the wireless detection of heavy metal ions
Accepted Manuscript Title: A bovine serum albumin-coated magnetoelastic biosensor for the wireless detection of heavy metal ions Authors: Xing Guo, Shengbo Sang, Aoqun Jian, Shuang Gao, Qianqian Duan, Jianlong Ji, Qiang Zhang, Wendong Zhang PII: DOI: Reference:
S0925-4005(17)31938-X https://doi.org/10.1016/j.snb.2017.10.040 SNB 23344
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
18-4-2017 4-10-2017 6-10-2017
Please cite this article as: { https://doi.org/ 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.
A bovine serum albumin-coated magnetoelastic biosensor for the wireless detection of heavy metal ions Xing Guo1†, Shengbo Sang1†*, Aoqun Jian1, Shuang Gao1, Qianqian Duan1, Jianlong Ji1, Qiang Zhang1, Wendong Zhang1 1
MicroNano System Research Center, Key Lab of Advanced Transducers and
Intelligent Control System of the Ministry of Education & College of Information Engineering, Taiyuan University of Technology, Jinzhong 030600, China.
† These authors contributed equally to this work. *Address correspondence to: [email protected] (Shengbo Sang)
Highlights 1． A BSA-coated ME biosensor with high-sensitive and low-cost features is presented for wireless detection of heavy metal ions. 2． The biosensor is used to detect Pb2+, Cd2+, Cu2+, and is more sensitive to the heavy metal with larger molecular weight. 3． The detection limits for Pb2+ and Cd2+ meet the standard limit (GB 8978-1996) currently available.
Abstract A magnetoelastic (ME) biosensor functionalized by bovine serum albumin (BSA) was firstly developed for the wireless detection of heavy metal ions in solution. The biosensor was fabricated by coating a gold-modified ME ribbon with BSA. Its resonance oscillation and resonance frequency can be actuated and wirelessly 1
monitored through magnetic fields. The combination of heavy metal ions and BSA produces the precipitation adhered to the biosensor surface, resulting in the increase of mass loading, and consequently leads to a decrease in the resonance frequency, corresponding to the concentrations of heavy metal ions. The biosensor was used to separately detect Pb2+, Cd2+ and Cu2+. Its response is linearly proportional to the low concentrations, becoming sub-linear at higher concentrations. Moreover, the SEM, fluorescence microscopy and EDS spectrum analysis proved that the modification and detection process were successful. The biosensor is more sensitive to the heavy metal with larger molecular weight, and has the sensitivity for Pb2+, Cd2+ and Cu2+ of about 9.4 × 107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. The detection limits for Pb2+ and Cd2+ of 3.3× 10−7 mol/L and 2.4× 10−7mol/L, respectively, are lower than the standard limit (GB 8978-1996).
Keywords: magnetoelastic biosensor; wireless; bovine serum albumin; detection; heavy metal ions
1. Introduction Among the health problems associated with industrial development, heavy metals released into the environment cause a significant threat to public health. Some metal ions, such as Pb2+, Cd2+ and Cu2+, are very toxic and can easily enter the food chain through multiple pathways [1]. Excessive amounts of heavy metal ions in drinking water result in several deadly diseases, such as cardiovascular disease, 2
neurodegenerative diseases, cancer and nervous system failure [2-4]. Accordingly, for health protection reasons, there is an obvious need to monitor heavy metal ions content in foodstuffs, medications, and the environment. A number of methods have been developed for heavy metal ion analysis, such as atomic absorption spectrometry [5], fluorescence spectroscopy [6], anodic stripping voltammetry [7]. Although these methods are well established, they are complex, time consuming and expensive. Furthermore, a magnetoelastic (ME) sensor based on the replacement reaction mechanism for lead ions detection has been recently investigated, but the detection limit of the sensor is not low enough [8]. Therefore, a high-sensitive, facile, inexpensive and precise method is needed for the detection of heavy metal ions at low concentrations. Due to its low cost feature and remote query ability, the wireless ME sensor made of amorphous, ferromagnetic metallic glass ribbon, such as Metglas 2826MB has been extensively investigated as a high-performance transducer for biosensors development [9-11]. In response to the superposition of both alternating and static magnetic field, the ME ribbon vibrates longitudinally at its resonance frequency f0 given by Eq. (1) [12]. 1
𝐸
𝑓0 = 2𝐿 √𝜌(1−𝑣2 ) (1) Where, E is the Young’s modulus of elasticity, ρ is the density of the sensor material, 𝑣 is the Poisson’s ratio and L is the length of the sensor. For a small extra mass load of ∆𝑚 on the sensor of mass M (∆𝑚 ≪ 𝑀), the shift in the frequency (∆𝑓) 3
is given by Eq. (2) [13]. 𝑓
∆𝑓 = − 20 ∙
∆𝑚 𝑀
(2)
It is apparent from Eq. (2) that ∆f is proportional to the mass loading. As the resonance frequency of the ME ribbon is easily affected by the mass change, the ME sensors coated with a chemical or biological sensing membrane can be used for the detection of a variety of particles, including endotoxin [14], glucose [15], Salmonella typhimurium [16], Escherichia coli [17], Staphylococcus aureus [18]. Since the ME sensor is also magnetostrictive, it can be wirelessly actuated and sensed using magnetic field/signal [19]. Magnetic field telemetry enables its contact-less, remote-query operation that is the principle advantage over other sensor platforms. The phenomenon of the precipitation of bovine serum albumin (BSA) induced by heavy metal ions has been widely investigated [20]. Proteins are amphoteric substances existing in solution as cations or anions, on the basis of whether the hydrogen ion concentration of the solution is larger or smaller than that at the isoelectric point. The theory for heavy metal salt precipitation is as follows: when a dilute solution of heavy metal salt is added in a protein (BSA) solution on the alkaline side of its isoelectric point, the anionic protein combines with the cation, leading to a precipitation, for example [21-22]: BSA + Pb (Ⅱ)→ Pb (Ⅱ)-BSA↓
(3)
BSA + Cd (Ⅱ)→ Cd (Ⅱ)-BSA↓
(4)
BSA + Cu (Ⅱ)→ Cu (Ⅱ)-BSA↓
(5)
In this study, an ME biosensor, a mass sensitive device, composed of an ME 4
ribbon as the transducer platform and BSA as the bio-recognition element, was demonstrated for target heavy metal ions detection. The performances of the wireless ME biosensor in Pb2+, Cd2+ and Cu2+ solution with different concentrations were evaluated here. The heavy metal ions-induced precipitation of BSA accumulated on the biosensor surface leads to a decrease in the biosensor resonance frequency, according to Eq. (2), which in turn is proportional to the amount of heavy metal ions. Moreover, based on its wireless nature, the BSA-coated ME biosensor can be used for remote detection of heavy metal ions.
2. Materials and methods 2.1. Materials BSA was obtained from Sigma-Aldrich (Saint Louis, MO, USA) and used without further purification. 6-Mercaptoundecanoic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS) and phosphate buffered saline (PBS buffer, pH= 7.4) were also purchased from Sigma-Aldrich. ME ribbons composed of Metglas alloy 2826 (Fe40Ni40P14B6) were purchased from
Honeywell
Corporation
(Morristown,
NJ,
USA)
and
cut
into
5mm×1mm×0.028mm using a computer-controlled laser cutting machine. All experiments were conducted at room temperature [20, 22]. 2.2 ME biosensor fabrication 2.2.1 Preparation of the ME sensor platform ME ribbons were consecutively cleaned in an ultrasonic bath with alcohol and 5
deionized water, for 10 min each, to remove any debris and organic film, and then dried in a stream of nitrogen. Afterwards, the cleaned sensor platforms were sputtered with chromium (100 nm thick), followed by gold (100 nm thick). The chromium layer was the middle layer to enhance the adhesion between the gold and the sensor platform. The gold layer was used to prevent the corrosion of the ME ribbon and to improve the bio-compatibility of the sensor surface for the immobilization of BSA. The gold-coated sensor platforms were annealed in a vacuum oven at 220℃ for 2 h to eliminate any residual stress in the sensor and improve adhesion of the gold layer to the ME substrate. The annealed gold-coated sensor platform (Fig. 1a) is ready for BSA immobilization to form the ME biosensor and its resonance frequency in air is approximately 450 kHz. Due to its coupling effect between magnetic energy and elastic energy, the ME sensor platform as a high-performance transducer used in the biosensor can provide the resonance frequency shift information reflecting the detection of heavy metal ions. 2.2.2 BSA immobilization The gold-coated sensor platforms were consecutively cleaned in an ultrasonic bath with acetone, isopropanol, deionized water and ethanol for 10 min each, and dried under a nitrogen stream. After the cleaning, the prepared sensor platforms were incubated, avoiding light, in a 10 mM/L ethanol solution of 6-mercaptoundecanoic acid for 24 h to form the self-assembled monolayer (SAM), as shown in Fig. 1b. The sensor platforms were then rinsed several times with ethanol to remove the unbonded thiols and dried under a stream of nitrogen. The thiol-modified sensor platforms were 6
immersed into a solution containing 40 mM EDC and 10 mM NHS in distilled water for 1 h to convert the terminal carboxylic groups to an active NHS ester. The sensor platforms were subsequently rinsed with deionized water and dried under a stream of nitrogen. BSA was diluted into 0.01 M/L PBS buffer to prepare a 4 mg/ml BSA solution. Afterwards, the sensor platforms were dipped into a 1 mL BSA solution for immobilization for 2 h, then rinsed with a PBS solution and deionized water to remove physically adsorbed BSA. Subsequently, the sensors were dried under a nitrogen stream. Then the ME biosensor functionalized by BSA is formed, as shown in Fig. 1c. Based on its ability to bind with heavy metal ions and low-cost characteristics, BSA is used as the bio-recognition element in the biosensor to combine with the target heavy metal ions, leading to an increase in the mass loading of the biosensor. 2.3 Signal measurement A bench-top vector network analyzer (AV3620A, the 41st Institute of CETC, Qingdao, China) was used to characterize the resonance frequency of the ME biosensor. The experimental setup for the wireless ME sensing measurement is shown in Fig. 2. Due to its wireless nature, the magnetic flux generated by vibrations can be detected using a remotely placed pickup coil [23]. The network analyzer was connected to a coil wound around a glass tube in which the biosensor was inserted vertically and wirelessly (without any wire connections with measurement system). The ME biosensor is wirelessly operated, containing no internal power sources such as a battery. The network analyzer operating in the S11 mode provides a swept 7
frequency signal to the coil and monitors the signal reflected from the coil. The coil is a 50-turn single-layer solenoid made of 32 AWG type wire which generates an alternating magnetic field actuating the biosensor to vibrate longitudinally. The frequency range of the alternating magnetic field was set from 430 to 460 kHz. In addition, a static magnetic field generated by a bar magnet (N35, 3cm × 2cm × 1cm, magnetization of thickness direction) was applied to enhance the resonance behavior of the biosensor [24]. The bar magnet was fixed on a stand so that the distance and direction of the magnet can be adjusted. Furthermore, we will consider using a Helmholtz coil in the future work so that the bias field can be easily measured and controlled. The resonance frequency (f0, f1) of the biosensor corresponding to the frequency of the lowest point in the S11 curve can then be measured wirelessly and remotely. In the test, the biosensors were placed into the test tubes, which contain 0.5 ml of various heavy metal ions solutions of different concentrations ranging from 0 to 9.6× 10−6mol/L. The various heavy metal ions solutions are the following: lead (II) nitrate, copper (II) nitrate and cadmium (II) nitrate dissolved in 0.9% brine, respectively. The resonance frequency of the biosensor was monitored at each concentration and recorded for 45 min with an interval of 5 min.
