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Mulberry-like [email protected] porous nanorods composites as signal amplifiers for sensitive detection of CEA
Biosensors and Bioelectronics 149 (2020) 111842
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Mulberry-like Au@PtPd porous nanorods composites as signal amplifiers for sensitive detection of CEA Yilei Jia , Yueyun Li *, Shuan Zhang , Ping Wang , Qing Liu , Yunhui Dong ** School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo, 255049, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrochemical immunosensor MoS2/CuS-Au nanomaterials Mulberry-like Au@PtPd porous nanorods Carcinoembryonic antigen
Effective detection of cancer biomarkers plays a crucial role in the prevention of early cancer. Here, a sandwichtype electrochemical immunosensor was successfully constructed for sensitive detection of carcinoembryonic antigen (CEA) using MoS2/CuS-Au as sensing platform and mulberry-like Au@PtPd porous nanorods (Au@PtPd MPs) as signal amplifiers. The large surface area and good biocompatibility of MoS2/CuS-Au increased the loading of primary antibody. And the good conductivity of MoS2/CuS-Au accelerated the electron transport rate of the electrode surface. Au@PtPd MPs with large specific surface area and a large number of catalytically active sites showed excellent electrocatalytic performance for hydrogen peroxide reduction. The sandwich-type immunosensor prepared by the signal amplification strategy exhibited a wide linear detection range (50 fg/ mL to 100 ng/mL) and a low detection limit of 16.7 fg/mL (S/N ¼ 3), featuring good selectivity, stability and reproducibility. Moreover, the satisfactory results in analysis of human serum samples indicated that it possessed a potential application promising in early clinical diagnoses.
1. Introduction Cancer, also known as malignant tumor, has seriously harmed human health (Garzon et al., 2009; Yang, L. et al., 2017). Importantly, the occurrence of cancer is tending to become younger due to changes in dietary structure, increased work pressure, and infection of various pathogens (Gangemi et al., 2010). Most early cancers have no obvious symptoms and are easily ignored. Therefore, effective early detection is very important for the patient (Kim et al., 2013; Wu et al., 2015). Cancer biomarkers are a promising tool for the diagnosis and prognosis of cancer, and carcinoembryonic antigen (CEA) is one of the most widely used cancer biomarkers (Labib et al., 2016). In general, CEA are asso ciated with many types of cancer when their levels in human serum exceed 5 ng/mL (Barton, 2014). So far, detection methods of CEA involved enzyme-linked immunoassay (Yang, W. et al., 2017), chem iluminescence immunoassay (Lee et al., 2012), radiological analysis (Lang et al., 2014), and so on. But these methods are usually time consuming and expensive, labor intensive, and use complex instruments (Chinen et al., 2015; Santharaman et al., 2016). Compared with these methods, electrochemical immunosensor have received extensive attention in CEA detection due to the advantages of simple operation,
low cost and affordability (Amani et al., 2018). And it has been widely used in drug screening, food testing, environmental monitoring and clinical diagnosis (Jia et al., 2015). As we all know, electrochemical immunosensor are analytical methods by determining the concentration of target antigen based on biological specificity recognition reaction, and converting biological signal into electrical signal (Du et al., 2010; Ke et al., 2018). However, antigen and antibody specific binding to form complexes on the elec trode surface could hinder electron transfer. Therefore, sensitive detection of immunosensor is still highly challenging. At present, building effective and sensitive immunosensor is usually improved by the following strategies: (i) designing new biocompatible and conduc tive substrate materials to increase the loading of antibodies and accelerate the electron transfer on the electrode surface; (ii) using new signal amplification markers to effectively improve the performance of the sensor (Zhou et al., 2016). In recent years, transition metal sulfides such as MoS2, CuS, CoS, and Cu7S4 have attracted attention of researchers (Barua et al., 2017; Lu et al., 2016; Wang et al., 2018). In particular, the layered transition metal MoS2 has a huge potential application in the manufacture of electrochemical immunosensor owing to large surface area, low cost and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Li), [email protected] (Y. Dong). https://doi.org/10.1016/j.bios.2019.111842 Received 31 July 2019; Received in revised form 17 October 2019; Accepted 1 November 2019 Available online 6 November 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
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high chemical stability (Garrett et al., 2016; Su et al., 2016). For example, Qian’s groups reported a MoS2-based nanocomposites immu nosensor, which exhibited high sensitivity for SCCA detection (Li et al., 2011). Jiang’s groups developed a platform based on MoS2-Thi com posites, achieving sensitive and rapid detection of ZEA (Wang et al., 2014). All of the above studies can demonstrate the excellent properties of MoS2. Inspired by the above works, a preliminary signal amplification strategy was designed by using MoS2/CuS-Au as sensing platform. The combination of MoS2 and CuS nanocomposites, could not only overcome the aggregation phenomenon caused by strong van der Waals force in the layered crystal of MoS2, but also increase the use properties of MoS2 (Tan et al., 2016). More importantly, the nanocomposites with tremella-like morphology possessed large surface area and exposed more active sites, which could improve the catalytic ability for hydrogen peroxide (H2O2) (Chaoliang and Hua, 2015). Furthermore, the gold nanoparticles (Au NPs) have sparked extensive concerns due to its excellent biocompatibility and good electrical conductivity. As expect, Au NPs introduced by the in-situ reduction method effectively improved the electron transporting ability of the electrode surface, coupled a large number of primary antibody (Ab1) by Au-N bond, and further enhanced the catalytic activity for H2O2 (Yang et al., 2015). Therefore, MoS2/CuS-Au as sensing platform improved the performance of the immunosensor and achieved preliminary signal amplification. New signal amplification strategy plays a crucial role in the con struction of sandwich-type immunosensor. Especially, compared to mono- and bi-metallic nanomaterials, trimetallic nanomaterials have better advantages in electrochemical immunosensor, such as superior biocompatibility, excellent electrical conductivity and catalytic prop erties (Lijuan et al., 2011; Teng et al., 2010). In addition to the electronic effects of multiple metals, these properties of nanomaterials depend to a large extent on morphology and structure. Recently, nanomaterials with core-shell rod-like structure have the most potential application in electrochemical immunosensor (Bu et al., 2017; Zheng et al., 2017). Core-shell rod-like nanomaterials have many advantages in terms of stability and synergy between compositions, and its complex electronic interactions can alter the surface electronic properties of core-shell rod-like nanomaterials (Chen et al., 2015, 2016). However, many pre viously reported shell regions are dense structures with limited catalytic sites (Su et al., 2017). In view of the problem, a metal nano-porous shell was designed to improve the electrocatalytic performance of the core-shell nanomaterial, thereby providing sufficient contactable active sites. In this work, a trimetallic mulberry-like Au@PtPd porous nanorods (Au@PtPd MPs) were synthesized in aqueous solution by a simple method as an ideal signal amplification label to detect CEA sensitively. Impressively, the fabrication of Au@PtPd MPs could not only increase the specific surface area and atomic utilization of Pt, but also provide abundant accessible pores for the reduction of H2O2 (Zhao et al., 2017). Remarkably, the composite had good biocompatibility and can immo bilized a large number of secondary antibodies (Ab2). In short, a different sandwich-type immunosensor was successfully constructed for sensitive detection of CEA. Using MoS2/CuS-Au as sensing platform to immobilize Ab1, improving the performance of the immunosensor. The mulberry-shaped Au@PtPd porous nanorods (Au@PtPd MPs) were used as signal amplifiers, and its unique porous core-shell rod-like structure and trimetallic component showed excel lent catalytic and stability. Therefore, signal amplification and sensi tivity detection could be achieved in the designed immunosensor for the detection of CEA in human serum.
