A simple label-free rhodamine 6G SERS probe for quantitative analysis of trace As³⁺ in an aptamer–nanosol

Abstract

Gold nanoparticles (NGs) were modified by the aptamer (ssDNA) to prepare a NGssDNA probe for As³⁺. In pH 8.0 HEPES buffer solution containing 50 mmol L⁻¹ NaCl, rhodamine 6G (Rh6G) molecules adsorbed on the NGssDNA sol substrate exhibited a strong surface-enhanced Raman scattering peak (SERS) at 1358 cm⁻¹. Upon addition of As³⁺, it reacts with the NGssDNA probe to form a stable As–ssDNA complex and release NGs that were aggregated to the NG aggregates (NGAs) as a substrate, in which Rh6G SERS activity is very weak. With the increase of As³⁺ concentration, the SERS peak decreased at 1358 cm⁻¹ due to more NGAs forming. The decreased SERS intensity responds linearly with the concentration of As³⁺ over 0.288–23.04 ng mL⁻¹, with a detection limit of 0.1 ng mL⁻¹.

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1. Introduction

Arsenic is a kind of toxic pollutant that can cause human cancer and other diseases. Arsenic mainly exists in the two forms As³⁺ and As⁵⁺ in environmental and biological samples. Among them, the toxicity of As³⁺ is greater than the As⁵⁺.¹ According to statistics,² the arsenic concentration of daily drinking water covering about 140 million peoples in the world exceed the safe drinking standard of 10 ng mL⁻¹. Therefore, it is great significance to establish a highly sensitive and selective analytical method to determine the concentration of arsenic ion in environmental, and food samples. At present, the main analysis methods for arsenic include atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry and high performance liquid chromatography.^3–12 Although these methods can be detected accurately trace arsenic, but they also have some disadvantages such as complicated process or high-cost equipment and low sensitivity.

Aptamer is a single strand nucleic acid that screened through the SELEX technology, and can combined with target molecules with strong specificity and selectivity.^13,14 Because aptamer has accurate identification, synthesis and modification, good stability, easy to in vitro to target specific binding features, it has been widely applied to analysis.^15–17 Nanogold has good stability, good biocompatibility and strong scattering effect, and has been applied in aptamer nanoanalysis.^18,19 Surface enhanced Raman scattering (SERS) is of abundant information, rapidity, sensitivity and selectivity, has been widely used in the qualitative detection in early stage.^20–24 With the improvement of preparation technology for SERS active substrate, SERS also has been utilized in quantitative detection.^25–27 Using the Langmuir–Blodgett assemblies of polyhedral Ag nanocrystals as highly active SERS substrates, as low as 1 ng mL⁻¹ As can be detected by SERS.²⁵ A label-free gold-nanoparticle-based SERS assay was reported for direct cyanide detection at the parts-per-trillion level.²⁶ A simple and reproducible surface-enhanced Raman scattering method has been developed for selective nano-mole iron(III) determination in aqueous solutions, using deserrioxamine B-functionalized silver nanoparticles as substrates.²⁷ In aptamer-modified nanoparticle SERS quantitative analysis, it focused on the detection of heavy metals, organic compounds and biological macromolecules.^28–34 A sensitive and selective single nanowire-on-film (SNOF) surface-enhanced resonance Raman scattering (SERRS) sensor was fabricated for Hg²⁺ detection, based on structure-switching double stranded DNAs (dsDNAs). The binding Hg²⁺ induces conformational changes of the dsDNAs and let a Raman reporter get close to the SNOF structure, thereby turning on SERRS signal.²⁸ Jiang et al.²⁹ used UO₂²⁺ cracking the double-stranded DNA to generate single-stranded DNA (ssDNA), the formed NGssDNA interacted with Rh6G to produce SERS effect, this method can be detected UO₂²⁺ as low as 1.6 nmol L⁻¹. Yao et al.³⁰ used gold nanoparticle sol as SERS substrate, a 10 μg mL⁻¹ BHA can be detected. Rinnert³¹ prepared gold nanoparticles that modified by polystyrene as SERS substrate, a 1–20 ppm naphthalene in water can be detected by SERS. Based on the complex of BCA-Cu⁺ with highly SERS activity³² and the quenching effect of protein in alkaline, a SERS method was set up for detection protein as low as 10 pg mL⁻¹. Rh6G has strong SERS activity, and it is an important SERS molecular probe which has been used for inorganic and organic analysis.^35,36 As far as we know, there are no reports about aptamer-modified nanogold as SERS substrate and label-free Rh6G as SERS molecular probes for detection of As³⁺. In this paper, using the aptamer gold/silver nanosol as substrate, the label-free Rh6G as SERS molecular probe, a new SERS quantitative method was developed for determination of trace As³⁺ in real sample.