3. Results and discussion 3.1 Assessment of the immobilization of BSA Fluorescence microscopy was used to confirm that BSA was successfully 8
immobilized on the biosensor surface. Fluorescein isothiocyanate (FITC) has the ability to react with amino groups and can be observed using a fluorescence microscope, thus staining with FITC can be used to confirm the presence of proteins [25-26]. In this study, the FITC-labelled BSA was covalently immobilized to the gold surface coated with a thiol self-assembled monolayer by the amine coupling method, as shown in Fig. 3a. Amine coupling introduces NHS esters on the surface of self-assembled monolayer with the EDC-NHS solution. The EDC is utilized as a coupling agent to activate carboxyl groups, while NHS provides a stable activated ester. Then the BSA is covalently linked to the activated NHS esters through the amide bond [27]. Finally, the BSA-FITC (Beijing Bo Sheng Bio-technology Co., Ltd.) immobilized on the biosensor surface was observed with a fluorescence microscope (DM 3000, Leica Microsystems Ltd.; Wetzlar, Germany). Numerous green fluoresce spots of BSA-FITC nanoparticles were observed on the biosensor surface as shown in Fig. 3b. In contrast, control experiment without BSA (0mg/mL) was conducted, as observed in Fig. 3c. The image is black background without any fluoresce spots. Similar observations have been made by Lademann et al. [28]. The results indicate that the BSA-FITC nanoparticles have been modified on the biosensor surface and additionally demonstrate that BSA can be successfully immobilized on the biosensor surface through the thiol self-assembly method. 3.2 Effect of pH values The effect of pH has been investigated by determining the ME biosensor responses to Pb2+ of 9.6× 10−6 mol/L at different pH values of 5.4, 6.1, 7, as shown 9
in Fig. 4. The isoelectric point of BSA is approximately pH 4.7 [29]. When the pH value of the solution is lower than 4.7, the BSA molecules exist as cations that cannot combine with heavy metal ions. Otherwise, when the pH value of the solution is higher than 4.7, the BSA remains anionic which can bind with heavy metal ions. Besides, considering that the three-dimensional structure of BSA would denature at higher pH values [29], we chose the pH values above 4.7 and below 7 to study in this work. From Fig. 4, we can see that with same concentration of heavy metal ions, the resonance frequency shift reached maximum at pH=6.1. Thus pH 6.1 was chosen for following determination. 3.3 The detection of lead ions Fig. 5 shows the dynamic frequency response of ME biosensor for Pb2+ detection at different concentrations ranging from 0 to 9.6× 10−6 mol/L. When the biosensor was immersed in Pb2+ solution (pH = 6.1), the Pb2+ from the solution were adsorbed to the active points in the BSA molecules on the biosensor surface. According to Eq. (2) and Eq. (3), the precipitation induced by Pb2+ causes an increase in mass on the biosensor surface, resulting in a decrease in the resonance frequency after a reaction period, as shown in Fig. 5, and thus the biosensor can be used for real-time monitoring. The reaction period is attributed to the time required for the combination. The results in Fig. 5 indicate that the steady-state response is achieved at 45 minutes, while the combination at higher concentration certainly needs more time to reach saturation. Additionally, the results in Fig. 5 reveal that the shift rate of the resonant frequency increases with the increasing Pb2+ concentration and a higher concentration 10
of Pb2+ can induce a larger resonance frequency shift. The phenomenon can be attributed to the fact that as more Pb2+ combine with BSA, then more precipitation adhere to the biosensor surface. Thus the wireless ME biosensor can be used to detect Pb2+. To confirm that the frequency response of the biosensor is only due to Pb2+, a control experiment was performed without Pb2+ (0 mol/L curve) that shows a noise level of ~18 Hz. However, there is a 60-Hz shift in resonance frequency for 0.6 × 10−6 mol/L Pb2+ over the same reaction period, as shown in Fig. 5. There is only a small frequency shift in the absence of interaction between Pb2+ and BSA, so the frequency response is caused by the Pb2+-induced BSA precipitate. The process of the BSA-Pb precipitate loaded on the biosensor surface was assessed by scanning electron microscopy (SEM) analysis and energy-dispersive spectroscopy (EDS) analysis. The SEM examination was performed using a SU3500 SEM (Hitachi corporation, Tokyo, Japan) operated at 5 keV. As observed in Fig. 6a, a naked surface image was from the biosensor gold surface without functionalization. The surface is smooth and has no impurity. After the biosensors were treated with BSA, spherical aggregates of the BSA nanoparticles were evenly distributed on the surface, as shown in Fig. 6b, indicating that the biosensors were immobilized with BSA successfully. Similar results have been described by others [30-31]. When the BSA-coated biosensors were exposed to Pb2+ solution (9.6× 10−6 mol/L), flake agglomerations were presented due to the aggregation by Pb2+, as shown in Fig. 6c. Furthermore, a typical EDS spectrum for elemental analysis before and after the 11
addition of Pb2+ is presented in Fig. 6d. It is evident that the accumulation of lead ions increased dramatically after the biosensors were immersed in Pb2+ solution. Given all this, the BSA-Pb precipitate adhered to the biosensor surface is demonstrated. Comparing the SEM images and the EDS spectrum, it can be concluded that the modification and detection procedures were successful. 3.4 The detection of cadmium ions In addition, similar kinds of experiments were performed for Cd2+ detection on the biosensor. The changes in resonance frequency versus time at different concentrations of Cd2+ ranging from 0 to 9.6× 10−6 mol/L are depicted in Fig. 7. These frequency responses are similar to those obtained for Pb2+. Due to the adherence of the BSA–Cd precipitate on the biosensor surface, the resonance frequency decreases with time according to Eq. (2) and Eq. (4). The shift in the resonance frequency increases with the rise of the Cd2+ concentration. Accordingly, Cd2+ can be detected wirelessly by the shift in resonance frequency of the ME biosensor. 3.5 The detection of copper ions Likewise, Cu2+ ions were detected through similar kinds of experiments with the ME biosensor. The chosen concentration range of Cu2+ was same as those of Cd2+ and Pb2+. As shown in Fig. 8, the frequency shift rises with the increasing concentration of Cu2+ in a similar manner as found above for the Cd2+ and Pb2+. This means that the mass effect resulting from the adherence of the BSA-Cu precipitate leads to the decrease in frequency according to Eq. (2) and Eq. (5). Consequently, the ME 12
biosensor is also suitable for the wireless detection of Cu2+. 3.6 The sensitivity and reproducibility of the biosensor The standard calibration curves for the detection of heavy metal ions on the biosensors during the first 45 min are shown in Fig. 9. For each heavy metal ion, the biosensor calibration experiments were performed five times under identical conditions. It is found that the shift in resonance frequency is linearly proportional to the concentration in the range of 0 to 4.8× 10−6 mol/L, however, with the increase of concentration the relationship becomes sub-linear, which indicates that the linearity holds better at low concentrations. The linear equations for Pb2+, Cd2+ and Cu2+ detection could be represented by ∆f = −1.5 × 108 𝐶𝑃𝑏2+ − 11 (𝑅 2 = 0.972),∆f = −7.4 × 107 𝐶𝐶𝑑2+ − 8.9(𝑅 2 = 0.989)
and
∆f = −4.9 × 107 𝐶𝐶𝑢2+ − 15(𝑅 2 =
