Synthesis of PtPb hollow nanoparticles and their application in an electrochemical immunosensor as signal tags for detection of dimethyl phthalate

Abstract

Platinum–lead (PtPb) hollow nanoparticles were used as signal tags for the first time to fabricate an electrochemical immunosensor, which was then used as the first electrochemical immunosensor to detect dimethyl phthalate (DMP). Pt nanoparticles possess good biocompatibility, highly efficient catalytic properties and good electrical conductivity. Pb was introduced to produce electrochemical signals, reduce the dosage of the noble metal and save costs. An AuPt–graphene sheet (AuPt–GS) was used to immobilize the DMP antibodies (Anti-DMP) due to its excellent conductivity and large surface area. A novel competitive immunoassay was proposed, using PtPb nanoparticles covalently conjugated with bovine serum albumin (BSA)–DMP for the sensitive detection of DMP concentrations. The quantitative detection was based on the competitive binding of DMP antibodies with PtPb-tagged DMP or free DMP. The electrochemical signal decreased with increasing concentration of the free DMP as the amount of PtPb–BSA–DMP tags decreased at the immunosensor. Electron microscopy, energy dispersive spectrometry, X-ray powder diffraction and a series of electrochemical techniques were used to characterize the nanoparticles. Differential pulse voltammetry was used to monitor the electrochemical response. The immunosensor exhibited a linear response from 1 to 1000 ng mL⁻¹ DMP with a detection limit of 0.33 ng mL⁻¹. The excellent performance of the PtPb nanoparticles showed promising applications as a sensor platform.

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Introduction

As a phthalate ester (PAE), dimethyl phthalate (DMP) is widespread in the environment because of its common usage as a plasticizer, or in insecticides, cosmetics, perfumes, agriculture plastics and agricultural detergents.¹ As a priority pollutant, DMP has obtained huge attention from environmental regulatory bodies owing to its endocrine disrupting antiandrogenic activity² and its refractory biodegradability. Among all of the PAEs, as a short-chained carboxylic diester, DMP is one of the most frequently detected plasticizers in the environment and it migrates easily into the food chain.³ Several analytical methods have been successfully proposed for the determination of these PAEs, involving high performance liquid chromatography-mass spectrometry (HPLC-MS),^4–6 gas chromatography-mass spectrometry (GC-MS),⁷ fluorescence,⁸ enzyme-linked immunoassay (ELISA),⁹ diffuse reflectance UV spectroscopy,¹⁰ molecular imprinted polymers (MIP)^11,12 and chemiluminescence (CL).^13,14 However, some of these techniques suffer from various drawbacks, involving labor-intensive sample preparation, expensive analysis settings or poor selectivity. Moreover, there are few reports that involve the detection of DMP¹⁵ using electrochemical methods. Therefore, the establishment of an electrochemical immunosensor for the detection of DMP is original.

Electrochemical immunosensors, due to their high sensitivity and selectivity, have recently gained growing interest and have been found to have wide applications in different fields, including environmental monitoring¹⁶ and clinical diagnosis.¹⁷ For the development of an electrochemical immunosensor, a signal tag is a key issue, which has a crucial effect on the performance of the immunosensor. Various nanomaterials such as gold nanoparticles,¹⁸ quantum dots,¹⁹ ferroferric oxide,²⁰ cuprous sulfide²¹ and graphene,²² have been used for the preparation of immunosensing signal tags, due to their high specific surface areas, ultrasensitive electrochemical sensing interface, or excellent electrical conductivity.

