A SERS biosensor with magnetic substrate CoFe2O4@Ag for sensitive detection of Hg2+

Accepted Manuscript Title: A SERS biosensor with magnetic substrate CoFe2 O4 @Ag for sensitive detection of Hg2+ Authors: Xia Yang, Yi He, Xueling Wang, Ruo Yuan PII: DOI: Reference:

S0169-4332(17)31123-6 http://dx.doi.org/doi:10.1016/j.apsusc.2017.04.106 APSUSC 35787

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APSUSC

Received date: Revised date: Accepted date:

19-12-2016 24-3-2017 14-4-2017

Please cite this article as: Xia Yang, Yi He, Xueling Wang, Ruo Yuan, A SERS biosensor with magnetic substrate CoFe2O4@Ag for sensitive detection of Hg2+, Applied Surface Sciencehttp://dx.doi.org/10.1016/j.apsusc.2017.04.106 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 SERS biosensor with magnetic substrate CoFe2O4@Ag for sensitive detection of Hg2+ Xia Yang, Yi He, Xueling Wang, Ruo Yuan Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China Graphical Abstract

*Corresponding author. Tel.: +852-34427826 E-mail address: [email protected] (R. Yuan) 1



Research highlights 

1． CoFe2O4@Ag can magnetically focus at one point to obtain a high Raman signal.



T-Hg2+-T structure is stable enough for detection Hg2+.



SWCNTs can act as a signal tag to avoid the labeling of Raman signal.



The SERS-based biosensor exhibited good performance of the detection of Hg2+.

Abstract: Mercuric ion (Hg2+) is one toxic metal ion existed in aquatic ecosystems which would seriously damage human central nervous system and other organs. So developing an approach to sensitively detect Hg2+ in our living environment is urgent and important. In this work, a novel surface enhancement Raman spectrum(SERS) sensor is fabricated for highly selective and ultrasensitive detection of Hg2+ in 2

aqueous solution, based on a stable thymine-Hg2+-thymine (T-Hg2+-T) structure and the - interaction between single-stranded DNA (ssDNA) and single walled carbon nanotubes (SWCNTs). Herein, SWCNTs act as Raman labels to produce characteristic Raman peaks which can be a beacon to quantitative detect Hg2+. In the presence of Hg2+, the ssDNA can capture Hg2+ forming T-Hg2+-T structure, which makes SWCNTs leave the hot spots of the SERS-based biosensor. With this design, the Raman intensity of SWCNTs decreased with the increasing concentration of Hg2+. At the same time, CoFe2O4@Ag as active SERS substrates can effectively enhance sensitivity and uniformity of the biosensor through aggregation by magnet. Under optimal conditions, this proposed biosensor can detect Hg2+ at a range from 1 pM to 100 nM with a detection limit of 0.84 pM. With the advantages of good sensitivity, selectivity, simplicity and rapidity, the biosensor is potentially suitable for monitoring of Hg2+ in environmental applications. Keywords: surface enhancement Raman spectrum; mercuric ion; magnetic substrate; thymine-Hg2+-thymine 1．Introduction Mercuric ion (Hg2+), as one of the most toxic heavy metals, is a serious detriment for environment.[1-6] Hg2+ often exists in water, forming stable water-soluble compounds with strong toxicity which can cause serious human health problems at a low concentration.[7,8] Therefore, it is necessary to search a rapid and sensitive method to detect Hg2+. Many methods have been developed to detect Hg2+, which

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mainly involve fluorophores [9], proteins [10] and metal nanoparticles [11-14]. Although these probes are simple and portable to bind or react with Hg2+, poor selectivity or water solubility often limited their applications in aqueous environments. To overcome the above mentioned shortcomings, oligonucleotides have been employed to specifically bind with Hg2+, forming stable thymine-Hg2+-thymine (THg2+-T) structures.[15-17] T-T mismatches in DNA duplexes can selectively and strongly capture Hg2+ with a higher binding constant than that of A-T.[18] Thus, this stable structure can be widely applied in selective and sensitive detection of Hg2+. So far, many traditional techniques have been developed to detect Hg2+ based on this THg2+-T structure, such as electrochemistry[19,20], electrochemiluminescence (ECL)[21] and fluorescence[22]. Although these methods possess good specificity, when detecting ultra-trace concentration of Hg2+, they often suffer from poor sensitivity and low precision. Surface enhancement Raman spectrum (SERS) technology can sensitively detect analytes at a level of femtomole which is widely applied in the field of biology, environments and medicine etc.[23] Recently, magnetic SERS substrates are arousing increasing attention for its fantastic enhanced capacity.[24,25] And these substrates are often composed of magnetic materials and noble metal (Au, Ag, Pt), which can enhance sensitivity and simplify the operation process by magnetic focusing at one point. In a word, it provides us a new consideration to sensitively and fast detect Hg2+.