(SM).
2. Experimental
3.2. Characterization of Au@PtPd MPs
2.1. Materials and apparatus
In this experiment, gold nanorods (Au NR) with high yield and uniform size was synthesized by seed growth method (Ye et al., 2013). Morphology and size of Au NR were characterized by TEM. As shown in Fig. 3A, we could clearly observe that almost all the particles were rod-like. The average diameter and length of the Au NR (Fig. 3B) were
2.2. Fabrication of the proposed immunosensor The detail fabrication process of the proposed immunosensor is outlined in Fig. 1. Before modified, the GCE (4 mm diameter) was pol ished by alumina powder (0.3 and 0.05 μm) to a mirror and then cleaned. Sequentially, MoS2/CuS-Au suspension (6 μL) were modified on the bare GCE and dried at ambient temperature. Following that, Ab1 (6 μL, 10 μg/mL) was incubated on the above GCE, and immobilized on the electrode surface by Au-N between Au in composite and amino group in antibody. After dried at 4 � C and washed with the PBS, BSA (3 μL, 1 wt%) was dripped on the above GCE to block the nonspecific binding sites. After that, the modified GCE was incubated with a series of different concentrations of CEA. At last, Au@PtPd MPs-Ab2 suspension (6 μL) was dropped on the above GCE for 50 min. The fabricated elec trode was cleaned completely with PBS to remove any unbound at tachments, and stored at 4 � C for later research. 2.3. Detection of the CEA The proposed immunosensor was tested by a conventional threeelectrode system (the fabricated electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and plat inum electrodes as the auxiliary electrode) in all electrochemical tests. Subsequently, amperometric i-t technique was conducted in PBS buffer (pH 7.1) with a scanning potential at 0.4 V. And after the current was in a steady state, H2O2 (10 μL, 5 mol/L) was injected into the PBS (10 mL) under mild stirring, following that the current response value was record. EIS was utilized to investigate the step-by-step fabrication of the proposed immunosensor, and recorded in solution with 2.5 mM [Fe (CN)6]3-/4- and 0.1 M KCl (frequency range: 10 1 to 105 Hz, and initial voltage: 0.24 V). 3. Results and discussion 3.1. Characterization of materials To verify the morphology of MoS2/CuS, SEM was helpful to char acterize the microstructure of MoS2, CuS and MoS2/CuS, respectively. As illustrated in Fig. 2, the SEM images showed that agglomerated spherical cluster of MoS2 (Fig. 2A) and Sheet-like CuS (Fig. 2B). When MoS2 and CuS were combined, the SEM images of MoS2/CuS with different magnification (Fig. 2C and Fig. 2D) presented a tremella-like structure with the continuous corrugated surface and rich wrinkle edges. The structure of MoS2/CuS could provide larger surface area for the loading of Au NPs, improving the sensitivity of the proposed immunosensor. The EDX spectrum (Fig. 2E) proved the occurrence of Mo, Cu and S elements. TEM images of MoS2/CuS-Au (Fig. 2F and Fig. 2G) demonstrated that a large amount of nanoparticles with the uniform size were load on the surface of MoS2/CuS. From the HRTEM image (Fig. 2H), we can observe that the nanoparticles had a uniform size about 10 nm. As shown in the EDX spectrum (Fig. 2I), the occur rence of Au, Mo, Cu and S elements could further testify that Au NPs had been introduced the surface of MoS2/CuS successfully. Element mapping is a typical analytical method that reveals element distribution. Fig. S1 confirmed that Au element was uniformly distributed throughout the surface of MoS2/CuS.
All chemicals are analytical reagent grades without further pro cessing. Ultrapure water (18.25 M, 24 � C) was used for all solution preparations. The other details are described in Supplementary material 2
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Biosensors and Bioelectronics 149 (2020) 111842
Fig. 1. (A) Preparation process of MoS2/CuS-Au. (B) Preparation process of Ab2 label. (C) The fabrication process of the sandwich-type immunosensor.
diffusion coefficient (cm2/s), the scan rate (V/s), the transferred electron number, the effective working area of the modified electrode (cm2) and the concentration of K3[Fe(CN)6] (mol/cm3), respectively. In view of above equation, AAu@PtPd MPs ¼ 0.5677 (calculated by anodic peak cur rent) and 0.5532 (calculated by cathodic peak current). The results implied that Au@PtPd MPs/GCE had a good electroactive surface area.