2. Experimental

A model of DXR smart Raman spectrometer (Thermo companies in the United States) was used, with a laser wavelength of 633 nm, power of 3.0 mW, average number of scanning of 2, collect exposure time of 5.0 s, preview exposure time of 1.0 s, sample exposures of 2, and correction of fluorescence. A model of F-7000 fluorescence spectrophotometer (Hitachi Company, Japan) was used to recording the resonance Rayleigh scattering spectrum, with a synchronous scanning technique (λ_ex − λ_em = Δλ = 0), detector voltage of 450 V and slit of 5.0 nm. A model of JSM-6380LV scanning electron microscope (Electronic Co. Ltd, Japan), a model of NaNo-ZS90 nanoparticle and zeta potentiometric analyzer (Malvern Company, England), a model of SK8200LH Ultrasonic reactor (Shanghai Guide Ultrasonic Instruments Co., LTD, China), and a magnetic stirrer (Jiangsu Jintan Cuhk Instrument Factory) were used. A 0.5 μmol L⁻¹ nucleic acid aptamer ssDNA1 with sequence of 5′-ACCATCGCGGAAGTCCAGTCTGCCATCAAAATCCGAAGTG-3′, 0.5 μmol L⁻¹ ssDNA2 with sequence of 5′-TTACAGAACAACCAACGTCGCTCCGGGTACTTCTTCATCG-3′, 0.5 μmol L⁻¹ ssDNA3 with sequence of 5′-ATGCAAACCCTTAAGAAAGTGGTCGTCCAAAAAACCATTG-3′ (Invitrogen Company, China), 1% HAuCl₄·4H₂O, 50 mmol L⁻¹ 4-hydroxyethyl piperazine ethyl sulfonic acid sodium solution (HEPES), 1 mol L⁻¹ NaCl and 1% sodium citrate solutions were prepared. As³⁺ standard solution was prepared as follows, taken 0.01 g NaAsO₂ and dissolved in water, diluted to 100 mL with water to obtain a 57.6 mg L⁻¹ As³⁺ stock solution. A 17.7 mg L⁻¹ As⁵⁺ standard solution was prepared to take 0.010 g Na₃AsO₄·12H₂O to dissolve in 100 mL water. HEPES buffer solution was prepared by 50 mmol L⁻¹ HEPES and 150 mmol L⁻¹ NaCl solutions. The gold and silver nanoparticles,^25,37 NGssDNA and NSssDNA probes were prepared. All reagents were of analytical grade and the water was doubly distilled.

The general detection procedure is, a 600 μL 83.3 nmol L⁻¹ NGssDNA solution, 150 μL pH 8.0 HEPES buffer solution, a certain amount of As³⁺ solution were added into a 5 mL marked test tube and mixed well. After 15 min at room temperature, 100 μL 1 mol L⁻¹ NaCl, 200 μL 5.23 × 10⁻⁵ mol L⁻¹ Rh6G was added in the mixture, and diluted to 2 mL. A part of the solution was transferred into a 1 cm quartz cell. The SERS intensity at 1358 cm⁻¹ (I₁₃₅₈) and the blank value I_b without As³⁺ were recorded. The value of ΔI₁₃₅₈ = (I₁₃₅₈)_b − I₁₃₅₈ was obtained.

3. Results and discussion

The ssDNA interacted with NGs to form a stable NGssDNA nanoprobe for As³⁺ by van der Waals force and intermolecular forces. In pH 8.0 HEPES buffer solution containing 50 mmol L⁻¹ NaCl, the NGssDNA probes are close to each other to form loose and stable aggregations in red color, the SERS probes of Rh6G molecules adsorbed on the surface produce strong SERS effect. Upon addition of As³⁺, it reacted with the ssDNA of the nanoprobe to form a stable complex of As–ssDNA and release the NGs that were aggregated to a larger tight aggregation (NGAs) with very weak SERS activity under the action of NaCl, and result in the SERS signals decreased. With the increase of As³⁺ concentration, the SERS intensity at 1358 cm⁻¹ decreased linearly due to more NGAs formation. Based on this, an aptamer–NG SERS method was set up to detect trace As³⁺ (Fig. 1).

Fig. 1 The principle of a label-free RhG SERS probe for detection of As³⁺ in aptamer-modified nanogold sol substrate.