4

Herein, a novel SERS biosensor was constructed based on a stable T-Hg2+-T structure with CoFe2O4@Ag magnetic substrate for the detection of Hg2+. As shown in Scheme 1, firstly, thiol group modified single-stranded DNA (ssDNA) was connected on magnetic substrate CoFe2O4@Ag through the Ag-S bond. Afterwards, ssDNA can stably bind with SWCNTs due to π-π stacking between nucleotide bases and nanotube walls, resulting in helical wrapping to the surface of SWCNT [26-28]. Because the SWCNT can act as a Raman label, the CoFe2O4@Ag@ssDNA@SWCNTs complexes can produce a strong Raman signal. In the presence of Hg2+, the ssDNA can capture Hg2+ forming T-Hg2+-T structure, which makes SWCNTs far away from the hot spots of the SERS-based biosensor, resulting in a decreased Raman signal. By this design, the Raman signal of SWCNTs decreased with the increasing concentration of Hg2+. And the corresponding signal intensity can have a linear relationship with the concentration of Hg2+, which indicate that the proposed SERS-based biosensor have promising potential in trace Hg2+ detection. 2．Materials and Methods 2.1 Materials and Regents Lysine was purchased from Ruji Biochemical Reagent Co., Ltd. (Shanghai, China). SWCNTs were obtained from Chengdu Organic Chemicals Co., Ltd. of the Chinese Academy of Science (Chengdu, China). (3-aminopropyl) triethoxysilane (ATPMS) was purchased from Shanghai Aladdin Biochem Technology (Shanghai, China). Ferric chloride (FeCl3·6H2O), cobaltous nitrate (Co(NO3)2·6H2O), sodium

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hydroxide (NaOH), ammonia aqueous (NH3·H2O), tetraethyl orthosilicate (TEOS), trisodium citrate, silver nitrate (AgNO3) and ethylene glycol were purchased from ChengDu Kelong Chemical Reagent Company (Chengdu, China). ssDNA: (5-SH(CH2)6-TCA TGT TTG TTT GTT GGC CCC CCT TCT TTC TTA-3) were synthesized by Sangon, Inc. (Shanghai, China). All other chemicals were of reagent grade and used as received. The solutions used in this experiment were prepared using ultrapure water (specific resistance of 18 MΩ·cm). 2.2 Apparatus The morphologies of nanoparticles were taken with scanning electronmicroscopy (SEM, S-4800, Japan). Transmission electron micrograph image was recorded on a Tecnai G2 F20 S-Twin 200 kV microscope (TEM, FEI, USA). The UV-vis absorption spectra were recorded in the range of 300-800 nm using a UV-vis spectrometer (UVvis, UV-vis 8500, China). The crystalline structures were characterized by X-ray diffraction (XRD, MAXima_X XRD-7000, Japan) with Cu Kα radiation at a scan speed of 5° min−1. SERS spectra were obtained using a Renishaw Invia Raman spectrometer (Renishaw Invia Raman spectrometer, Invia, UK) with 50-objective. The excitation wavelength was 532 nm from a 50 mW laser. Raman spectrometer was calibrated by a silicon wafer at 520 cm−1 Raman shift before SERS measurement. All the spectra were the results of a single 10 s accumulation. 2.3 Preparation of CoFe2O4@Ag magnetic SERS substrate

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Magnetic nanoparticles CoFe2O4 were prepared according to the literature.[29] Firstly, 0.88 g lysine were added into 25 mL ethylene glycol with a magnetic stirring, then 0.27 g FeCl3·6H2O and 0.29 g Co (NO3)2·6H2O were added into above solution and marked as A. 0.36 g NaOH was dissolved in 15 mL ethylene glycol to form homogeneous solution B. Then solution B was dropwise added into solution A with a magnetic stirring for 20 min. Nextly, the mixed solution were transferred to a Teflonlined stainless steel autoclave and heated at 180 °C for 10 h. Afterwards, the obtained deposits were collected by centrifugation at 8000 rpm for 15 min, washed alternately with ultrapure water and ethanol, and then dispersed in ultrapure water for further use. CoFe2O4@Ag were synthesized as followings: Firstly, 0.01 g CoFe2O4 were dissolved into a solution which already containing 28 mL C2H5OH, 7 mL H2O and 0.4 mL NH3·H2O. Then 0.08 mL TEOS were dropwise added into above solution with magnetic stirring until producing milky white precipitates. The obtained products were centrifuged three times and then dispersed into ultrapure water. Nextly, 500 μL APTMS were added into the above 2 mL obtained solution and magnetic stirring 8 h to introduce amino group. Finally, 1 mL 0.01 M AgNO3 was added into the amino modified CoFe2O4 with 30 min stirring, followed by adding 1 mL 2 mM trisodium citrate and 1 mL 10 mM NaBH4 to produce the Ag nanoparticles. The CoFe2O4@Ag were washed by ethanol and ultrapure water alternately, then dispersed in 1 mL ultrapure water. 2.4 Fabrication of SERS-based biosensor