0.991) respectively. The limit of detection (LOD) for Pb2+, Cd2+, Cu2+ is calculated to be 3.3× 10−7mol/L, 2.4× 10−7 mol/L, 2.3× 10−7mol/L, respectively , according to the Eq. (6) [32]: LOD = 3𝑆𝐵 /𝑏
(6)
Where 𝑆𝐵 is the standard deviation of the blank solution and 𝑏 is the slope of the fitted curve. The detection limits for Pb2+ and Cd2+ are converted to 0.107mg/L and 0.074mg/L, respectively, are both lower than the maximum permissible discharge standards for Pb2+ and Cd2+ of 1mg/L and 0.1mg/L given in the Integrated Waste Water Discharge Standard (GB 8978-1996), which show the LOD of the biosensor meets the standard currently available. The sensitivity of the biosensor for Pb2+, Cd2+ and Cu2+ is calculated from the linear region of the curves in Fig. 9, and is 9.4 × 13
107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. By contrast, various published methods for detection of heavy metal ions are summarized in Table 1, showing the superior performance of our biosensor. Therefore, the sensitivity and detection limit of the ME BSA-coated biosensor are better than those reported in the previous researches.To calibrate the responses from different sensors, by our experience,
𝑓𝑟 =450 kHz is set for the standard reference
frequency of the biosensor in the air. Then the result of resonance frequency shift ∆F after calibration is given by Eq. (7) and Eq. (8). ∆𝑓𝑟 = 𝑓 − 𝑓𝑟
(7)
∆F = ∆𝑓 − ∆𝑓𝑟
(8)
Where f is the biosensor’ resonance frequency measured in the air before use and ∆𝑓 is the resonance frequency shift measured in the detection process. In this way, the deviation from different biosensors can be calibrated. Furthermore, as shown in Fig. 9, the resonance frequency shift of the biosensor for the detection of Pb2+ at the same concentration with Cd2+ and Cu2+ is the largest. In addition, it is clear from Fig. 9 that the shift in resonance frequency for Cd2+ is larger than that of Cu2+ at the same concentration. The molecular weights of lead, cadmium and copper are 207.2, 112.4 and 64 respectively. Since the molecular weight of lead is the largest among the three heavy metals, the mass of precipitate adhered to the biosensor surface induced by Pb2+ is the largest, while that induced by Cd2+ takes second place. According to Eq. (2), the shift in resonance frequency for all three metal ions is in decreasing order: Pb2+> Cd2+> Cu2+. Thus, the biosensor responds to all 14
three metal ions with decreasing sensitivity in the order: Pb2+> Cd2+> Cu2+. This reveals that the biosensor is more sensitive to the heavy metal with larger molecular weight. 3.7 Interference experiment The mutual interference between Cu2+ and Pb2+ on the biosensor was investigated. Fig. 10 shows the frequency response of the biosensor towards Pb2+ in the presence of Cu2+ with different blending ratio in the solution. It is evident from the curves in Fig. 10 that the shift rate of the resonant frequency decreases with the addition of Cu2+. Besides, in the presence of Cu2+, the frequency shift is less than that of the individual determination of Pb2+ at a certain total mole number of ions. The results illustrate that the Cu2+ coexisting in the solution would decrease the response to Pb2+. As the molecular weight of Cu is smaller than that of Pb, the mass of BSA-Cu precipitate is smaller than that of BSA-Pb precipitate loaded on the biosensor. Likewise, the addition of Pb2+ could enhance the response of the coexisting Cu2+.
4. Conclusions For the first time, a BSA-coated ME biosensor for the wireless detection of heavy metal ions is presented. BSA is immobilized onto the ME ribbon surface to form an ME biosensor to detect Pb2+, Cd2+, Cu2+. The adherence of the BSA precipitate induced by heavy metal ions leads to an increase in the mass loading of the biosensor, which results in the decrease of the resonance frequency of the biosensor. The shift in resonance frequency increases with the increasing concentration of heavy metal ions, 15
which is linearly proportional to low concentrations, but becomes sub-linear at higher concentrations. The sensitivity of the biosensor for Pb2+, Cd2+ and Cu2+ is 9.4 × 107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. The results indicate that the biosensor can be used as a high-sensitive device to detect heavy metal ions and is more sensitive to the heavy metal with larger molecular weight. Additionally, the detection limit of the ME biosensor for Pb2+, Cd2+ and Cu2+ is approximately 3.3× 10−7mol/L, 2.4 × 10−7 mol/L and 2.3 × 10−7 mol/L, respectively. Considering that the elastic property, liquid viscosity and replacement reaction mechanism of the Pb2+ in solution with Fe and Ni in the ME ribbon may all affect the ME biosensor’s response, its respective contribution to frequency shift will be investigated in detail in the next study. In addition, the present biosensor is not specific for any particular metal ions but can be utilized for bulk detection of several heavy metal ions. Future work will be focused on the specific protein to detect a particular kind of heavy metal ion and a portable detector for the consistent measurement of the resonance frequency.
Acknowledgements The authors are grateful for the support by the National Natural Science Foundation of China (No. 51622507, 61471255, 61474079, 61501316, 51505324), Doctoral
Fund
of
MOE
of
China
(No.
20131402110013),
863
project
(2015AA042601), Excellent Talents Technology Innovation Program of Shanxi Province of China (201605D211023). 16
References [1] F. Ge, M.M. Li, H. Ye, B.X. Zhao. Effective removal of heavy metal ions Cd2+, Zn2+, Pb2+, Cu2+ from aqueous solution by polymer-modified magnetic nanoparticles. J. Hazard. Mater. 211 (2012) 366-372. [2] Z. Dong, L. Wang, J. Xu, Y. Li, Y. Zhang, S. Zhang, J. Miao. Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells. Toxicology in Vitro. 23 (2009) 105–110. [3] J. Liu, W. Qu, M.B. Kadiiska. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol. Appl. Pharmacol. 238 (2009) 209–214. [4] R. Verma, B.D. Gupta. Detection of heavy metal ions in contaminated water by surface plasmon resonance based optical fibre sensor using conducting polymer and chitosan. Food Chem. 166 (2015) 568-575. [5] D.I. Bannon, J.J. Chisolm. Anodic stripping voltammetry compared with graphite furnace atomic absorption spectrophotometry for blood lead analysis.
Clin.
Chem. 47 (2001), 1703-1704. [6] Q. He, E.W. Miller, A.P. Wong, C.J. Chang. A selective fluorescent sensor for detecting lead in living cells. Journal of American Chemical Society. 128 (2006) 9316–9317. [7] W.R. Yang, E. Chow, G.D. Willett, D.B. Hibbert, J.J. Gooding. Exploring the use of the tripeptide Gly-Gly-His as a selective recognition element for the fabrication of electrochemical copper sensors. Analyst. 128 (2003) 712-718. 17
[8] S. Sang, S. Gao, X. Guo, P. Cheng, W. Zhang. The detection of Pb2+ in solution using bare magnetoelastic resonator. Appl. Phys. Lett. 108 (2016) 054102. [9] L. Fu, S. Li, K. Zhang, I. Chen, V.A. Petrenko, Z. Cheng. Magnetostrictive microcantilever as an advanced transducer for biosensors. Sensors. 7(11) (2007) 2929-2941. [10] S.J. Huang, P.F. Pang, X.L. Xiao, L.E. He, Q.Y. Cai, C.A. Grimes. A wireless, remote query sensor for real-time detection of Escherichia coli O157:H7 concentrations. Sens. Actuators B 131 (2008) 489–495. [11] H. Lin, Q. Lu, S. Ge, Q. Cai, C.A. Grimes. Detection of pathogen Escherichia coli O157: H7 with a wireless magnetoelastic-sensing device amplified by using chitosan-modified magnetic Fe 3 O 4 nanoparticles. Sensors and Actuators B: Chemical. 147(1) (2010) 343-349. [12] P.G. Stoyanov, C.A. Grimes. A remote query magnetostrictive viscosity sensor. Sens. Actuators A 80 (2000) 8–14. [13] W. Shen, L.C. Mathison, V.A. Petrenko, B.A. Chin. Design and characterization of a magnetoelastic sensor for the detection of biological agents.