Pt nanoparticles^23,24 have shown exceptional performance in catalysis, sensors, optics and biomedicine owing to their large surface area, large number of active sites, high catalytic activity and excellent biocompatibility.^25,26 Pt-based bimetallic nanomaterials (Pt–M, where M = Au, Pd, Ag, Ru and so on) in place of pure Pt will not only retain the beneficial features of Pt, but also modify the crystallographic and electronic structures of Pt, and enhance the catalytic performance.^27–30 They also have the advantages of saving materials and reduced cost.³¹ Combining the advantages of both a hollow structure and alloying, here we developed a facile way to synthesize platinum–lead hollow nanospheres (PtPb). When the size of a nanomaterial decreases, the atoms will gradually collect on the surface of the nanomaterial. This will endow the nanomaterial with strong chemical activity. Importantly, it was found that Pb could produce obvious anodic peak currents at −0.58 V (vs. a saturated calomel electrode). PtPb has the advantages of both elements. This bimetallic signal tag typically provides a rapid response, good stability and electrocatalytic capability for target detection. To our knowledge, this is the first report about using PtPb hollow nanospheres in an electrochemical immunosensor as signal tags.

Herein, we report a novel electrochemical immunosensor for the determination of DMP, using bovine serum albumin (BSA)–DMP conjugates incubated with PtPb (PtPb–BSA–DMP), based on a competitive immunoassay mode. An AuPt–graphene sheet (AuPt–GS) was used to modify the materials due to the large surface area, fast rate of electron transfer and large amount of biomolecule binding sites. The electrochemical signal was generated by the redox reactions of PtPb under differential pulse voltammetry (DPV). The concentration of DMP can be reflected directly because the decreased free DMP concentration in the sample will increase the binding sites of PtPb–BSA–DMP captured with the DMP antibody (Anti-DMP).

Experimental

Reagents and materials

Anti-DMP and BSA–DMP were purchased from Beijing Kwinbon Biotechnology Co., Ltd. (Beijing, China). Free DMP was purchased from Aladdin. Other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). A phosphate buffer solution (PBS) (0.1 M, pH 7.4) was prepared by mixing stock solutions of KH₂PO₄ and Na₂HPO₄. A HAc–NaAc (CH₃COOH–CH₃COONa) buffer solution (pH 5.5) was prepared as working solution by mixing stock solutions of HAc and NaAc. Ultrapure water was used throughout the experiment. All of the chemicals were of analytical reagent grade.

All electrochemical experiments were performed using a traditional three-electrode system. It contained a glassy carbon electrode (GCE, 4 mm diameter) as the working electrode, a platinum wire electrode as the auxiliary electrode and a saturated calomel electrode as the reference electrode. All electrochemical measurements were carried out using a CHI 760D electrochemical workstation (Chenhua Instrument Shanghai Co. Ltd, China). Scanning electron microscope (SEM) images were obtained using a field emission SEM (Zeiss, Germany). Transmission electron microscope (TEM) images were obtained from an H-800 microscope (Hitachi, Japan). X-ray powder diffraction (XRD) was gathered from a Bruker D8 Focus diffractometer (Germany) using CuKα radiation (40 kV, 30 mA) of wavelength 0.154 nm.

Preparation of AuPt–GS

AuPt–GS was synthesized as described by Zhang et al. with slight modifications.³² Briefly, graphene oxide (GO) used herein was synthesized using a modified Hummers’ method.³³ To synthesize the nanocomposites of the bimetallic alloyed Au–Pt particles on the GS surface, 0.1 g GO was first ultrasonicated in 100 mL of ultrapure water to make it disperse well. Then solutions containing 0.015 mmol HAuCl₄ and 0.025 mmol H₂PtCl₆ were added drop by drop and the reaction stirred for 30 min. Subsequently, 50 mL of a 0.15 M NaBH₄ solution was added drop by drop, and the mixing solution was heated to 100 °C in an oil bath with stirring for 24 h. Afterward, the solution was cooled down to room temperature. The products were separated by filtration, washed by ultrapure water, and fully dried at 60 °C in an oven to get the final AuPt–GS.