7

Firstly, 200 μL 1 μM ssDNA was incubated with the prepared 200 μL CoFe2O4@Ag in 1 μM NaCl solution overnight at room temperature. Then, 100 μL SWCNTs (0.2 mg mL-1) were added into 100 μL CoFe2O4@Ag@ssDNA compounds for 30 min in order to form CoFe2O4@Ag@ssDNA@SWCNTs complexes. The complexes were washed by magnetic separate three times with Tris-HCl buffer (pH=8.0). 2.5 SERS-based biosensor assay procedure Different concentrations of Hg2+ were incubated into the prepared biosensor for 1 h and washed by PBS (pH=7.0) twice. Nextly, the biosensor was measured by Renishaw InVia Raman microscope equipped with a 532 nm laser. 3. Results and Discussion 3.1 Characterization of materials As shown in Figure 1A, the SEM image of CoFe2O4 revealed that the uniform spherical nanoparticles were with the size of about 50 nm. TEM image (Figure 1B) proved further evidence for sphere shape and size of CoFe2O4. Figure 1C was the SEM image of SWCNTs which displayed a well-dispersed tubular structure. Figure 1D was the SEM of CoFe2O4@Ag@ssDNA@SWCNTs complexes, from which we can see the SWCNTs were successfully connected on the biosensor. The X-ray diffraction (XRD) was also characterized to prove the properties of Co@C particles. As shown in Figure 2A, all peaks can be assigned to the 8

corresponding phase of CoFe2O4 (JCPDS card No. 030864) which indicated the successful synthesis of CoFe2O4. And the UV-Vis spectrum of CoFe2O4 was shown in Figure 2B (curve a). When Ag nanoparticles were in situ reduced onto CoFe2O4, there is an obvious absorption peak at 480 nm (Figure 2B, curve b), which indicated Ag nanoparticles have already been reduced on the CoFe2O4 nanospheres. 3.2 Optimization of the assay condition To increase the sensitivity of the SERS-based biosensor, the optimization of the concentration of SWCNTs was investigated. The concentration of SWCNTs is an important parameter affecting the analytical performance of biosensor. At room temperature, the Raman intensity increased with the increasing concentration of SWCNTs up to 0.20 mg mL-1 and then kept in a balance status (Figure 3A), which indicated that the combination between SWCNTs and ssDNA have achieved saturation. Therefore, 0.2 mg mL-1 SWCNTs were chosen for the detection of Hg2+. Incubation time play a critical role during this detection process, so we have done the optimization of incubation time of the biosensor with 1 nM Hg2+, which is shown in Figure 3B. We can see that with the increasing of incubation time, the intensity of the SERS biosensor decrease. After 60 min, the intensity tended to be stable, indicating the Hg2+ is saturated at that time. So we choose 60 min as the optimized incubation time. 3.3 Analytical performance of the SERS biosensor for detection of Hg2+

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To exploit the sensitivity and potential quantitative application of the proposed biosensor, we measured standard samples of Hg2+ with different concentrations by employing a stable T-Hg2+-T structure. Under optimum condition, the Raman signal intensity decreased with the increasing concentration of Hg2+ as shown in Figure 4A. The calibration plot showed a good linear relationship between the Raman peak which is located at 1600 cm-1 from SWCNTs and the logarithm of the Hg2+ concentration in the range from 1 pM to 100 nM (Figure 4B). And the regression equation is y = 13558.9 – 2367.1 lgc (where y is the Raman signal intensity at 1600 cm-1, c is the concentration of Hg2+) with a correlation coefficient squares of 0.99. The limit of detection (LOD) was calculated by LOD = kSb/m, where Sb is the standard deviation of the blank signals (nB = 11), m is the analytical sensitivity, which can be estimated as the slope of the calibration plot at lower concentration ranges and k is the numerical factor chosen in accordance with the desired confidence level (k =3). This SERS-based biosensor for the detection of Hg2+ was much more sensitive than other biosensors which were listed in the Table 1. These good results may owe to the magnetic SERS substrate (CoFe2O4@Ag) which can magnetically aggregate the biosensor to enhance Raman intensity, and thus the sensitivity of the biosensor was further improved. 3.4 Selectivity of the SERS biosensor To evaluate the selectivity of the SERS-based biosensor, we evaluated the proposed biosensor with other possible interferences such as Ag+, Pb2+, Cu2+, Zn2+, Ca2+, Co2+, Mn2+, Fe3+ and Cr3+ in the same detecting conditions. As shown in Figure 10