J. Phys. D:
Appl. Phys. 43 (2009) 015004. [14] K.G. Ong, J.M. Leland, K.F. Zeng, G. Barrett, M. Zourob, C.A. Grimes. A rapid highly-sensitive endotoxin detection system. Biosens. Bioelectron. 21 (2006) 2270–2274. [15] P. Pang, Y. Zhang, S. Ge, Q. Cai, S. Yao, C.A. Grimes. Determination of glucose using bienzyme layered assembly magnetoelastic sensing device. Sens. Actuators, 18
B 136 (2009), 310-314. [16] M.K. Park, S. Li, B.A. Chin. Detection of Salmonella typhimurium grown directly on tomato surface using phage-based magnetoelastic biosensors. Food and Bioprocess Technology. 6 (2013), 682-689. [17] A.L. Possan, C. Menti, M. Beltrami, A.D. Santos, M. Roesch-Ely, F.P. Missell. Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli. Mater. Sci. Eng., C 58 (2016) 541-547. [18] N. Hiremath, R. Guntupalli, V. Vodyanoy, B.A. Chin, M.K. Park. Detection of methicillin-resistant Staphylococcus aureus using novel lytic phage-based magnetoelastic biosensors. Sens. Actuators, B 210 (2015) 129-136. [19] S. Li, Y. Li, H. Chen, S. Horikawa, W. Shen, A. Simonian, B.A. Chin. Direct detection of Salmonella typhimurium on fresh produce using phage-based magnetoelastic biosensors. Biosens. Bioelectron. 26 (2010) 1313-1319. [20] Y. Yuan, Y. Cai, Q. Xie, S. Yao. Piezoelectric quartz crystal impedance study of the Pb2+-induced precipitation of bovine serum albumin and its dissolution with EDTA in an aqueous solution. Anal. Sci. 18 (2002) 767-771. [21] J. Yin, W. Wei, X. Liu, B. Kong, L. Wu, S. Gong. Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance. Anal. Biochem. 360 (2007) 99-104. [22] Y. Cai, Q. Xie, A. Zhou, Y. Zhang, S. Yao. A piezoelectric quartz crystal impedance study on Cu 2+-induced precipitation of bovine serum albumin in 19
aqueous solution. J. Biochem. Biophys. Methods. 47 (2001) 209-219. [23] L. Chen, J. Li, T.T. ThanhThuy, L. Zhou, L. Yuan, Q. Cai. A wireless and sensitive
detection
of
octachlorostyrene
using
modified
AuNPs
as
signal-amplifying tags. Biosens. Bioelectron. 52 (2014) 427-432. [24] K. Zhang, L. Zhang, L. Fu, S. Li, H. Chen, Z.Y. Cheng. Magnetostrictive resonators as sensors and actuators. Sens. Actuators, A 200 (2013) 2–10. [25] Y. Zhang, C. Wang. Micropatterning of Proteins on 3D Porous Polymer Film Fabricated by Using the Breath‐Figure Method. Advanced Materials. 19 (2007) 913-916. [26] S. Kreitz, C. Penache, M. Thomas, C.P. Klages. Patterned DBD treatment for area-selective metallization of polymers–plasma printing. Surf. Coat. Technol. 200 (2005) 676-679. [27] R. Pei, Z. Cheng, E. Wang, X. Yang. Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy. Biosens. Bioelectron. 16 (2001) 355-361. [28] J. Lademann, H. Richter, F. Knorr, A. Patzelt, M.E. Darvin, E. Rühl, K.Y. Cheung, K.K. Lai, R. Renneberg, W.C. Mak. Triggered release of model drug from AuNP-doped BSA nanocarriers in hair follicles using IRA radiation. Acta biomaterialia. 30 (2016) 388-396. [29] T. Kopac, K. Bozgeyik, J. Yener. Effect of pH and temperature on the adsorption of bovine serum albumin onto titanium dioxide. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 322(1) (2008) 19-28. 20
[30] H.H. Nguyen, J. Ryu, J.H. Che, T.S. Kang, J.K. Lee, C.W. Song, S. Ko. Robust size control of bovine serum albumin (BSA) nanoparticles by intermittent addition of a desolvating agent and the particle formation mechanism. Food Chem. 141 (2013) 695-701. [31] J.Y. Jun, H.H. Nguyen, S.Y.R. Paik, H.S. Chun, B.C. Kang, S. Ko. Preparation of size-controlled bovine serum albumin (BSA) nanoparticles by a modified desolvation method. Food Chem. 127 (2011) 1892-1898. [32] I. Krull, M. Swartz. Determining limits of detection and quantitation. LC–GC 16 (1998) 922–924. [33] Y. Liu, L. Zhang, R. Liu, P. Zhang. Spectroscopic identification of interactions of Pb2+ with bovine serum albumin. Journal of fluorescence. 22(1) (2012) 239-245. [34] D. Zhao, X. Guo, T. Wang, N. Alvarez, V.N. Shanov, W.R. Heineman. Simultaneous Detection of Heavy Metals by Anodic Stripping Voltammetry Using Carbon Nanotube Thread. Electroanalysis. 26 (2014) 488. [35] Y.F. Sun, L.J. Zhao, T.J. Jiang, S.S. Li, M. Yang, X.J. Huang. Sensitive and selective
electrochemical
amino-functionalized
carbon
detection
of
microspheres.
heavy
metal
Journal
of
ions
using
Electroanalytical
Chemistry. 760 (2016)143-150.
Author Biographies Xing Guo earned the B.Eng. degree in 2014 at Taiyuan University of Technology. She is currently a Ph.D. student at MicroNano System Research Center, Key Lab of Advanced Transducers and Intelligent Control System of the Ministry of Education & 21
College of Information Engineering. Her main research interest is in biosensors. Shengbo Sang received the B.Eng. degree in communication engineering and the M.Eng. degree in electronic science and technology from the North University of China, Taiyuan, China, in 2000 and 2006, respectively. He received the Ph.D. degree from the school of mechanical engineering, Ilmenau, Ilmenau University of Technology, Germany, in 2010. His current research interests include MEMS/NEMS, optical devices and biosensors. Aoqun Jian received the B.Eng. degree in electronic and information technology and the M.Eng. degree in microelectronics and solid electronics from the North University of China, Taiyuan, China, in 2005 and 2008, respectively. He received the Ph.D. degree in the Department of Applied Physics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, in 2013. His current research interests include nanooptics, particularly surface plasmon resonance resonant, optical tunneling effect and optofluidics. Shuang Gao received the B.Eng. degree in 2016 at Taiyuan University of Technology. He mainly engaged in MEMS and sensors. Qianqian Duan received the Bachelor of Engineer degree in Optical Information Science & Technology from Dalian University of Technology, Dalian, China, in 2007. She received Master of Science degree and Doctor of Science degree in Optics from Harbin Institute of Technology, Harbin,China, in 2009 and 2013, respectively. Her current research interests include nanooptics, nanowaveguide, nanometer fluorescence probe. Jianlong Ji received his PhD in Taiyuan University of technology and joined the Micro-nano system research center in 2014. He has united research experience in Tsinghua University, current research focus mainly on micro/nano-electromechanical systems (MEMS/NEMS) and microfluidic devices. Qiang Zhang received the Ph.D. degree in School of Instruments and Electronics, North University of China, Tai Yuan, China, in 2015. Her current research interests include nanomaterials, particularly soft strain sensors based on nanomaterials and photocatalytic performance of semiconductor nanomaterials. 22
Wendong Zhang received the Ph.D. degree from Beijing Institute of Technology, Beijing, China, in 1995. He went to the école normale supérieure (ENS) and Massachusetts Institute of Technology (MIT) as a senior visiting scholar in 2006 and 2009, respectively. He served the North University of China and Taiyuan University of Technology as the headmaster in 2004 and 2010, respectively. He is the technical chief of Project of National Security Key Basic Research. He was awarded the Second Prize of State Technological Invention Award three times and the Third Prize of State Technological Invention Award once. His current research interests include MEMS/NEMS and optical gyroscope.
Figure captions Fig. 1. Schematic diagram of the modification procedure of the biosensor.
Fig. 2. Schematic of the experimental setup for the wireless ME biosensor measurement.
23
Fig. 3. Schematic diagram (a) and fluorescence image (b) of the BSA-FITC modified on the biosensor surface. (c) Blank control image without BSA.
Fig. 4. The biosensor’s frequency response to Pb2+ of 9.6× 10−6 mol/L at different pH conditions.
Fig. 5. Real-time frequency response curves for Pb2+ detection at different concentrations ranging from 0 to 9.6× 10−6 mol/L.
24
Fig. 6. (a) SEM image of the gold-coated biosensor; (b) SEM image of the biosensor surface after the BSA immobilization; (c) SEM image of the biosensor surface after the Pb2+ detection; (d) EDS spectrum of the biosensor surface before and after the addition of Pb2+.
25
Fig. 7. The time-dependent frequency shift as a function of Cd2+ concentrations ranging from 0 to 9.6× 10−6 mol/L.
Fig. 8. Real-time frequency changes of the biosensor to Cu2+ concentrations ranging 26
from 0 to 9.6× 10−6 mol/L.
Fig. 9. The calibration curve: the 45 min shift in resonance frequency versus concentrations ranging from 0 to 9.6× 10−6 mol/L of different heavy metal ions.
27
Fig. 10. The frequency response of the biosensor in the simultaneous presence of Cu2+ and Pb2+ in solution.
28
Methods ME BSA-coated biosensor ME sensor based on replacement reaction mechanism PQCI study based on BSA PQCI study based on BSA Spectroscopic Identification SWASV based on Carbon Nanotube Thread SWASV based on NH2-CMS
Sensitivity (Hz per mol/L) 2+ Pb :9.4× 107
Pb2+: 8.0 × 103
Detection limit (mol/L) 2+ Pb : 3.3× 10−7 Cu2+: 2.3× 10−7 Cd2+: 2.4× 10−7 Pb2+: 1.5× 10−2
References This work
[8]
Cu2+: 9.98× 10−5
[21]
Pb2+: 4.8× 10−4
[19]
Pb2+: 1.0× 10−4
[33]
Cd2+: 1.0×10−6
[34]
Pb2+: 3.8× 10−7 Cu2+: 2.5× 10−7 Cd2+: 1.1× 10−6
[35]
Table 1. Comparisons of performances between various methods for the detection of heavy metal ions
29
S0925-4005(17)31938-X https://doi.org/10.1016/j.snb.2017.10.040 SNB 23344
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
18-4-2017 4-10-2017 6-10-2017
Please cite this article as: { https://doi.org/ 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.