about 20 nm and 80 nm, respectively. After that, the mulberry-like Au@PtPd porous nanorods were further synthesized by using Au NR as seed. It was obvious that Au@PtPd MPs (Fig. 3C) had a hole-like rough surface with uniform shape and particle size. The TEM image of Au@PtPd MPs (Fig. 3D) was clearly observed that all the particles were mulberry-like porous nanorods with an average diameter of 50 nm and an average length of 100 nm. The TEM image (Fig. 3E) revealed the morphology of Au@PtPd MPs, which was composed of a rod-like core surrounded by a porous shell of about 30 nm thickness. From the HRTEM in Fig. 3F, the clear lattice fringes indicated that PtPd nano particles have good crystallinity. To further explore the distribution of elements, the elemental mapping was helped to study the Au@PtPd MPs. As demonstrated in Fig. S2, Pd was mainly distributed on the surface of Au NR, and Pt was mostly on the outer layer of the rod-like structure, indicating that the Pd precursor was easier to reduce. From the line-scanning profile of Au@PtPd MPs (Fig. S2F), Au@PtPd MPs were core-shell structure, and the element composition was consistent with the element mapping result. Fig. 4A showed that CV of Au@PtPd MPs/GCE was performed at different scan rates (0.01–0.1 V/s) in ferricyanide solution, and recorded to calculate the effective electrochemically active surface area of the modified electrodes. Obviously, the peak current value increased with the elevated scan rate. Most importantly, a good linear relationship occurred between the peak current values of the cathode and anode and the square root of the scan rate, indicating the redox reaction was diffusion-controlled process (Wang et al., 2016). The fitting curves of the anode peak and the cathode peak (Fig. 4B): ipa ¼ 395.86 V1/2 þ 9.68, ipc ¼ 385.75 V1/2–10.64, respectively. Electrochemical surface area was calculated by the Randles-Sevick equation (Bard et al., 1980; Zhang et al., 2016): ip ¼ 2.69∙105D1/2V1/2n3/2AC where ip, D, V, n, A and C refers to the redox peak current (A), the
3.3. Signal amplification mechanism of the immunosensor The catalytic properties of materials were critical for sensitive detection of immunosensor. Here, a comparative study of the electro catalytic properties for hydrogen peroxide (H2O2) reduction by different materials was carried out to study the signal amplification mechanism. Different materials of the same concentration were modified on the bare electrode to conduct test by i-t technique. As shown in Fig. 4C, the bare electrode did not have any catalytic effect on the reduction of H2O2. When MoS2 and MoS2/CuS were modified on the bare electrode respectively, the current increased sequentially, indicating that the combination of MoS2 and metal sulfide improved conductivity and optimized the catalytic properties of MoS2. When MoS2/CuS-Au was modified on the bare electrode, the current was further increased, confirming that good conductivity of Au NPs accelerated the electron transport rate on the electrode surface. In particular, MoS2/CuS-Au can immobilize antibodies better than MoS2/CuS due to the excellent biocompatibility of Au NPs. Subsequently, when Au@PtPd MPs were coated on the bare electrode, the current response was the largest. This was ascribed to the large surface area of the mulberry-like Au@PtPd porous nanorods structure, provided many catalytically active sites and exhibited excellent catalytic performance for the reduction of H2O2. According to the literature, the electrocatalytic mechanism towards the reduction of H2O2 can be illustrated as follows (Hui and Erkang, 2013; 3
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Fig. 2. SEM image of: MoS2 (A), CuS (B); MoS2/CuS (C) and (D); EDX image of MoS2/CuS (E); TEM image of MoS2/CuS-Au (F) and (G); HRTEM image of MoS2/CuSAu (H); EDX image of MoS2/CuS-Au (I).
Fig. 3. TEM image of: Au NR (A) and (B); SEM image of Au@PtPd MPs (C); TEM image of Au@PtPd MPs (D) and (E); HRTEM image of Au@PtPd MPs (F).
Venrooij and Koper, 1995): H2O2 þ e- → OHad þ OH-
OHad þ e → OH -
þ
-
2OH þ2H → 2H2O
3.4. The immunosensor construction process (1)
In this study, EIS was used to monitor the preparation of the immunosensor. In the Nyquist diagram, the high frequency semicircular part corresponds to the electron transfer process; the low frequency linear part is related to the diffusion process (Ho et al., 2012). Also, the diameter of the semicircular part is associated with the charge transfer resistance (Rct). Their values were fitted by ZSimWin software, and the results were shown in Table SI. Fig. 4D showed EIS Nyquist plot of
(2) (3)
4
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Biosensors and Bioelectronics 149 (2020) 111842
Fig. 4. (A) CV of the electrodes modified Au@PtPd MPs; (B) The relationship between redox peak currents versus square root of scan rates of Au@PtPd MPs. (C) Current responses of different nanocomposites to 5 mol/L H2O2: (a) bare GCE, (b) MoS2, (c) MoS2/CuS, (d) MoS2/CuS-Au, (e) Au@PtPd MPs. (D) Nyquist diagram of A.C. impedance: (a) bare GCE, (b) MoS2/CuS-Au/GCE, (c) Ab1/MoS2/CuS-Au/GCE, (d) BSA/Ab1/MoS2/CuS-Au/GCE, (e) CEA/BSA/Ab1/MoS2/CuS-Au/GCE, and (f) Au@PtPd MPs-Ab2/CEA/BSA/Ab1/MoS2/CuS-Au/GCE.
different modified electrodes in 2.5 mM [Fe(CN)6]3-/4- and 0.1 M KCl. As observed in Fig. 4D, the charge transfer resistance (Rct) value decreased when MoS2/CuS-Au was modified on the electrode, which was attrib uted to the good conductive performance of MoS2/CuS-Au. However, when Ab1, BSA, CEA (curve c, d and e, respectively) and Au@PtPd MPs-Ab2 (curve f) were continuously immobilized on the electrode, the charge transfer resistance (Rct) value was significantly increased, reflecting their effective fixation. This was because biomolecules occupy and block a portion of the electron transport channel and increase resistance when ferricyanide passes through the membrane, indicating that the proposed immunosensor layer modification was successful.
1.5 mg/mL in the range of 0.5–2.5 mg/mL. Therefore, 1.5 mg/mL was used as the best concentration in this experiment. In addition, the con centration of Au@PtPd MPs was also studied. As observed in Fig. S3C, the peak current continued to increase with the concentration of Au@PtPd MPs-Ab2 from 0.5 to 4.0 mg/mL, reached a maximum at 2.5 mg/mL, following that the peak current began to decrease with increasing concentration. Because the proper concentration not only ensured successful specific binding between the antigen and the anti body, but also effectively improved the electrocatalytic behavior. However, at high concentrations, the excessive thickness of the Au@PtPd MPs-Ab2 film hindered electron transfer due to increased interfacial resistance. Therefore, the optimal concentration of Au@PtPd MPs-Ab2 was 2.5 mg/mL.