SERS activity of NGs is closely related with its preparation methods. Using NG sol as substrate that prepared by sodium borohydride reduction method, no SERS effect of Rh6G was observed. Using the NG sol prepared by sodium citrate reduction method as substrate, Rh6G showed strong SERS effect, so it was chosen for use. In the HEPES buffer solution, Rh6G molecular probes exhibited SERS peaks at 608 cm⁻¹, 773 cm⁻¹, 1122 cm⁻¹, 1174 cm⁻¹, 1303 cm⁻¹, 1358 cm⁻¹, 1508 cm⁻¹ and 1644 cm⁻¹. Among them, the SERS peak at 1358 cm⁻¹ is big, and the SERS peak intensity linearly decreased with the concentration of As³⁺ increasing. Thus, the peak was chosen for the determination of As³⁺ (Fig. 2). Using the NSssDNA nanoprobes as substrate, the SERS peaks are similar to the NGssDNA, the sensitivity for As³⁺ is higher than the NGssDNA system, but the accuracy is inferior to the NGssDNA probe. A peak at 1358 cm⁻¹ was also chosen for the determination of As³⁺ (Fig. 1S†).

Fig. 2 SERS spectra of the NGssDNA–As³⁺–Rh6G system. (a) 25 nmol L⁻¹ NGssDNA + pH 8.0 HEPES + 50 mmol L⁻¹ NaCl + 5.23 μmol L⁻¹ Rh6G; (b) a + 0.72 ng mL⁻¹ As³⁺; (c) a + 5.76 ng mL⁻¹ As³⁺; (d) a + 11.52 ng mL⁻¹ As³⁺; (e) a + 23.04 ng mL⁻¹ As³⁺.

Resonance Rayleigh scattering spectrum is a simple and sensitive technology to study nanoparticles and its aggregations. In general, both the particle size increasing and aggregation forming result in the scattering signal enhanced.^38–42 Because the ssDNA combine with NG to form a stable NGssDNA probes that are stable in high concentrations of salt, the RRS signal at 375 nm is about 2400 (Fig. 2Sa†). In the presence of As³⁺, it interacted with NGssDNA probe to form stable As–ssDNA compounds and release NGs that were aggregated to big particles leading RRS intensity increased to 3000 (Fig. 2Se†). The RRS enhanced value of 600 indicated more NGAs formed in the system. Furthermore, the size distribution was recorded by laser scattering technique (Fig. 3). Fig. 3a showed that the average size is about 60 nm and most NGssDNAs were dispersed in the solution. Upon addition of As³⁺, the average size is 160 nm (Fig. 3b), this indicated that big NGAs exist in the system. NGs exhibited a surface plasmon resonance (SPR) absorption peak at 525 nm. When the NG was modified by ssDNA, the SPR absorption peak holds constant. In pH 8.0 HEPES buffer solution and 50 mmol L⁻¹ NaCl, NGssDNA also had a SPR peak at 525 nm (Fig. 3S†) owing to no formation of NGA in the system. Upon adding of As³⁺, the color of solution change from red to blue, the SPR absorption peak at 525 nm decreased due to the released NGs aggregating.

Fig. 3 Laser scattering of the NGssDNA–As³⁺ system. (a) 25 nmol L⁻¹ NGssDNA + pH 8.0 HEPES + 50 mmol L⁻¹ NaCl; (b) a + 23.04 ng mL⁻¹ As³⁺.

According to the procedure to get the aptamer reaction solution, a 1.0 mL the solution was taken into a 1.5 mL centrifuge tube, centrifuged in 15 000 rpm for 20 min to abandon the supernatant. A 1.0 mL water was add in the centrifuge tube and dispersed by ultrasonic 30 min, and centrifuged again. The operation was repeated two times, the dispersed sample solution was dropped onto a silicon wafers and dried naturally, the scanning electron microscopy (SEM) was recorded. In the absence of As³⁺, the NGssDNA nanoprobes are stable in the presence of NaCl, and the particle size is small (Fig. 4a). Upon addition of As³⁺, it reacted with the NGssDNA nanoprobes to form stable ssDNA–As³⁺ compounds and release the NGs that were aggregated to big aggregations (NGAs) under the action of NaCl (Fig. 4b). This is consistent with the previous experimental results and the analysis principle.

Fig. 4 SEM of the NGssDNA–As³⁺ system. (a) 25 nmol L⁻¹ NGssDNA + pH 8.0 HEPES+50 mmol L⁻¹ NaCl; (b) a + 23.04 ng mL⁻¹ As³⁺.