Preparation of PtPb

PtPb was fabricated using Co nanoparticles as sacrificial templates.^31,34 Briefly, 1.5 mL of 0.1 M CoCl₂ was added rapidly to 20 mL of a deaerated aqueous solution containing 0.25 mM citric acid. After adding 2 mmol NaBH₄ rapidly, the solution turned brown, indicating the formation of Co nanoparticles. It was obvious to see that bubbles were evolved during the reaction and continued for several minutes. When the bubbles ceased, a solution containing 0.1 mmol H₂PtCl₄ and 0.025 mmol Pb(CH₃COO)₂ was added under stirring. The solution turned blackish and was stirred for 2 h. Finally the solution became transparent with some black grains in it. The products were centrifuged and washed with ultrapure water. Then the PtPb hollow nanospheres were obtained.

Preparation of the competitive immunoassay

Protein molecules could be loaded onto PtPb alloys and it has been proven that amino groups in proteins can be strongly bound to Pt.^35,36 The conjugation procedure of PtPb to BSA–DMP was as follows: PtPb (2.5 mg) was dispersed in 1.0 mL of the phosphate buffer at pH 7.4. Then, 4 μg of BSA–DMP was added into the solution. The mixture was allowed to react at 4 °C under stirring for 24 h. The reaction mixture was then centrifuged and the supernatant was removed. The PtPb–BSA–DMP bioconjugates were treated with 1 mL of a 1 wt% BSA solution for 30 min to block non-specific binding. The mixture was centrifuged, using PBS to remove any uncombined BSA–DMP. The final mixture was re-dispersed in 1.0 mL PBS (pH 7.4) and stored at 4 °C before use.

A GCE was polished carefully with alumina powder of 1.0, 0.3 and 0.05 μm respectively, to a mirror-like surface, then cleaned with ultrapure water. 4.0 μL of AuPt–GS (1.5 mg mL⁻¹) was added onto the GCE. Then, 4 μL Anti-DMP (1 μg mL⁻¹) was immobilized onto the AuPt–GS. Subsequently, incubation with 2 μL 1 wt% BSA solution for 1 h was used to eliminate nonspecific binding between the analyte and the electrode surface. After drying, the electrodes were washed with pH 7.4 PBS. Then, 3 μL containing an unknown amount of free DMP was dropped onto the electrode and reacted with the Anti-DMP for another 1 h at 4 °C. Finally, 3 μL of the preparative PtPb–BSA–DMP bioconjugates was incubated with the electrode to combine with the remaining Anti-DMP. The procedures used for the construction of the immunosensor are depicted in Fig. 1.

Fig. 1 Schematic diagram of the detection principle of DMP with a competitive immunoassay mode.

Measurement procedure

The HAc–NaAc buffer solution (pH 5.5) was used for all of the electrochemical measurements. Electrochemical signals were measured using the DPV technique. The applied DPV parameters were 0.004 V increments, 50 mV pulse amplitude, 50 ms pulse width, 0.0167 s sampling width, 0.2 s pulse period and a voltage range from −0.9 V to −0.3 V.