3.5. Optimization of conditions for the immunosensor
3.6. Analysis and detection of the immunosensor
In order to obtain the best analytical performance of immunosensor, the key parameters were optimized, including pH, concentration of MoS2/CuS-Au and Au@PtPd MPs. The pH in the buffer solution was key parameter for analyzing the property of the immunosensor. It had strong impact on the biological activity and stability. As shown in Fig. S3A, the peak current continued to increase with pH from 5.3 to 7.1, reached a maximum at 7.1, and then decreased as the pH increased to 8.0. Obviously, strong acid or strong alkali solution can severely damage antigen-antibody attachment and greatly affect the biological activity of the attached protein. Therefore, the pH of optimized conditions was chosen as 7.1 for subsequent experiments. The concentration of the substrate material will affect the immobi lized amount of Ab1, the charge transfer efficiency and the H2O2 reduction catalytic process. As shown in Fig. S3B, the concentration of MoS2/CuS-Au was studied. The best peak current value appeared at
To examine the sensitivity of the proposed immunosensor, developed immunosensor incubated with different concentrations of CEA were studied based on the optimal working condition by i-t technique. Fig. 5A showed that the current value of the proposed immunosensor increased as the elevated CEA concentration ranging from 50 fg/mL to 100 ng/mL. As observed in Fig. 5B, the current value had a good linear relationship with the logarithm of the CEA concentration. The regression equation was I ¼ 20.07 log C þ 114.88, R2 ¼ 0.9981. The calculated detection limit was approximately 16.7 fg/mL when the S/N was 3. Furthermore, the proposed immunosensor showed better sensitivity than the previous CEA test report (Table SII).
5
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Fig. 5. (A) Amperometric response of immunosensor for different concentrations of CEA detection: (a–k) 50 fg/mL, 100 fg/mL, 500 fg/mL, 1.0 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL, 1.0 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL. (B) Calibration curves of the immunosensor to different concentrations of CEA. Error bar ¼ RSD (n ¼ 5).
3.7. Reproducibility, specificity and stability of the proposed immunosensor
infirmary. Firstly, the human serum was diluted 10 times with PBS buffer until it reached the level within the calibration range before detection. Then, the initial concentrations of CEA in human serum samples was calculated by the standard addition methods. In the end, CEA solutions with concentrations of 1.5, 3.0, and 4.5 ng/mL were spiked into diluted human serum samples, respectively, and the recov ery experiments were analyzed by the designed immunosensor. As shown in Table 1, the RSD was in the range from 2.35% to 3.89% and the recovery was in the range from 96.9% to 104.3%. The results suggested that the immunoassay for detecting CEA had good sensitivity and ac curacy in real serum sample analysis, which provided a promising approach for clinical research and diagnostic applications. To further demonstrate the reliability and precision of the proposed analytical methods, the commercialized available enzyme-linked immunosorbent assay (ELISA) method was used as a reference method to detect the same CEA samples. As shown in Table 1, compared with the values of reference method, the relative error between the two methods was less than 5%. The detection results of the two methods were basi cally consistent, demonstrating that the prepared immunosensor was promising for clinical application.
For evaluating property of the designed immunosensor, reproduc ibility, specificity and stability were indispensable test step. The good reproducibility was an important indicator to certify the precision of the designed immunosensor. As illustrated in Fig. S4A, five groups working electrodes (six working electrodes each group) were prepared to detect CEA (1 pg/mL, 10 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL) in 10 mL PBS buffer via i-t technique under the optimal conditions. The relative standard deviation (RSD) was less than 5.0%, indicating that the pro posed immunosensor exhibited good reproducibility. The specificity of the designed immunosensor was a vital analytical process for detecting CEA, which was investigated in the presence of nonspecific molecules. 100-fold higher concentrations of interfering substances including prostate-specific antigen (PSA), human immuno globulin G (HIgG), and alpha fetoprotein (AFP) were measured compared to target CEA. As shown in Fig. S4B, there is almost no current signal in the sample of the interfering substance compared with the background. However, the current signal was increased in the presence of target CEA. The results implied that the immunosensor had a good anti-interference ability and specificity. To access the stability of the proposed immunosensor, we prepared the optimized immunosensor and stored at 4 � C before use. Fig. S4C showed that the detection current response was decreased to 86% of its initial response after 4 weeks, which implied that the immunosensor had good stability.
4. Conclusions In this work, a sandwich-type electrochemical immunosensor for the detection of CEA was successfully developed by using MoS2/CuS-Au as sensing platform and Au@PtPd MPs as signal amplifiers. Under optimal experimental conditions, the designed immunosensor showed wide linear range (50 fg/mL to 100 ng/mL), low detection limit (16.7 fg/mL), good reproducibility, selectivity and stability. Compared with ELISA methods, the immunosensor exhibited good accuracy for CEA detection, which had a promising way for clinical diagnosis. However, there are still some areas where work restrictions need to be improved, such as simplifying the preparation of immunosensors, miniaturizing equipment and portability, which is the direction of further research.
3.8. Real sample analysis To certify the reliability and the applicability of the designed immunosensor in real sample analysis, recovery experiments were conducted by study the different concentrations of CEA in human serum sample. The human serum sample were provided from the school