5, when these interfering ions were separately added in the biosensor, the intensity was obviously higher than that of the mixed sample (interfering ions and Hg2+ existed together) and the Hg2+ exist alone (1 nM), which indicated that the interfering ions would not change the signal of the biosensor and the SERS-based biosensor owned good selectivity. 3.5 Reproducibility of the SERS biosensor SERS spectra of the CoFe2O4@Ag magnetic substrate-based biosensor at 15 different spots were collected and the intensity at 1600 cm-1 was used to test the reproducibility. From Figure 6, we can see that the coefficient of variation was less than 4% which indicated the proposed biosensor possess good reproducibility. 3.6 Analysis of actual application ability The proposed assay was used for detection Hg2+ in the water of Jialing River with the standard addition method. Different concentration of Hg2+ was tested three times and the results were shown in Table 2. The recovery and relative standard deviation values were ranging from 90.50% to 116.7% and 5.7% to 9.7%, respectively, suggesting the potentiality of this SERS-based biosensor for Hg2+ detection in real samples. 4. Conclusion In conclusion, with a stable T-Hg2+-T stable structure, we put forward a sensitive SERS-based biosensor utilizing magnetic compounds CoFe2O4@Ag as active SERS substrate for the detection of Hg2+. This proposed biosensor owns the advantage of 11

high sensitivity, good reproducibility and simple analysis procedure, which is attributed to the following reasons: firstly, CoFe2O4@Ag could magnetically focus at one point to obtain a high Raman signal which can improve the sensitivity of the biosensor. Besides, T-T base pair can specific capture Hg2+ forming stable structure which can enhance the specificity of the biosensor. In addition, employing SWCNTs as a signal tag can avoid the labeling of Raman signal. Based on these advantages, we anticipate this sensitive and selective SERS-based biosensor can be applied in the future for detection of Hg2+. Acknowledgements This project has been financially supported by National Natural Science Foundation of China (No. 51602263), Fundamental Research Funds for the Central Universities (XDJK2015C099,

SWU114079),

China

Postdoctoral

Science

Foundation

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Scheme 1. The construction of SERS-based biosensor for detection of Hg2+.

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Figure 1. (A) SEM image of CoFe2O4; (B) TEM image of CoFe2O4; (C) SEM image of SWCNTs; (D) SEM image of CoFe2O4@Ag@ssDNA@SWCNTs complexes.

Figure 2. (A) XRD of CoFe2O4; (B) UV-Vis of CoFe2O4 (curve a), CoFe2O4@Ag (curve b).

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Figure 3. Optimization of (A) the concentration of SWCNTs; (B) incaubation time of the biosensor with with 1 nM Hg2+ .

Figure 4. (A) SERS-based biosensor with the increasing concentration of Hg2+ (pM) from a to h: (a) 105; (b) 5×104; (c) 104; (d) 5×103; (e) 103; (f) 102; (g) 10; (h) 1. (B) The calibration plot of Raman intensity at 1600 cm−1vs lgc (error bars = SD, n = 3).

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Figure 5. Selectivity of the proposed SERS biosensor. The concentrations of Ag+, Pb2+, Cu2+, Zn2+, Ca2+, Co2+, Mn2+, Fe3+ and Cr3+ were each 100 nM. The mixture contains Ag+ (100 nM), Pb2+ (100 nM), Cu2+ (100 nM), Zn2+ (100 nM), Ca2+ (100 nM), Co2+ (100 nM), Mn2+ (100 nM), Fe3+ (100 nM) , Cr3+ (100 nM) and Hg2+ (1 nM).

Figure 6. The Raman intensity at 1600 cm-1 from 15 different spots (cHg2+ = 1 nM).

Table 1. Proposed SERS biosensor performance compared with other biosensors for Hg2+ detection. Analytical method

Detection limit

Linear range

refs

Electrochemistry

0.094 nM

1 nM-5 μM

[30]

18

Fluorescent

0.083 μM

0.1μM-100 μM

[31]

Fluorescent

0.33 nM

1.2 nM-14 nM

[32]

Fluorescent

8.6 nM

37.5 nM-3750 nM

[33]

ECL

5.1 pM

0.05 nM -10 nM

[34]

SERS

0.84 pM

1 pM-100 nM

This work

Table 2. Determination of Hg2+ added in water of Jialing River (n = 3) with the proposed SERS-based biosensor. Serum sample

Concentration of Hg2+ added /(pM)

Concentration obtained with biosensor/(pM)

Recovery RSD /%

/%

1

10

11.67

116.7

9.7

2

100

90.50

90.50

8.9

3

1000

1065.82

106.6

8.1

4

10000

9567.081

95.67

5.7

19