Results and discussion

Characterization of the nanomaterials

The morphology of the AuPt–GS was observed by SEM. As shown in Fig. 2A, the graphene nanosheets are randomly compacted and stacked together, and the AuPt nanoparticles that are well dispersed on the surface of graphene may enhance the conductivity. TEM observation of PtPb (Fig. 2B) shows a bright center surrounded by a much darker edge, confirming their hollow architecture. Those hollow nanospheres formed on the basis of the replacement reaction between the Co nanoparticles and the metallic ions.³¹ The formation mechanism can be attributed to a so-called Kirkendall effect, in which pores form because of the difference in the diffusion rates of these components.^37,38 Because of the subsized and hollow architecture, PtPb nanoparticles remain suspended in water and do not aggregate for a long time. Fig. 2C shows the dispersibility of the PtPb nanoparticles in water. After 4 hours (b), a slight settlement had occurred. In order to confirm the elemental composition of PtPb, energy dispersive spectrometry (EDS) analysis was carried out and the results are displayed in Fig. 2D. The PtPb nanoparticles were dropped onto the aluminum foil for the test. It shows three major elements in the sample: Pt, Pb and Al. Obviously, Pt and Pb derived from the PtPb nanoparticles, while Al originated from the aluminum foil. Co was thoroughly sacrificed, so that pure PtPb nanoparticles were obtained. XRD analysis (Fig. 2E) was used to characterize the crystalline structure of the PtPb nanoparticles. The peaks around 39.8°, 46.2°, 67.4° and 81.2° can be attributed to the diffraction peaks of the Pt crystal faces (111), (200), (220) and (311), respectively, which are characteristic of the Pt face-centered cubic phase. All of the diffraction peaks matched well with those from the Jade PDF card (65-2868) and were indicated by the vertical lines. By contrast, in the inset, the peaks for PtPb shifted to lower 2θ values, indicating an enlargement in the PtPb interatomic distances. Both Pt and PtPb show typical diffraction peaks that match well with the standard lines. It can be seen that there are no Pb peaks, indicating that the Pb was alloyed with Pt, which was observed in agreement with the literature.³⁴ As shown in Fig. 2F, a redox cycle of the electrical probes was recorded using cyclic voltammetry (CV) in the acetate buffer solution (pH 5.5). The anodic peak at −0.54 V belonged to the Pb of the PtPb nanoparticles. The amperometric responses of Pt and PtPb (4 μL, 2.5 mg mL⁻¹) toward H₂O₂ are shown in Fig. 2G. Both Pt and PtPb exhibit remarkable catalytic performance towards H₂O₂. A similar amperometric response was generated by the same dosage of Pt and PtPb, or put another way, it economized the noble metal and reduced the cost. Basically there is no signal attenuation of PtPb catalyzing the reduction of H₂O₂. This may have promising applications in an electrochemical immunosensor using PtPb nanoparticles as signal tags for amperometric methods. Electrochemical methods are suitable for investigating the interaction between biological molecules and other materials.³⁹ Due to the fact that proteins are intrinsically unable to act as redox partners, the combined biological molecules will obviously weaken the electrochemical signals. From Fig. 2H, the PtPb shows an enormous peak (curve b). However, after BSA–DMP combined with PtPb, the PtPb–BSA–DMP bioconjugate shows a relatively small peak (curve a). The results show that the BSA–DMP was conjugated onto PtPb successfully.

Fig. 2 SEM (A) of the AuPt–GS; TEM (B) of PtPb; photograph (C) of (a) a suspension of PtPb and (b) after 4 hours sedimentation; EDS (D) of PtPb; XRD spectra (E) of Pt and PtPb; CV measurement (F) of PtPb in the acetate buffer solution (pH = 5.5); amperometric response (G) of Pt and PtPb (4 μL, 2.5 mg mL⁻¹) at −0.4 V after the successive addition of 1.0 mmol L⁻¹ H₂O₂; electrochemical characterization (H) of the bioconjugation between PtPb and BSA–DMP: (a) DPV of PtPb–BSA–DMP, (b) DPV of PtPb.

Optimization of the experimental conditions

All of the steps involved in the immunoassay were optimized. We investigated the influence of pH and the concentration of AuPt–GS on the performance of the immunosensor. Fig. 3A shows that the best DPV current response of the immunosensor was obtained at pH 5.5. The acidic buffer solution will be beneficial to metal ion activity, but overacidity will be harmful to the protein molecule. Thus, pH 5.5 with the HAc–NaAc buffer was selected as one of the optimized conditions. The electrical signal increases with the increase of AuPt–GS concentration from 0.5 to 2.5 mg mL⁻¹ and reaches a maximum at 1.5 mg mL⁻¹ (Fig. 3B), but decreases when the concentration further increases. The increase of the AuPt–GS film thickness may lead to an increase of the interfacial electron transfer resistance, and the electron transfer will become more difficult. Therefore, 1.5 mg mL⁻¹ of AuPt–GS was chosen for all subsequent experiments.

Fig. 3 Effect of pH (A), and the concentration of AuPt–GS (B) on the response of the competitive immunoassay to 10 ng mL⁻¹ DMP. Error bar = RSD (n = 5).