Table 1 Detection of CEA in human serum samples with the proposed immunosensor and ELISA methods. Initial CEA concentration in sample (ng/mL)a
2.50
a b
Added CEA concentration (ng/mL)
1.50 3.00 4.50
The detection Content (ng/ mL)b
RSD (%, n ¼ 5)
Immunosensor
ELISA
Immunosensor
ELISA
Immunosensor
ELISA
3.98 � 0.24 5.63 � 0.19 6.86 � 0.24
3.97 � 0.18 5.60 � 0.19 6.87 � 0.23
3.89 2.35 2.41
3.82 2.61 2.36
99.20 104.3 96.9
98.10 103.47 97.3
: The human serum samples were provided from the school infirmary. : Each value is the average of five measurements. 6
Recovery (%, n ¼ 5)
Relative deviation (%)
4.03 4.28 2.33
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Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Yilei Jia: Conceptualization, Methodology, Software, Data curation, Writing - original draft. Yueyun Li: Visualization, Investigation, Vali dation. Shuan Zhang: Software, Validation. Ping Wang: Supervision, Investigation. Qing Liu: Supervision, Investigation. Yunhui Dong: Re sources, Supervision. Acknowledgements This study was supported by the Key Research and Development Program of Shandong Province (No.2018GSF120001), National Natural Science Foundation of China (No. 21575079). All of the authors express their deep thanks. We also thank the instrument testing support pro vided by the analysis and testing center of Shandong University of technology. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2019.111842. References Amani, J., Maleki, M., Khoshroo, A., Sobhani-Nasab, A., Rahimi-Nasrabadi, M., 2018. An electrochemical immunosensor based on poly p-phenylenediamine and graphene nanocomposite for detection of neuron-specific enolase via electrochemically amplified detection. Anal. Biochem. 548, 53–59. Bard, A.J., Faulkner, L.R., Leddy, J., Zoski, C.G., 1980. Electrochemical Methods: Fundamentals and Applications. wiley, New York. Barton, M.K., 2014. Early outpatient referral to palliative care services improves end-oflife care. Ca - Cancer J. Clin. 64 (4), 223. Barua, S., Dutta, H.S., Gogoi, S., Devi, R., Khan, R., 2017. Nanostructured MoS2 based advanced biosensors: a review. ACS Appl. Nano Mater. acsanm.7b00157. Bu, L., Shao, Q., B, E., Guo, J., Yao, J., Huang, X., 2017. PtPb/PtNi intermetallic core/ atomic layer shell octahedra for efficient oxygen reduction electrocatalysis. J. Am. Chem. Soc. 139 (28) jacs.7b03510. Chaoliang, T., Hua, Z., 2015. Two-dimensional transition metal dichalcogenide nanosheet-based composites. Chem. Soc. Rev. 44 (9), 2713–2731. Chen, D., Li, C., Liu, H., Ye, F., Yang, J., 2015. Core-shell Au@Pd nanoparticles with enhanced catalytic activity for oxygen reduction reaction via core-shell Au@Ag/Pd constructions. Sci. Rep. 5 (JUL), 11949. Chen, Y., Fu, G., Li, Y., Gu, Q., Lin, X., Sun, D., Tang, Y., 2016. L-Glutamic acid derived PtPd@Pt core/satellite nanoassemblies as an effectively cathodic electrocatalyst. J. Mater. Chem. 5 (8), 10.1039.C1036TA09451A. Chinen, A.B., Guan, C.M., Ferrer, J.R., Barnaby, S.N., Merkel, T.J., Mirkin, C.A., 2015. Nanoparticle probes for the detection of cancer biomarkers, cells, and tissues by fluorescence. Chem. Rev. 115 (19), 150827151416009. Du, D., Zou, Z., Shin, Y., Wang, J., Wu, H., Engelhard, M.H., Liu, J., Aksay, I.A., Lin, Y., 2010. Sensitive immunosensor for cancer biomarker based on dual signal amplification strategy of graphene sheets and multi-enzyme functionalized carbon nanospheres. Anal. Chem. 82 (7), 2989–2995. Gangemi, A., Salehi, P., Hatipoglu, B., Martellotto, J., Barbaro, B., Kuechle, J.B., Qi, M., Wang, Y., Pallan, P., Owens, C., 2010. Islet transplantation for brittle type 1 diabetes: the UIC protocol. Am. J. Transplant. 8 (6), 1250–1261. Garrett, B.R., Polen, S.M., Click, K.A., He, M., Huang, Z., Hadad, C.M., Wu, Y., 2016. Tunable molecular MoS2 edge-site mimics for catalytic hydrogen production. Inorg. Chem. 55 (8), 3960. Garzon, R., Liu, S., Fabbri, M., Liu, Z., Heaphy, C.E., Callegari, E., Schwind, S., Pang, J., Yu, J., Muthusamy, N., 2009. MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood 113 (25), 6411–6418. Ho, M.Y., D’Souza, N., Migliorato, P., 2012. Electrochemical aptamer-based sandwich assays for the detection of explosives. Anal. Chem. 84 (84), 4245–4247. Hui, W., Erkang, W., 2013. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 42 (14), 6060–6093. Jia, H., Gao, P., Ma, H., Wu, D., Du, B., Wei, Q., 2015. Preparation of Au-Pt nanostructures by combining top-down with bottom-up strategies and application in
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Biosensors and Bioelectronics journal homepage: http://www.elsevier.com/locate/bios
Mulberry-like Au@PtPd porous nanorods composites as signal amplifiers for sensitive detection of CEA Yilei Jia , Yueyun Li *, Shuan Zhang , Ping Wang , Qing Liu , Yunhui Dong ** School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo, 255049, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrochemical immunosensor MoS2/CuS-Au nanomaterials Mulberry-like Au@PtPd porous nanorods Carcinoembryonic antigen
Effective detection of cancer biomarkers plays a crucial role in the prevention of early cancer. Here, a sandwichtype electrochemical immunosensor was successfully constructed for sensitive detection of carcinoembryonic antigen (CEA) using MoS2/CuS-Au as sensing platform and mulberry-like Au@PtPd porous nanorods (Au@PtPd MPs) as signal amplifiers. The large surface area and good biocompatibility of MoS2/CuS-Au increased the loading of primary antibody. And the good conductivity of MoS2/CuS-Au accelerated the electron transport rate of the electrode surface. Au@PtPd MPs with large specific surface area and a large number of catalytically active sites showed excellent electrocatalytic performance for hydrogen peroxide reduction. The sandwich-type immunosensor prepared by the signal amplification strategy exhibited a wide linear detection range (50 fg/ mL to 100 ng/mL) and a low detection limit of 16.7 fg/mL (S/N ¼ 3), featuring good selectivity, stability and reproducibility. Moreover, the satisfactory results in analysis of human serum samples indicated that it possessed a potential application promising in early clinical diagnoses.