A bovine serum albumin-coated magnetoelastic biosensor for the wireless detection of heavy metal ions Xing Guo1†, Shengbo Sang1†*, Aoqun Jian1, Shuang Gao1, Qianqian Duan1, Jianlong Ji1, Qiang Zhang1, Wendong Zhang1 1
MicroNano System Research Center, Key Lab of Advanced Transducers and
Intelligent Control System of the Ministry of Education & College of Information Engineering, Taiyuan University of Technology, Jinzhong 030600, China.
† These authors contributed equally to this work. *Address correspondence to: [email protected] (Shengbo Sang)
Highlights 1． A BSA-coated ME biosensor with high-sensitive and low-cost features is presented for wireless detection of heavy metal ions. 2． The biosensor is used to detect Pb2+, Cd2+, Cu2+, and is more sensitive to the heavy metal with larger molecular weight. 3． The detection limits for Pb2+ and Cd2+ meet the standard limit (GB 8978-1996) currently available.
Abstract A magnetoelastic (ME) biosensor functionalized by bovine serum albumin (BSA) was firstly developed for the wireless detection of heavy metal ions in solution. The biosensor was fabricated by coating a gold-modified ME ribbon with BSA. Its resonance oscillation and resonance frequency can be actuated and wirelessly 1
monitored through magnetic fields. The combination of heavy metal ions and BSA produces the precipitation adhered to the biosensor surface, resulting in the increase of mass loading, and consequently leads to a decrease in the resonance frequency, corresponding to the concentrations of heavy metal ions. The biosensor was used to separately detect Pb2+, Cd2+ and Cu2+. Its response is linearly proportional to the low concentrations, becoming sub-linear at higher concentrations. Moreover, the SEM, fluorescence microscopy and EDS spectrum analysis proved that the modification and detection process were successful. The biosensor is more sensitive to the heavy metal with larger molecular weight, and has the sensitivity for Pb2+, Cd2+ and Cu2+ of about 9.4 × 107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. The detection limits for Pb2+ and Cd2+ of 3.3× 10−7 mol/L and 2.4× 10−7mol/L, respectively, are lower than the standard limit (GB 8978-1996).
Keywords: magnetoelastic biosensor; wireless; bovine serum albumin; detection; heavy metal ions
1. Introduction Among the health problems associated with industrial development, heavy metals released into the environment cause a significant threat to public health. Some metal ions, such as Pb2+, Cd2+ and Cu2+, are very toxic and can easily enter the food chain through multiple pathways [1]. Excessive amounts of heavy metal ions in drinking water result in several deadly diseases, such as cardiovascular disease, 2
neurodegenerative diseases, cancer and nervous system failure [2-4]. Accordingly, for health protection reasons, there is an obvious need to monitor heavy metal ions content in foodstuffs, medications, and the environment. A number of methods have been developed for heavy metal ion analysis, such as atomic absorption spectrometry [5], fluorescence spectroscopy [6], anodic stripping voltammetry [7]. Although these methods are well established, they are complex, time consuming and expensive. Furthermore, a magnetoelastic (ME) sensor based on the replacement reaction mechanism for lead ions detection has been recently investigated, but the detection limit of the sensor is not low enough [8]. Therefore, a high-sensitive, facile, inexpensive and precise method is needed for the detection of heavy metal ions at low concentrations. Due to its low cost feature and remote query ability, the wireless ME sensor made of amorphous, ferromagnetic metallic glass ribbon, such as Metglas 2826MB has been extensively investigated as a high-performance transducer for biosensors development [9-11]. In response to the superposition of both alternating and static magnetic field, the ME ribbon vibrates longitudinally at its resonance frequency f0 given by Eq. (1) [12]. 1
𝐸
𝑓0 = 2𝐿 √𝜌(1−𝑣2 ) (1) Where, E is the Young’s modulus of elasticity, ρ is the density of the sensor material, 𝑣 is the Poisson’s ratio and L is the length of the sensor. For a small extra mass load of ∆𝑚 on the sensor of mass M (∆𝑚 ≪ 𝑀), the shift in the frequency (∆𝑓) 3
is given by Eq. (2) [13]. 𝑓
∆𝑓 = − 20 ∙
∆𝑚 𝑀
(2)
It is apparent from Eq. (2) that ∆f is proportional to the mass loading. As the resonance frequency of the ME ribbon is easily affected by the mass change, the ME sensors coated with a chemical or biological sensing membrane can be used for the detection of a variety of particles, including endotoxin [14], glucose [15], Salmonella typhimurium [16], Escherichia coli [17], Staphylococcus aureus [18]. Since the ME sensor is also magnetostrictive, it can be wirelessly actuated and sensed using magnetic field/signal [19]. Magnetic field telemetry enables its contact-less, remote-query operation that is the principle advantage over other sensor platforms. The phenomenon of the precipitation of bovine serum albumin (BSA) induced by heavy metal ions has been widely investigated [20]. Proteins are amphoteric substances existing in solution as cations or anions, on the basis of whether the hydrogen ion concentration of the solution is larger or smaller than that at the isoelectric point. The theory for heavy metal salt precipitation is as follows: when a dilute solution of heavy metal salt is added in a protein (BSA) solution on the alkaline side of its isoelectric point, the anionic protein combines with the cation, leading to a precipitation, for example [21-22]: BSA + Pb (Ⅱ)→ Pb (Ⅱ)-BSA↓
(3)
BSA + Cd (Ⅱ)→ Cd (Ⅱ)-BSA↓
(4)
BSA + Cu (Ⅱ)→ Cu (Ⅱ)-BSA↓
(5)
In this study, an ME biosensor, a mass sensitive device, composed of an ME 4
ribbon as the transducer platform and BSA as the bio-recognition element, was demonstrated for target heavy metal ions detection. The performances of the wireless ME biosensor in Pb2+, Cd2+ and Cu2+ solution with different concentrations were evaluated here. The heavy metal ions-induced precipitation of BSA accumulated on the biosensor surface leads to a decrease in the biosensor resonance frequency, according to Eq. (2), which in turn is proportional to the amount of heavy metal ions. Moreover, based on its wireless nature, the BSA-coated ME biosensor can be used for remote detection of heavy metal ions.
2. Materials and methods 2.1. Materials BSA was obtained from Sigma-Aldrich (Saint Louis, MO, USA) and used without further purification. 6-Mercaptoundecanoic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS) and phosphate buffered saline (PBS buffer, pH= 7.4) were also purchased from Sigma-Aldrich. ME ribbons composed of Metglas alloy 2826 (Fe40Ni40P14B6) were purchased from
Honeywell
Corporation
(Morristown,
NJ,
USA)
and
cut
into
5mm×1mm×0.028mm using a computer-controlled laser cutting machine. All experiments were conducted at room temperature [20, 22]. 2.2 ME biosensor fabrication 2.2.1 Preparation of the ME sensor platform ME ribbons were consecutively cleaned in an ultrasonic bath with alcohol and 5
deionized water, for 10 min each, to remove any debris and organic film, and then dried in a stream of nitrogen. Afterwards, the cleaned sensor platforms were sputtered with chromium (100 nm thick), followed by gold (100 nm thick). The chromium layer was the middle layer to enhance the adhesion between the gold and the sensor platform. The gold layer was used to prevent the corrosion of the ME ribbon and to improve the bio-compatibility of the sensor surface for the immobilization of BSA. The gold-coated sensor platforms were annealed in a vacuum oven at 220℃ for 2 h to eliminate any residual stress in the sensor and improve adhesion of the gold layer to the ME substrate. The annealed gold-coated sensor platform (Fig. 1a) is ready for BSA immobilization to form the ME biosensor and its resonance frequency in air is approximately 450 kHz. Due to its coupling effect between magnetic energy and elastic energy, the ME sensor platform as a high-performance transducer used in the biosensor can provide the resonance frequency shift information reflecting the detection of heavy metal ions. 2.2.2 BSA immobilization The gold-coated sensor platforms were consecutively cleaned in an ultrasonic bath with acetone, isopropanol, deionized water and ethanol for 10 min each, and dried under a nitrogen stream. After the cleaning, the prepared sensor platforms were incubated, avoiding light, in a 10 mM/L ethanol solution of 6-mercaptoundecanoic acid for 24 h to form the self-assembled monolayer (SAM), as shown in Fig. 1b. The sensor platforms were then rinsed several times with ethanol to remove the unbonded thiols and dried under a stream of nitrogen. The thiol-modified sensor platforms were 6
immersed into a solution containing 40 mM EDC and 10 mM NHS in distilled water for 1 h to convert the terminal carboxylic groups to an active NHS ester. The sensor platforms were subsequently rinsed with deionized water and dried under a stream of nitrogen. BSA was diluted into 0.01 M/L PBS buffer to prepare a 4 mg/ml BSA solution. Afterwards, the sensor platforms were dipped into a 1 mL BSA solution for immobilization for 2 h, then rinsed with a PBS solution and deionized water to remove physically adsorbed BSA. Subsequently, the sensors were dried under a nitrogen stream. Then the ME biosensor functionalized by BSA is formed, as shown in Fig. 1c. Based on its ability to bind with heavy metal ions and low-cost characteristics, BSA is used as the bio-recognition element in the biosensor to combine with the target heavy metal ions, leading to an increase in the mass loading of the biosensor. 2.3 Signal measurement A bench-top vector network analyzer (AV3620A, the 41st Institute of CETC, Qingdao, China) was used to characterize the resonance frequency of the ME biosensor. The experimental setup for the wireless ME sensing measurement is shown in Fig. 2. Due to its wireless nature, the magnetic flux generated by vibrations can be detected using a remotely placed pickup coil [23]. The network analyzer was connected to a coil wound around a glass tube in which the biosensor was inserted vertically and wirelessly (without any wire connections with measurement system). The ME biosensor is wirelessly operated, containing no internal power sources such as a battery. The network analyzer operating in the S11 mode provides a swept 7
frequency signal to the coil and monitors the signal reflected from the coil. The coil is a 50-turn single-layer solenoid made of 32 AWG type wire which generates an alternating magnetic field actuating the biosensor to vibrate longitudinally. The frequency range of the alternating magnetic field was set from 430 to 460 kHz. In addition, a static magnetic field generated by a bar magnet (N35, 3cm × 2cm × 1cm, magnetization of thickness direction) was applied to enhance the resonance behavior of the biosensor [24]. The bar magnet was fixed on a stand so that the distance and direction of the magnet can be adjusted. Furthermore, we will consider using a Helmholtz coil in the future work so that the bias field can be easily measured and controlled. The resonance frequency (f0, f1) of the biosensor corresponding to the frequency of the lowest point in the S11 curve can then be measured wirelessly and remotely. In the test, the biosensors were placed into the test tubes, which contain 0.5 ml of various heavy metal ions solutions of different concentrations ranging from 0 to 9.6× 10−6mol/L. The various heavy metal ions solutions are the following: lead (II) nitrate, copper (II) nitrate and cadmium (II) nitrate dissolved in 0.9% brine, respectively. The resonance frequency of the biosensor was monitored at each concentration and recorded for 45 min with an interval of 5 min.