Characterization of the immunosensor

CV was used for further characterization of the modified electrode. Fig. 4A demonstrates the cyclic voltammetric behavior of the bare electrode and the different modified electrodes in a Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ solution containing 0.1 mM KCl with a scan rate of 100 mV s⁻¹. The GCE (curve a) had a pair of well-defined voltammetric peaks. After being modified with AuPt–GS (curve b), the peak current was much higher than that of the GCE. Afterwards, the anodic and cathodic peak currents decreased gradually with the addition of Anti-DMP (curve c), BSA (curve d), DMP and PtPb–BSA–DMP (curve e). As a result, the immunosensor has been fabricated successfully.

Fig. 4 CVs (A) and EIS (B) obtained for different modified electrodes in Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ containing 0.1 mM KCl solution: (a) GCE, (b) AuPt–GS/GCE, (c) Anti-DMP/AuPt–GS/GCE, (d) BSA/Anti-DMP/AuPt–GS/GCE, (e) PtPb–BSA–DMP and free DMP/BSA/Anti-DMP/AuPt–GS/GCE.

Electrochemical impedance spectroscopy (EIS) is a powerful tool to investigate the interface properties of surface-modified electrodes and it was carried out to illustrate that all of the fabricating steps of the immunosensor were effective. Curve (a) in Fig. 4B shows the EIS plot of the bare GCE. It possesses a very small semicircle domain and implies a very low electron transfer resistance in the electrolyte solution. Curve (b) displays a much lower interfacial electron transfer resistance than curve (a), indicating that the AuPt–GS accelerated electron transfer at the electrode. After Anti-DMP covalently binds, an increased semicircle at a higher frequency region was observed (curve c). The reason for R_et increasing was that the nonconductive antibody obstructed the electron transfer of the redox probe from the solution to the electrode surface. The adsorption of BSA at the Anti-DMP/AuPt–GS electrode resulted in a dramatically increased diameter of the semicircle (curve d), indicating a higher electron transfer resistance at the electrode interface. After incorporating PtPb–BSA–DMP and free DMP onto the electrode, the electron transfer resistance further increased (curve e). These results were consistent with the changes observed in the electron transfer resistance by CV. The above results clearly confirmed that the immunosensor had been fabricated successfully.

Analytical performance characteristics

DPV was used to evaluate the performance of the immunosensor. The DPV peak currents decreased with increasing concentration of free DMP under optimized conditions. Fig. 5Ashows the DPV response for 1 ng mL⁻¹, 5 ng mL⁻¹, 10 ng mL⁻¹, 50 ng mL⁻¹, 100 ng mL⁻¹, 250 ng mL⁻¹, 500 ng mL⁻¹ and 1000 ng mL⁻¹ (a–h, in order) concentrations of free DMP, respectively. The currents changed linearly with the logarithm of the concentrations of DMP in the range from 1 ng mL⁻¹ to 1000 ng mL⁻¹ with a detection limit of 0.33 ng mL⁻¹, based on S/N = 3. The linear regression equation was I (μA) = 31.2428 − 9.1264 log C (ng mL⁻¹) with a statistically significant correlation coefficient of 0.9939 (Fig. 5B), and the sensitivity was 9.1264 μA/log (ng mL⁻¹). Additionally, the analytical performance of this method has been compared with those of other methods for the detection of DMP (Table 1). As can be seen, the proposed immunosensor exhibited a lower detection limit. Compared with conventional methods, the sensitivity of this method is greatly improved due to the following reasons: (1) the high specificity of the combination of antigen and antibody will decrease the nonspecific interference. (2) The electrochemical signal of PtPb produced at −0.58 V will avoid the effect of other electroactive materials. (3) The AuPt–GS not only provided a large specific surface area for the immunomobilization of abundant antibodies but also facilitated electron transfer. These results indicated that the electrode which was constructed using AuPt–GS and PtPb nanoparticles was more beneficial for the electrochemical immunosensing platform.