1. Introduction Cancer, also known as malignant tumor, has seriously harmed human health (Garzon et al., 2009; Yang, L. et al., 2017). Importantly, the occurrence of cancer is tending to become younger due to changes in dietary structure, increased work pressure, and infection of various pathogens (Gangemi et al., 2010). Most early cancers have no obvious symptoms and are easily ignored. Therefore, effective early detection is very important for the patient (Kim et al., 2013; Wu et al., 2015). Cancer biomarkers are a promising tool for the diagnosis and prognosis of cancer, and carcinoembryonic antigen (CEA) is one of the most widely used cancer biomarkers (Labib et al., 2016). In general, CEA are asso ciated with many types of cancer when their levels in human serum exceed 5 ng/mL (Barton, 2014). So far, detection methods of CEA involved enzyme-linked immunoassay (Yang, W. et al., 2017), chem iluminescence immunoassay (Lee et al., 2012), radiological analysis (Lang et al., 2014), and so on. But these methods are usually time consuming and expensive, labor intensive, and use complex instruments (Chinen et al., 2015; Santharaman et al., 2016). Compared with these methods, electrochemical immunosensor have received extensive attention in CEA detection due to the advantages of simple operation,
low cost and affordability (Amani et al., 2018). And it has been widely used in drug screening, food testing, environmental monitoring and clinical diagnosis (Jia et al., 2015). As we all know, electrochemical immunosensor are analytical methods by determining the concentration of target antigen based on biological specificity recognition reaction, and converting biological signal into electrical signal (Du et al., 2010; Ke et al., 2018). However, antigen and antibody specific binding to form complexes on the elec trode surface could hinder electron transfer. Therefore, sensitive detection of immunosensor is still highly challenging. At present, building effective and sensitive immunosensor is usually improved by the following strategies: (i) designing new biocompatible and conduc tive substrate materials to increase the loading of antibodies and accelerate the electron transfer on the electrode surface; (ii) using new signal amplification markers to effectively improve the performance of the sensor (Zhou et al., 2016). In recent years, transition metal sulfides such as MoS2, CuS, CoS, and Cu7S4 have attracted attention of researchers (Barua et al., 2017; Lu et al., 2016; Wang et al., 2018). In particular, the layered transition metal MoS2 has a huge potential application in the manufacture of electrochemical immunosensor owing to large surface area, low cost and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Li), [email protected] (Y. Dong). https://doi.org/10.1016/j.bios.2019.111842 Received 31 July 2019; Received in revised form 17 October 2019; Accepted 1 November 2019 Available online 6 November 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
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high chemical stability (Garrett et al., 2016; Su et al., 2016). For example, Qian’s groups reported a MoS2-based nanocomposites immu nosensor, which exhibited high sensitivity for SCCA detection (Li et al., 2011). Jiang’s groups developed a platform based on MoS2-Thi com posites, achieving sensitive and rapid detection of ZEA (Wang et al., 2014). All of the above studies can demonstrate the excellent properties of MoS2. Inspired by the above works, a preliminary signal amplification strategy was designed by using MoS2/CuS-Au as sensing platform. The combination of MoS2 and CuS nanocomposites, could not only overcome the aggregation phenomenon caused by strong van der Waals force in the layered crystal of MoS2, but also increase the use properties of MoS2 (Tan et al., 2016). More importantly, the nanocomposites with tremella-like morphology possessed large surface area and exposed more active sites, which could improve the catalytic ability for hydrogen peroxide (H2O2) (Chaoliang and Hua, 2015). Furthermore, the gold nanoparticles (Au NPs) have sparked extensive concerns due to its excellent biocompatibility and good electrical conductivity. As expect, Au NPs introduced by the in-situ reduction method effectively improved the electron transporting ability of the electrode surface, coupled a large number of primary antibody (Ab1) by Au-N bond, and further enhanced the catalytic activity for H2O2 (Yang et al., 2015). Therefore, MoS2/CuS-Au as sensing platform improved the performance of the immunosensor and achieved preliminary signal amplification. New signal amplification strategy plays a crucial role in the con struction of sandwich-type immunosensor. Especially, compared to mono- and bi-metallic nanomaterials, trimetallic nanomaterials have better advantages in electrochemical immunosensor, such as superior biocompatibility, excellent electrical conductivity and catalytic prop erties (Lijuan et al., 2011; Teng et al., 2010). In addition to the electronic effects of multiple metals, these properties of nanomaterials depend to a large extent on morphology and structure. Recently, nanomaterials with core-shell rod-like structure have the most potential application in electrochemical immunosensor (Bu et al., 2017; Zheng et al., 2017). Core-shell rod-like nanomaterials have many advantages in terms of stability and synergy between compositions, and its complex electronic interactions can alter the surface electronic properties of core-shell rod-like nanomaterials (Chen et al., 2015, 2016). However, many pre viously reported shell regions are dense structures with limited catalytic sites (Su et al., 2017). In view of the problem, a metal nano-porous shell was designed to improve the electrocatalytic performance of the core-shell nanomaterial, thereby providing sufficient contactable active sites. In this work, a trimetallic mulberry-like Au@PtPd porous nanorods (Au@PtPd MPs) were synthesized in aqueous solution by a simple method as an ideal signal amplification label to detect CEA sensitively. Impressively, the fabrication of Au@PtPd MPs could not only increase the specific surface area and atomic utilization of Pt, but also provide abundant accessible pores for the reduction of H2O2 (Zhao et al., 2017). Remarkably, the composite had good biocompatibility and can immo bilized a large number of secondary antibodies (Ab2). In short, a different sandwich-type immunosensor was successfully constructed for sensitive detection of CEA. Using MoS2/CuS-Au as sensing platform to immobilize Ab1, improving the performance of the immunosensor. The mulberry-shaped Au@PtPd porous nanorods (Au@PtPd MPs) were used as signal amplifiers, and its unique porous core-shell rod-like structure and trimetallic component showed excel lent catalytic and stability. Therefore, signal amplification and sensi tivity detection could be achieved in the designed immunosensor for the detection of CEA in human serum.
(SM).
2. Experimental
3.2. Characterization of Au@PtPd MPs
2.1. Materials and apparatus
In this experiment, gold nanorods (Au NR) with high yield and uniform size was synthesized by seed growth method (Ye et al., 2013). Morphology and size of Au NR were characterized by TEM. As shown in Fig. 3A, we could clearly observe that almost all the particles were rod-like. The average diameter and length of the Au NR (Fig. 3B) were
2.2. Fabrication of the proposed immunosensor The detail fabrication process of the proposed immunosensor is outlined in Fig. 1. Before modified, the GCE (4 mm diameter) was pol ished by alumina powder (0.3 and 0.05 μm) to a mirror and then cleaned. Sequentially, MoS2/CuS-Au suspension (6 μL) were modified on the bare GCE and dried at ambient temperature. Following that, Ab1 (6 μL, 10 μg/mL) was incubated on the above GCE, and immobilized on the electrode surface by Au-N between Au in composite and amino group in antibody. After dried at 4 � C and washed with the PBS, BSA (3 μL, 1 wt%) was dripped on the above GCE to block the nonspecific binding sites. After that, the modified GCE was incubated with a series of different concentrations of CEA. At last, Au@PtPd MPs-Ab2 suspension (6 μL) was dropped on the above GCE for 50 min. The fabricated elec trode was cleaned completely with PBS to remove any unbound at tachments, and stored at 4 � C for later research. 2.3. Detection of the CEA The proposed immunosensor was tested by a conventional threeelectrode system (the fabricated electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and plat inum electrodes as the auxiliary electrode) in all electrochemical tests. Subsequently, amperometric i-t technique was conducted in PBS buffer (pH 7.1) with a scanning potential at 0.4 V. And after the current was in a steady state, H2O2 (10 μL, 5 mol/L) was injected into the PBS (10 mL) under mild stirring, following that the current response value was record. EIS was utilized to investigate the step-by-step fabrication of the proposed immunosensor, and recorded in solution with 2.5 mM [Fe (CN)6]3-/4- and 0.1 M KCl (frequency range: 10 1 to 105 Hz, and initial voltage: 0.24 V). 3. Results and discussion 3.1. Characterization of materials To verify the morphology of MoS2/CuS, SEM was helpful to char acterize the microstructure of MoS2, CuS and MoS2/CuS, respectively. As illustrated in Fig. 2, the SEM images showed that agglomerated spherical cluster of MoS2 (Fig. 2A) and Sheet-like CuS (Fig. 2B). When MoS2 and CuS were combined, the SEM images of MoS2/CuS with different magnification (Fig. 2C and Fig. 2D) presented a tremella-like structure with the continuous corrugated surface and rich wrinkle edges. The structure of MoS2/CuS could provide larger surface area for the loading of Au NPs, improving the sensitivity of the proposed immunosensor. The EDX spectrum (Fig. 2E) proved the occurrence of Mo, Cu and S elements. TEM images of MoS2/CuS-Au (Fig. 2F and Fig. 2G) demonstrated that a large amount of nanoparticles with the uniform size were load on the surface of MoS2/CuS. From the HRTEM image (Fig. 2H), we can observe that the nanoparticles had a uniform size about 10 nm. As shown in the EDX spectrum (Fig. 2I), the occur rence of Au, Mo, Cu and S elements could further testify that Au NPs had been introduced the surface of MoS2/CuS successfully. Element mapping is a typical analytical method that reveals element distribution. Fig. S1 confirmed that Au element was uniformly distributed throughout the surface of MoS2/CuS.