3. Results and discussion 3.1 Assessment of the immobilization of BSA Fluorescence microscopy was used to confirm that BSA was successfully 8
immobilized on the biosensor surface. Fluorescein isothiocyanate (FITC) has the ability to react with amino groups and can be observed using a fluorescence microscope, thus staining with FITC can be used to confirm the presence of proteins [25-26]. In this study, the FITC-labelled BSA was covalently immobilized to the gold surface coated with a thiol self-assembled monolayer by the amine coupling method, as shown in Fig. 3a. Amine coupling introduces NHS esters on the surface of self-assembled monolayer with the EDC-NHS solution. The EDC is utilized as a coupling agent to activate carboxyl groups, while NHS provides a stable activated ester. Then the BSA is covalently linked to the activated NHS esters through the amide bond [27]. Finally, the BSA-FITC (Beijing Bo Sheng Bio-technology Co., Ltd.) immobilized on the biosensor surface was observed with a fluorescence microscope (DM 3000, Leica Microsystems Ltd.; Wetzlar, Germany). Numerous green fluoresce spots of BSA-FITC nanoparticles were observed on the biosensor surface as shown in Fig. 3b. In contrast, control experiment without BSA (0mg/mL) was conducted, as observed in Fig. 3c. The image is black background without any fluoresce spots. Similar observations have been made by Lademann et al. [28]. The results indicate that the BSA-FITC nanoparticles have been modified on the biosensor surface and additionally demonstrate that BSA can be successfully immobilized on the biosensor surface through the thiol self-assembly method. 3.2 Effect of pH values The effect of pH has been investigated by determining the ME biosensor responses to Pb2+ of 9.6× 10−6 mol/L at different pH values of 5.4, 6.1, 7, as shown 9
in Fig. 4. The isoelectric point of BSA is approximately pH 4.7 [29]. When the pH value of the solution is lower than 4.7, the BSA molecules exist as cations that cannot combine with heavy metal ions. Otherwise, when the pH value of the solution is higher than 4.7, the BSA remains anionic which can bind with heavy metal ions. Besides, considering that the three-dimensional structure of BSA would denature at higher pH values [29], we chose the pH values above 4.7 and below 7 to study in this work. From Fig. 4, we can see that with same concentration of heavy metal ions, the resonance frequency shift reached maximum at pH=6.1. Thus pH 6.1 was chosen for following determination. 3.3 The detection of lead ions Fig. 5 shows the dynamic frequency response of ME biosensor for Pb2+ detection at different concentrations ranging from 0 to 9.6× 10−6 mol/L. When the biosensor was immersed in Pb2+ solution (pH = 6.1), the Pb2+ from the solution were adsorbed to the active points in the BSA molecules on the biosensor surface. According to Eq. (2) and Eq. (3), the precipitation induced by Pb2+ causes an increase in mass on the biosensor surface, resulting in a decrease in the resonance frequency after a reaction period, as shown in Fig. 5, and thus the biosensor can be used for real-time monitoring. The reaction period is attributed to the time required for the combination. The results in Fig. 5 indicate that the steady-state response is achieved at 45 minutes, while the combination at higher concentration certainly needs more time to reach saturation. Additionally, the results in Fig. 5 reveal that the shift rate of the resonant frequency increases with the increasing Pb2+ concentration and a higher concentration 10
of Pb2+ can induce a larger resonance frequency shift. The phenomenon can be attributed to the fact that as more Pb2+ combine with BSA, then more precipitation adhere to the biosensor surface. Thus the wireless ME biosensor can be used to detect Pb2+. To confirm that the frequency response of the biosensor is only due to Pb2+, a control experiment was performed without Pb2+ (0 mol/L curve) that shows a noise level of ~18 Hz. However, there is a 60-Hz shift in resonance frequency for 0.6 × 10−6 mol/L Pb2+ over the same reaction period, as shown in Fig. 5. There is only a small frequency shift in the absence of interaction between Pb2+ and BSA, so the frequency response is caused by the Pb2+-induced BSA precipitate. The process of the BSA-Pb precipitate loaded on the biosensor surface was assessed by scanning electron microscopy (SEM) analysis and energy-dispersive spectroscopy (EDS) analysis. The SEM examination was performed using a SU3500 SEM (Hitachi corporation, Tokyo, Japan) operated at 5 keV. As observed in Fig. 6a, a naked surface image was from the biosensor gold surface without functionalization. The surface is smooth and has no impurity. After the biosensors were treated with BSA, spherical aggregates of the BSA nanoparticles were evenly distributed on the surface, as shown in Fig. 6b, indicating that the biosensors were immobilized with BSA successfully. Similar results have been described by others [30-31]. When the BSA-coated biosensors were exposed to Pb2+ solution (9.6× 10−6 mol/L), flake agglomerations were presented due to the aggregation by Pb2+, as shown in Fig. 6c. Furthermore, a typical EDS spectrum for elemental analysis before and after the 11
addition of Pb2+ is presented in Fig. 6d. It is evident that the accumulation of lead ions increased dramatically after the biosensors were immersed in Pb2+ solution. Given all this, the BSA-Pb precipitate adhered to the biosensor surface is demonstrated. Comparing the SEM images and the EDS spectrum, it can be concluded that the modification and detection procedures were successful. 3.4 The detection of cadmium ions In addition, similar kinds of experiments were performed for Cd2+ detection on the biosensor. The changes in resonance frequency versus time at different concentrations of Cd2+ ranging from 0 to 9.6× 10−6 mol/L are depicted in Fig. 7. These frequency responses are similar to those obtained for Pb2+. Due to the adherence of the BSA–Cd precipitate on the biosensor surface, the resonance frequency decreases with time according to Eq. (2) and Eq. (4). The shift in the resonance frequency increases with the rise of the Cd2+ concentration. Accordingly, Cd2+ can be detected wirelessly by the shift in resonance frequency of the ME biosensor. 3.5 The detection of copper ions Likewise, Cu2+ ions were detected through similar kinds of experiments with the ME biosensor. The chosen concentration range of Cu2+ was same as those of Cd2+ and Pb2+. As shown in Fig. 8, the frequency shift rises with the increasing concentration of Cu2+ in a similar manner as found above for the Cd2+ and Pb2+. This means that the mass effect resulting from the adherence of the BSA-Cu precipitate leads to the decrease in frequency according to Eq. (2) and Eq. (5). Consequently, the ME 12
biosensor is also suitable for the wireless detection of Cu2+. 3.6 The sensitivity and reproducibility of the biosensor The standard calibration curves for the detection of heavy metal ions on the biosensors during the first 45 min are shown in Fig. 9. For each heavy metal ion, the biosensor calibration experiments were performed five times under identical conditions. It is found that the shift in resonance frequency is linearly proportional to the concentration in the range of 0 to 4.8× 10−6 mol/L, however, with the increase of concentration the relationship becomes sub-linear, which indicates that the linearity holds better at low concentrations. The linear equations for Pb2+, Cd2+ and Cu2+ detection could be represented by ∆f = −1.5 × 108 𝐶𝑃𝑏2+ − 11 (𝑅 2 = 0.972),∆f = −7.4 × 107 𝐶𝐶𝑑2+ − 8.9(𝑅 2 = 0.989)
and
∆f = −4.9 × 107 𝐶𝐶𝑢2+ − 15(𝑅 2 =
0.991) respectively. The limit of detection (LOD) for Pb2+, Cd2+, Cu2+ is calculated to be 3.3× 10−7mol/L, 2.4× 10−7 mol/L, 2.3× 10−7mol/L, respectively , according to the Eq. (6) [32]: LOD = 3𝑆𝐵 /𝑏