The conditions of preparation of NGssDNA probe was considered (Fig. 4S and 5S†), the procedure is as follows, piped 10 mL 58.0 μg mL⁻¹ NGs into a conical flask, added 2.0 mL 2.0 mL 0.5 μmol L⁻¹ ssDNA solution under the stirring slowly, continued to stir 15 min, and stored at 4 °C. In terms of ssDNA, the NGssDNA concentration is 83.3 nmol L⁻¹. The effect of pH was considered. The ΔI value reached its maximum when the pH was 8.0 (Fig. 6S†). The ΔI value reached its maximum when the concentration was 0.75 mmol L⁻¹. Thus, 0.75 mmol L⁻¹ pH 8 HEPES buffer solution was chosen for use. The effect of NGssDNA concentration was considered. The ΔI value reached its maximum when the concentration was 25 × 10⁻⁹ mol L⁻¹ (Fig. 7S†). Thus, a 25 × 10⁻⁹ mol L⁻¹ of NGssDNA solution was chosen for use. The effect of Rh6G concentration was considered. The ΔI value reached its maximum when the concentration was 5.23 μmol L⁻¹ (Fig. 8S†). Thus, a 5.23 μmol L⁻¹ Rh6G was chosen for use. According to the procedure, the effect of foreign substances on the determination of 88 nmol L⁻¹ (11.25 ng mL⁻¹) As³⁺ was tested, with a relative error within ±10%. Results (Table 1S†) showed that common ions, amino acids and blood albumin etc. did not interfere with the determination, which indicated that this method had good selectivity.

Under the optimal conditions, the SERS intensity for different As³⁺ concentrations (C) were recorded and the working curves were drawn according the relationship between C and their corresponding ΔI values. We have investigated the influence of different aptamer on the working curve (Fig. 9S†). Results (Table 2S†) shows that the ssDNA3 system is best, with the widest linear range and lowest detection limit, the ssDNA3 was selected for the SERS determination of As³⁺. When NSssDNA3 was used as substrate, the decreased SERS intensity at 1358 cm⁻¹ responds linearly with the concentration of As³⁺ over 0.0288–17.28 ng mL⁻¹, with a linear regression equation of ΔI = 22.3C + 15, coefficient R² of 0.9862 and a detection limit of 0.01 ng mL⁻¹ (Fig. 10S†). The accuracy is very important for the SERS quantitative analysis, and the relative standard deviation (RSD) of five determinations was examined for the NSssDNA3 system. The RSD is 10.7%, 7.5.2% and 5.4% for 0.1, 1.0 and 5.0 ng mL⁻¹ As³⁺ respectively. At the same time we also inspected the analysis feature of As⁵⁺–ssDNA3 reaction system. Results (Fig. 11S†) show that the As³⁺ system is more sensitive than the As⁵⁺ system. The decreased SERS intensity at 1358 cm⁻¹ responds linearly with the concentration of As³⁺ over 0.288–23.04 ng mL⁻¹, the linear regression equation is ΔI_{1358 cm⁻¹} = 3.2C + 7.0, and detection limit is 0.1 ng mL⁻¹. The RSD of five determinations is 5.7%, 4.2% and 3.7% for 1.0, 5.0 and 10.0 ng mL⁻¹ As³⁺, respectively. This indicated that the accuracy of the NGssDNA3 system is super to the NSssDNA3 system, and was chosen for use.

The natural water samples including Li river and Rong lake, and waste water were filtered to obtain water sample solutions. It was analyzed according to the procedure, and no As was detected in the natural water samples (Table 3S†). Then, a known amount of As³⁺ was added into the water sample to obtain the recovery. The relative standard deviation was in the range of 4.2–6.8%, and the recovery was in the range of 95.2–97.9% that indicated this method is accuracy. The two waste water samples were analyzed by this SERS method and hydride generation-atomic absorption spectrometry (HQ-AAS), both results are consistent.

A 1.00 g milk sample was taken into a 200 mL flask, then about 10 mL concentrated HNO₃, 1 mL 30% H₂O₂ and 5 mL concentrated HClO₄ solutions were added. The flask was heated on an electric furnace to dissolve the sample to generate white fog. About 2 mL concentrated H₂SO₄ solution was added, and heated to near dry. The mixture was removed from the furnace to cool, and diluted to 10 mL with water to obtain the milk sample solutions.⁴³ According to the procedure, the samples were used to detect As. The results are agreement with that of HG-AAS (Table 3S†).

4. Conclusion

An aptamer-modified nanogold probes (NGssDNA) were prepared, and were used as SERS substrate, in which Rh6G molecular probes show strong SERS effect, but there is weak SERS effect in the NGA substrate. Based on the NGssDNA aptamer reaction between NGssDN and As³⁺, and NGs aggregation reaction, a new method was developed for the detection of arsenic by the label-free RhG SERS molecular probe, with advantages of high sensitivity, good selectivity, simplicity and rapidity.

Acknowledgements

This work supported by the National Natural Science Foundation of China (no. 21267004, 21367005, 21307017, 21365011), the Research Funds of Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, the Natural Science Foundation of Guangxi (no. 2013GXNSFFA019003, 2013GXNSFAA019046), and the Research Funds of Guangxi Education Department (no. 2013YB234, 2013YB035).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra04416a

‡ Author contributions, these authors (GQ Wen and LL Ye) contributed equally to this work.

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