All chemicals are analytical reagent grades without further pro cessing. Ultrapure water (18.25 M, 24 � C) was used for all solution preparations. The other details are described in Supplementary material 2
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Fig. 1. (A) Preparation process of MoS2/CuS-Au. (B) Preparation process of Ab2 label. (C) The fabrication process of the sandwich-type immunosensor.
diffusion coefficient (cm2/s), the scan rate (V/s), the transferred electron number, the effective working area of the modified electrode (cm2) and the concentration of K3[Fe(CN)6] (mol/cm3), respectively. In view of above equation, AAu@PtPd MPs ¼ 0.5677 (calculated by anodic peak cur rent) and 0.5532 (calculated by cathodic peak current). The results implied that Au@PtPd MPs/GCE had a good electroactive surface area.
about 20 nm and 80 nm, respectively. After that, the mulberry-like Au@PtPd porous nanorods were further synthesized by using Au NR as seed. It was obvious that Au@PtPd MPs (Fig. 3C) had a hole-like rough surface with uniform shape and particle size. The TEM image of Au@PtPd MPs (Fig. 3D) was clearly observed that all the particles were mulberry-like porous nanorods with an average diameter of 50 nm and an average length of 100 nm. The TEM image (Fig. 3E) revealed the morphology of Au@PtPd MPs, which was composed of a rod-like core surrounded by a porous shell of about 30 nm thickness. From the HRTEM in Fig. 3F, the clear lattice fringes indicated that PtPd nano particles have good crystallinity. To further explore the distribution of elements, the elemental mapping was helped to study the Au@PtPd MPs. As demonstrated in Fig. S2, Pd was mainly distributed on the surface of Au NR, and Pt was mostly on the outer layer of the rod-like structure, indicating that the Pd precursor was easier to reduce. From the line-scanning profile of Au@PtPd MPs (Fig. S2F), Au@PtPd MPs were core-shell structure, and the element composition was consistent with the element mapping result. Fig. 4A showed that CV of Au@PtPd MPs/GCE was performed at different scan rates (0.01–0.1 V/s) in ferricyanide solution, and recorded to calculate the effective electrochemically active surface area of the modified electrodes. Obviously, the peak current value increased with the elevated scan rate. Most importantly, a good linear relationship occurred between the peak current values of the cathode and anode and the square root of the scan rate, indicating the redox reaction was diffusion-controlled process (Wang et al., 2016). The fitting curves of the anode peak and the cathode peak (Fig. 4B): ipa ¼ 395.86 V1/2 þ 9.68, ipc ¼ 385.75 V1/2–10.64, respectively. Electrochemical surface area was calculated by the Randles-Sevick equation (Bard et al., 1980; Zhang et al., 2016): ip ¼ 2.69∙105D1/2V1/2n3/2AC where ip, D, V, n, A and C refers to the redox peak current (A), the
3.3. Signal amplification mechanism of the immunosensor The catalytic properties of materials were critical for sensitive detection of immunosensor. Here, a comparative study of the electro catalytic properties for hydrogen peroxide (H2O2) reduction by different materials was carried out to study the signal amplification mechanism. Different materials of the same concentration were modified on the bare electrode to conduct test by i-t technique. As shown in Fig. 4C, the bare electrode did not have any catalytic effect on the reduction of H2O2. When MoS2 and MoS2/CuS were modified on the bare electrode respectively, the current increased sequentially, indicating that the combination of MoS2 and metal sulfide improved conductivity and optimized the catalytic properties of MoS2. When MoS2/CuS-Au was modified on the bare electrode, the current was further increased, confirming that good conductivity of Au NPs accelerated the electron transport rate on the electrode surface. In particular, MoS2/CuS-Au can immobilize antibodies better than MoS2/CuS due to the excellent biocompatibility of Au NPs. Subsequently, when Au@PtPd MPs were coated on the bare electrode, the current response was the largest. This was ascribed to the large surface area of the mulberry-like Au@PtPd porous nanorods structure, provided many catalytically active sites and exhibited excellent catalytic performance for the reduction of H2O2. According to the literature, the electrocatalytic mechanism towards the reduction of H2O2 can be illustrated as follows (Hui and Erkang, 2013; 3
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Fig. 2. SEM image of: MoS2 (A), CuS (B); MoS2/CuS (C) and (D); EDX image of MoS2/CuS (E); TEM image of MoS2/CuS-Au (F) and (G); HRTEM image of MoS2/CuSAu (H); EDX image of MoS2/CuS-Au (I).
Fig. 3. TEM image of: Au NR (A) and (B); SEM image of Au@PtPd MPs (C); TEM image of Au@PtPd MPs (D) and (E); HRTEM image of Au@PtPd MPs (F).