(6)
Where 𝑆𝐵 is the standard deviation of the blank solution and 𝑏 is the slope of the fitted curve. The detection limits for Pb2+ and Cd2+ are converted to 0.107mg/L and 0.074mg/L, respectively, are both lower than the maximum permissible discharge standards for Pb2+ and Cd2+ of 1mg/L and 0.1mg/L given in the Integrated Waste Water Discharge Standard (GB 8978-1996), which show the LOD of the biosensor meets the standard currently available. The sensitivity of the biosensor for Pb2+, Cd2+ and Cu2+ is calculated from the linear region of the curves in Fig. 9, and is 9.4 × 13
107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. By contrast, various published methods for detection of heavy metal ions are summarized in Table 1, showing the superior performance of our biosensor. Therefore, the sensitivity and detection limit of the ME BSA-coated biosensor are better than those reported in the previous researches.To calibrate the responses from different sensors, by our experience,
𝑓𝑟 =450 kHz is set for the standard reference
frequency of the biosensor in the air. Then the result of resonance frequency shift ∆F after calibration is given by Eq. (7) and Eq. (8). ∆𝑓𝑟 = 𝑓 − 𝑓𝑟
(7)
∆F = ∆𝑓 − ∆𝑓𝑟
(8)
Where f is the biosensor’ resonance frequency measured in the air before use and ∆𝑓 is the resonance frequency shift measured in the detection process. In this way, the deviation from different biosensors can be calibrated. Furthermore, as shown in Fig. 9, the resonance frequency shift of the biosensor for the detection of Pb2+ at the same concentration with Cd2+ and Cu2+ is the largest. In addition, it is clear from Fig. 9 that the shift in resonance frequency for Cd2+ is larger than that of Cu2+ at the same concentration. The molecular weights of lead, cadmium and copper are 207.2, 112.4 and 64 respectively. Since the molecular weight of lead is the largest among the three heavy metals, the mass of precipitate adhered to the biosensor surface induced by Pb2+ is the largest, while that induced by Cd2+ takes second place. According to Eq. (2), the shift in resonance frequency for all three metal ions is in decreasing order: Pb2+> Cd2+> Cu2+. Thus, the biosensor responds to all 14
three metal ions with decreasing sensitivity in the order: Pb2+> Cd2+> Cu2+. This reveals that the biosensor is more sensitive to the heavy metal with larger molecular weight. 3.7 Interference experiment The mutual interference between Cu2+ and Pb2+ on the biosensor was investigated. Fig. 10 shows the frequency response of the biosensor towards Pb2+ in the presence of Cu2+ with different blending ratio in the solution. It is evident from the curves in Fig. 10 that the shift rate of the resonant frequency decreases with the addition of Cu2+. Besides, in the presence of Cu2+, the frequency shift is less than that of the individual determination of Pb2+ at a certain total mole number of ions. The results illustrate that the Cu2+ coexisting in the solution would decrease the response to Pb2+. As the molecular weight of Cu is smaller than that of Pb, the mass of BSA-Cu precipitate is smaller than that of BSA-Pb precipitate loaded on the biosensor. Likewise, the addition of Pb2+ could enhance the response of the coexisting Cu2+.
4. Conclusions For the first time, a BSA-coated ME biosensor for the wireless detection of heavy metal ions is presented. BSA is immobilized onto the ME ribbon surface to form an ME biosensor to detect Pb2+, Cd2+, Cu2+. The adherence of the BSA precipitate induced by heavy metal ions leads to an increase in the mass loading of the biosensor, which results in the decrease of the resonance frequency of the biosensor. The shift in resonance frequency increases with the increasing concentration of heavy metal ions, 15
which is linearly proportional to low concentrations, but becomes sub-linear at higher concentrations. The sensitivity of the biosensor for Pb2+, Cd2+ and Cu2+ is 9.4 × 107 Hz per mol/L, 7.1 × 107 Hz per mol/L and 4.7 × 107 Hz per mol/L, respectively. The results indicate that the biosensor can be used as a high-sensitive device to detect heavy metal ions and is more sensitive to the heavy metal with larger molecular weight. Additionally, the detection limit of the ME biosensor for Pb2+, Cd2+ and Cu2+ is approximately 3.3× 10−7mol/L, 2.4 × 10−7 mol/L and 2.3 × 10−7 mol/L, respectively. Considering that the elastic property, liquid viscosity and replacement reaction mechanism of the Pb2+ in solution with Fe and Ni in the ME ribbon may all affect the ME biosensor’s response, its respective contribution to frequency shift will be investigated in detail in the next study. In addition, the present biosensor is not specific for any particular metal ions but can be utilized for bulk detection of several heavy metal ions. Future work will be focused on the specific protein to detect a particular kind of heavy metal ion and a portable detector for the consistent measurement of the resonance frequency.
Acknowledgements The authors are grateful for the support by the National Natural Science Foundation of China (No. 51622507, 61471255, 61474079, 61501316, 51505324), Doctoral
Fund
of
MOE
of
China
(No.
20131402110013),
863
project
(2015AA042601), Excellent Talents Technology Innovation Program of Shanxi Province of China (201605D211023). 16
References [1] F. Ge, M.M. Li, H. Ye, B.X. Zhao. Effective removal of heavy metal ions Cd2+, Zn2+, Pb2+, Cu2+ from aqueous solution by polymer-modified magnetic nanoparticles. J. Hazard. Mater. 211 (2012) 366-372. [2] Z. Dong, L. Wang, J. Xu, Y. Li, Y. Zhang, S. Zhang, J. Miao. Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells. Toxicology in Vitro. 23 (2009) 105–110. [3] J. Liu, W. Qu, M.B. Kadiiska. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol. Appl. Pharmacol. 238 (2009) 209–214. [4] R. Verma, B.D. Gupta. Detection of heavy metal ions in contaminated water by surface plasmon resonance based optical fibre sensor using conducting polymer and chitosan. Food Chem. 166 (2015) 568-575. [5] D.I. Bannon, J.J. Chisolm. Anodic stripping voltammetry compared with graphite furnace atomic absorption spectrophotometry for blood lead analysis.
Clin.
Chem. 47 (2001), 1703-1704. [6] Q. He, E.W. Miller, A.P. Wong, C.J. Chang. A selective fluorescent sensor for detecting lead in living cells. Journal of American Chemical Society. 128 (2006) 9316–9317. [7] W.R. Yang, E. Chow, G.D. Willett, D.B. Hibbert, J.J. Gooding. Exploring the use of the tripeptide Gly-Gly-His as a selective recognition element for the fabrication of electrochemical copper sensors. Analyst. 128 (2003) 712-718. 17
[8] S. Sang, S. Gao, X. Guo, P. Cheng, W. Zhang. The detection of Pb2+ in solution using bare magnetoelastic resonator. Appl. Phys. Lett. 108 (2016) 054102. [9] L. Fu, S. Li, K. Zhang, I. Chen, V.A. Petrenko, Z. Cheng. Magnetostrictive microcantilever as an advanced transducer for biosensors. Sensors. 7(11) (2007) 2929-2941. [10] S.J. Huang, P.F. Pang, X.L. Xiao, L.E. He, Q.Y. Cai, C.A. Grimes. A wireless, remote query sensor for real-time detection of Escherichia coli O157:H7 concentrations. Sens. Actuators B 131 (2008) 489–495. [11] H. Lin, Q. Lu, S. Ge, Q. Cai, C.A. Grimes. Detection of pathogen Escherichia coli O157: H7 with a wireless magnetoelastic-sensing device amplified by using chitosan-modified magnetic Fe 3 O 4 nanoparticles. Sensors and Actuators B: Chemical. 147(1) (2010) 343-349. [12] P.G. Stoyanov, C.A. Grimes. A remote query magnetostrictive viscosity sensor. Sens. Actuators A 80 (2000) 8–14. [13] W. Shen, L.C. Mathison, V.A. Petrenko, B.A. Chin. Design and characterization of a magnetoelastic sensor for the detection of biological agents.