Fig. 5 DP voltammogram (A) of 1, 5, 10, 50, 100, 250, 500 and 1000 ng mL⁻¹ (a–h, in order); the calibration curve (B) of the current values vs. the DMP concentration. Error bar = RSD (n = 5).

Table 1 Comparison of the different methods presented for the detection of DMP

Method	Linear range (ng mL^−1)	Detection limit (ng mL^−1)	Reference
a DLLME-HPLC-VWD, dispersive liquid–liquid microextraction-high-performance liquid chromatography-variable wavelength detector.b AALLME-GC-FID, air-assisted liquid–liquid microextraction-gas chromatography-flame ionization detection.
DLLME-HPLC-VWD^a	5.00–5000	1.8	4
Surface enhanced Raman scattering	—	19.4	15
AALLME-GC-FID^b	3.00–10 000	1.15	40
Electrochemical immunosensor	1.00–1000	0.33	This work

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Moreover, the repeatability, selectivity, and stability of the immunosensor were also investigated. The repeatability of the proposed immunosensor was investigated by inter- and intra-assays. All of the relative standard deviations (RSD) for the inter- and intra-assays were not more than 5%. The experimental results suggested the acceptable repeatability of the proposed immunosensor.

Selectivity is a very important characteristic and it is necessary to check the binding selectivity of the proposed immunosensor to DMP (5 ng mL⁻¹ was chosen as the sample). Owing to the structural similarity to DMP, several other small organic chemicals (each 500 ng mL⁻¹) were selected: estradiol, bisphenol A, toluidine blue and ascorbic acid. The presence of high concentrations of interfering components did not interfere with the assay for DMP (Fig. 6A). These results clearly indicated the selectivity of the immunosensor was acceptable. That may be attributed to two factors. On the one hand, the specificity of the antigen–antibody binding complex results in no interference in the determination of analytes. On the other hand, the negative response current near −0.6 V could avoid an interference peak generated by another electroactive material.

Fig. 6 Selectivity of the immunosensor (A) and stability of the immunosensor (B). Error bar = RSD (n = 5).

The stability of immunosensors is also a key factor in their application and development (Fig. 6B). When the prepared electrodes were not in use, they were stored at 4 °C. It can be seen that 94.3% of the initial response remained after one week and 89.2% of the initial response remained after two weeks (5 ng mL⁻¹ was chosen as the sample). The decrease in the current response may be due to the gradual denaturation of the antibodies.

Application in analysis of water samples

In order to assess the reliability of the proposed immunosensor for DMP detection, the proposed immunosensor was investigated for practical analysis by adding different concentrations of DMP into a water sample. The results are shown in Table 2. 10, 50 or 250 ng mL⁻¹ of DMP was added to each of the water samples. These data showed that the recovery (between 92% and 108%) was acceptable, and the results demonstrated excellent promise for determining DMP in real water samples.

Table 2 Detection of DMP in water samples

Water samples	DMP added (ng mL^−1)	DMP found (n = 5, ng mL^−1)	Recovery (%)
Tap water	10	9.6	96
	50	47	94
	250	238	95.2
Lake water	10	9.2	92
	50	54	108
	250	262	104.8

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Conclusions

In summary, a novel electrochemical immunosensor for the determination of DMP using PtPb–BSA–DMP bioconjugates was fabricated. It is worth mentioning that PtPb nanoparticles were used for the first time in immunosensor fabrication and obtained good results. Based on a competitive immunoassay strategy and the excellent electrochemical signal of PtPb, the electrochemical immunosensor shows a large linear response and a low detection limit. Due to the good precision, high sensitivity and acceptable stability, the electrochemical immunosensor has potential application for rapid and simple measurement of DMP in real samples.

Acknowledgements

This study was supported by the Natural Science Foundation of China (no. 21175057, 21375047, 21377046), the Science and Technology Plan Project of Jinan (no. 201307010), the Science and Technology Development Plan of Shandong Province (no. 2014GSF120004), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (no. ts20130937).

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