J. Phys. D:
Appl. Phys. 43 (2009) 015004. [14] K.G. Ong, J.M. Leland, K.F. Zeng, G. Barrett, M. Zourob, C.A. Grimes. A rapid highly-sensitive endotoxin detection system. Biosens. Bioelectron. 21 (2006) 2270–2274. [15] P. Pang, Y. Zhang, S. Ge, Q. Cai, S. Yao, C.A. Grimes. Determination of glucose using bienzyme layered assembly magnetoelastic sensing device. Sens. Actuators, 18
B 136 (2009), 310-314. [16] M.K. Park, S. Li, B.A. Chin. Detection of Salmonella typhimurium grown directly on tomato surface using phage-based magnetoelastic biosensors. Food and Bioprocess Technology. 6 (2013), 682-689. [17] A.L. Possan, C. Menti, M. Beltrami, A.D. Santos, M. Roesch-Ely, F.P. Missell. Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli. Mater. Sci. Eng., C 58 (2016) 541-547. [18] N. Hiremath, R. Guntupalli, V. Vodyanoy, B.A. Chin, M.K. Park. Detection of methicillin-resistant Staphylococcus aureus using novel lytic phage-based magnetoelastic biosensors. Sens. Actuators, B 210 (2015) 129-136. [19] S. Li, Y. Li, H. Chen, S. Horikawa, W. Shen, A. Simonian, B.A. Chin. Direct detection of Salmonella typhimurium on fresh produce using phage-based magnetoelastic biosensors. Biosens. Bioelectron. 26 (2010) 1313-1319. [20] Y. Yuan, Y. Cai, Q. Xie, S. Yao. Piezoelectric quartz crystal impedance study of the Pb2+-induced precipitation of bovine serum albumin and its dissolution with EDTA in an aqueous solution. Anal. Sci. 18 (2002) 767-771. [21] J. Yin, W. Wei, X. Liu, B. Kong, L. Wu, S. Gong. Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance. Anal. Biochem. 360 (2007) 99-104. [22] Y. Cai, Q. Xie, A. Zhou, Y. Zhang, S. Yao. A piezoelectric quartz crystal impedance study on Cu 2+-induced precipitation of bovine serum albumin in 19
aqueous solution. J. Biochem. Biophys. Methods. 47 (2001) 209-219. [23] L. Chen, J. Li, T.T. ThanhThuy, L. Zhou, L. Yuan, Q. Cai. A wireless and sensitive
detection
of
octachlorostyrene
using
modified
AuNPs
as
signal-amplifying tags. Biosens. Bioelectron. 52 (2014) 427-432. [24] K. Zhang, L. Zhang, L. Fu, S. Li, H. Chen, Z.Y. Cheng. Magnetostrictive resonators as sensors and actuators. Sens. Actuators, A 200 (2013) 2–10. [25] Y. Zhang, C. Wang. Micropatterning of Proteins on 3D Porous Polymer Film Fabricated by Using the Breath‐Figure Method. Advanced Materials. 19 (2007) 913-916. [26] S. Kreitz, C. Penache, M. Thomas, C.P. Klages. Patterned DBD treatment for area-selective metallization of polymers–plasma printing. Surf. Coat. Technol. 200 (2005) 676-679. [27] R. Pei, Z. Cheng, E. Wang, X. Yang. Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy. Biosens. Bioelectron. 16 (2001) 355-361. [28] J. Lademann, H. Richter, F. Knorr, A. Patzelt, M.E. Darvin, E. Rühl, K.Y. Cheung, K.K. Lai, R. Renneberg, W.C. Mak. Triggered release of model drug from AuNP-doped BSA nanocarriers in hair follicles using IRA radiation. Acta biomaterialia. 30 (2016) 388-396. [29] T. Kopac, K. Bozgeyik, J. Yener. Effect of pH and temperature on the adsorption of bovine serum albumin onto titanium dioxide. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 322(1) (2008) 19-28. 20
[30] H.H. Nguyen, J. Ryu, J.H. Che, T.S. Kang, J.K. Lee, C.W. Song, S. Ko. Robust size control of bovine serum albumin (BSA) nanoparticles by intermittent addition of a desolvating agent and the particle formation mechanism. Food Chem. 141 (2013) 695-701. [31] J.Y. Jun, H.H. Nguyen, S.Y.R. Paik, H.S. Chun, B.C. Kang, S. Ko. Preparation of size-controlled bovine serum albumin (BSA) nanoparticles by a modified desolvation method. Food Chem. 127 (2011) 1892-1898. [32] I. Krull, M. Swartz. Determining limits of detection and quantitation. LC–GC 16 (1998) 922–924. [33] Y. Liu, L. Zhang, R. Liu, P. Zhang. Spectroscopic identification of interactions of Pb2+ with bovine serum albumin. Journal of fluorescence. 22(1) (2012) 239-245. [34] D. Zhao, X. Guo, T. Wang, N. Alvarez, V.N. Shanov, W.R. Heineman. Simultaneous Detection of Heavy Metals by Anodic Stripping Voltammetry Using Carbon Nanotube Thread. Electroanalysis. 26 (2014) 488. [35] Y.F. Sun, L.J. Zhao, T.J. Jiang, S.S. Li, M. Yang, X.J. Huang. Sensitive and selective
electrochemical
amino-functionalized
carbon
detection
of
microspheres.
heavy
metal
Journal
of
ions
using
Electroanalytical
Chemistry. 760 (2016)143-150.
Author Biographies Xing Guo earned the B.Eng. degree in 2014 at Taiyuan University of Technology. She is currently a Ph.D. student at MicroNano System Research Center, Key Lab of Advanced Transducers and Intelligent Control System of the Ministry of Education & 21
College of Information Engineering. Her main research interest is in biosensors. Shengbo Sang received the B.Eng. degree in communication engineering and the M.Eng. degree in electronic science and technology from the North University of China, Taiyuan, China, in 2000 and 2006, respectively. He received the Ph.D. degree from the school of mechanical engineering, Ilmenau, Ilmenau University of Technology, Germany, in 2010. His current research interests include MEMS/NEMS, optical devices and biosensors. Aoqun Jian received the B.Eng. degree in electronic and information technology and the M.Eng. degree in microelectronics and solid electronics from the North University of China, Taiyuan, China, in 2005 and 2008, respectively. He received the Ph.D. degree in the Department of Applied Physics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, in 2013. His current research interests include nanooptics, particularly surface plasmon resonance resonant, optical tunneling effect and optofluidics. Shuang Gao received the B.Eng. degree in 2016 at Taiyuan University of Technology. He mainly engaged in MEMS and sensors. Qianqian Duan received the Bachelor of Engineer degree in Optical Information Science & Technology from Dalian University of Technology, Dalian, China, in 2007. She received Master of Science degree and Doctor of Science degree in Optics from Harbin Institute of Technology, Harbin,China, in 2009 and 2013, respectively. Her current research interests include nanooptics, nanowaveguide, nanometer fluorescence probe. Jianlong Ji received his PhD in Taiyuan University of technology and joined the Micro-nano system research center in 2014. He has united research experience in Tsinghua University, current research focus mainly on micro/nano-electromechanical systems (MEMS/NEMS) and microfluidic devices. Qiang Zhang received the Ph.D. degree in School of Instruments and Electronics, North University of China, Tai Yuan, China, in 2015. Her current research interests include nanomaterials, particularly soft strain sensors based on nanomaterials and photocatalytic performance of semiconductor nanomaterials. 22
Wendong Zhang received the Ph.D. degree from Beijing Institute of Technology, Beijing, China, in 1995. He went to the école normale supérieure (ENS) and Massachusetts Institute of Technology (MIT) as a senior visiting scholar in 2006 and 2009, respectively. He served the North University of China and Taiyuan University of Technology as the headmaster in 2004 and 2010, respectively. He is the technical chief of Project of National Security Key Basic Research. He was awarded the Second Prize of State Technological Invention Award three times and the Third Prize of State Technological Invention Award once. His current research interests include MEMS/NEMS and optical gyroscope.
Figure captions Fig. 1. Schematic diagram of the modification procedure of the biosensor.
Fig. 2. Schematic of the experimental setup for the wireless ME biosensor measurement.
23
Fig. 3. Schematic diagram (a) and fluorescence image (b) of the BSA-FITC modified on the biosensor surface. (c) Blank control image without BSA.
Fig. 4. The biosensor’s frequency response to Pb2+ of 9.6× 10−6 mol/L at different pH conditions.
Fig. 5. Real-time frequency response curves for Pb2+ detection at different concentrations ranging from 0 to 9.6× 10−6 mol/L.
24
Fig. 6. (a) SEM image of the gold-coated biosensor; (b) SEM image of the biosensor surface after the BSA immobilization; (c) SEM image of the biosensor surface after the Pb2+ detection; (d) EDS spectrum of the biosensor surface before and after the addition of Pb2+.
25
Fig. 7. The time-dependent frequency shift as a function of Cd2+ concentrations ranging from 0 to 9.6× 10−6 mol/L.
Fig. 8. Real-time frequency changes of the biosensor to Cu2+ concentrations ranging 26
from 0 to 9.6× 10−6 mol/L.
Fig. 9. The calibration curve: the 45 min shift in resonance frequency versus concentrations ranging from 0 to 9.6× 10−6 mol/L of different heavy metal ions.
27
Fig. 10. The frequency response of the biosensor in the simultaneous presence of Cu2+ and Pb2+ in solution.
28
Methods ME BSA-coated biosensor ME sensor based on replacement reaction mechanism PQCI study based on BSA PQCI study based on BSA Spectroscopic Identification SWASV based on Carbon Nanotube Thread SWASV based on NH2-CMS
Sensitivity (Hz per mol/L) 2+ Pb :9.4× 107
Pb2+: 8.0 × 103
Detection limit (mol/L) 2+ Pb : 3.3× 10−7 Cu2+: 2.3× 10−7 Cd2+: 2.4× 10−7 Pb2+: 1.5× 10−2
References This work
[8]
Cu2+: 9.98× 10−5
[21]
Pb2+: 4.8× 10−4
[19]
Pb2+: 1.0× 10−4
[33]
Cd2+: 1.0×10−6
[34]
Pb2+: 3.8× 10−7 Cu2+: 2.5× 10−7 Cd2+: 1.1× 10−6
[35]
Table 1. Comparisons of performances between various methods for the detection of heavy metal ions
29