Venrooij and Koper, 1995): H2O2 þ e- → OHad þ OH-
OHad þ e → OH -
þ
-
2OH þ2H → 2H2O
3.4. The immunosensor construction process (1)
In this study, EIS was used to monitor the preparation of the immunosensor. In the Nyquist diagram, the high frequency semicircular part corresponds to the electron transfer process; the low frequency linear part is related to the diffusion process (Ho et al., 2012). Also, the diameter of the semicircular part is associated with the charge transfer resistance (Rct). Their values were fitted by ZSimWin software, and the results were shown in Table SI. Fig. 4D showed EIS Nyquist plot of
(2) (3)
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Fig. 4. (A) CV of the electrodes modified Au@PtPd MPs; (B) The relationship between redox peak currents versus square root of scan rates of Au@PtPd MPs. (C) Current responses of different nanocomposites to 5 mol/L H2O2: (a) bare GCE, (b) MoS2, (c) MoS2/CuS, (d) MoS2/CuS-Au, (e) Au@PtPd MPs. (D) Nyquist diagram of A.C. impedance: (a) bare GCE, (b) MoS2/CuS-Au/GCE, (c) Ab1/MoS2/CuS-Au/GCE, (d) BSA/Ab1/MoS2/CuS-Au/GCE, (e) CEA/BSA/Ab1/MoS2/CuS-Au/GCE, and (f) Au@PtPd MPs-Ab2/CEA/BSA/Ab1/MoS2/CuS-Au/GCE.
different modified electrodes in 2.5 mM [Fe(CN)6]3-/4- and 0.1 M KCl. As observed in Fig. 4D, the charge transfer resistance (Rct) value decreased when MoS2/CuS-Au was modified on the electrode, which was attrib uted to the good conductive performance of MoS2/CuS-Au. However, when Ab1, BSA, CEA (curve c, d and e, respectively) and Au@PtPd MPs-Ab2 (curve f) were continuously immobilized on the electrode, the charge transfer resistance (Rct) value was significantly increased, reflecting their effective fixation. This was because biomolecules occupy and block a portion of the electron transport channel and increase resistance when ferricyanide passes through the membrane, indicating that the proposed immunosensor layer modification was successful.
1.5 mg/mL in the range of 0.5–2.5 mg/mL. Therefore, 1.5 mg/mL was used as the best concentration in this experiment. In addition, the con centration of Au@PtPd MPs was also studied. As observed in Fig. S3C, the peak current continued to increase with the concentration of Au@PtPd MPs-Ab2 from 0.5 to 4.0 mg/mL, reached a maximum at 2.5 mg/mL, following that the peak current began to decrease with increasing concentration. Because the proper concentration not only ensured successful specific binding between the antigen and the anti body, but also effectively improved the electrocatalytic behavior. However, at high concentrations, the excessive thickness of the Au@PtPd MPs-Ab2 film hindered electron transfer due to increased interfacial resistance. Therefore, the optimal concentration of Au@PtPd MPs-Ab2 was 2.5 mg/mL.
3.5. Optimization of conditions for the immunosensor
3.6. Analysis and detection of the immunosensor
In order to obtain the best analytical performance of immunosensor, the key parameters were optimized, including pH, concentration of MoS2/CuS-Au and Au@PtPd MPs. The pH in the buffer solution was key parameter for analyzing the property of the immunosensor. It had strong impact on the biological activity and stability. As shown in Fig. S3A, the peak current continued to increase with pH from 5.3 to 7.1, reached a maximum at 7.1, and then decreased as the pH increased to 8.0. Obviously, strong acid or strong alkali solution can severely damage antigen-antibody attachment and greatly affect the biological activity of the attached protein. Therefore, the pH of optimized conditions was chosen as 7.1 for subsequent experiments. The concentration of the substrate material will affect the immobi lized amount of Ab1, the charge transfer efficiency and the H2O2 reduction catalytic process. As shown in Fig. S3B, the concentration of MoS2/CuS-Au was studied. The best peak current value appeared at
To examine the sensitivity of the proposed immunosensor, developed immunosensor incubated with different concentrations of CEA were studied based on the optimal working condition by i-t technique. Fig. 5A showed that the current value of the proposed immunosensor increased as the elevated CEA concentration ranging from 50 fg/mL to 100 ng/mL. As observed in Fig. 5B, the current value had a good linear relationship with the logarithm of the CEA concentration. The regression equation was I ¼ 20.07 log C þ 114.88, R2 ¼ 0.9981. The calculated detection limit was approximately 16.7 fg/mL when the S/N was 3. Furthermore, the proposed immunosensor showed better sensitivity than the previous CEA test report (Table SII).
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Fig. 5. (A) Amperometric response of immunosensor for different concentrations of CEA detection: (a–k) 50 fg/mL, 100 fg/mL, 500 fg/mL, 1.0 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL, 1.0 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL. (B) Calibration curves of the immunosensor to different concentrations of CEA. Error bar ¼ RSD (n ¼ 5).
3.7. Reproducibility, specificity and stability of the proposed immunosensor
infirmary. Firstly, the human serum was diluted 10 times with PBS buffer until it reached the level within the calibration range before detection. Then, the initial concentrations of CEA in human serum samples was calculated by the standard addition methods. In the end, CEA solutions with concentrations of 1.5, 3.0, and 4.5 ng/mL were spiked into diluted human serum samples, respectively, and the recov ery experiments were analyzed by the designed immunosensor. As shown in Table 1, the RSD was in the range from 2.35% to 3.89% and the recovery was in the range from 96.9% to 104.3%. The results suggested that the immunoassay for detecting CEA had good sensitivity and ac curacy in real serum sample analysis, which provided a promising approach for clinical research and diagnostic applications. To further demonstrate the reliability and precision of the proposed analytical methods, the commercialized available enzyme-linked immunosorbent assay (ELISA) method was used as a reference method to detect the same CEA samples. As shown in Table 1, compared with the values of reference method, the relative error between the two methods was less than 5%. The detection results of the two methods were basi cally consistent, demonstrating that the prepared immunosensor was promising for clinical application.
For evaluating property of the designed immunosensor, reproduc ibility, specificity and stability were indispensable test step. The good reproducibility was an important indicator to certify the precision of the designed immunosensor. As illustrated in Fig. S4A, five groups working electrodes (six working electrodes each group) were prepared to detect CEA (1 pg/mL, 10 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL) in 10 mL PBS buffer via i-t technique under the optimal conditions. The relative standard deviation (RSD) was less than 5.0%, indicating that the pro posed immunosensor exhibited good reproducibility. The specificity of the designed immunosensor was a vital analytical process for detecting CEA, which was investigated in the presence of nonspecific molecules. 100-fold higher concentrations of interfering substances including prostate-specific antigen (PSA), human immuno globulin G (HIgG), and alpha fetoprotein (AFP) were measured compared to target CEA. As shown in Fig. S4B, there is almost no current signal in the sample of the interfering substance compared with the background. However, the current signal was increased in the presence of target CEA. The results implied that the immunosensor had a good anti-interference ability and specificity. To access the stability of the proposed immunosensor, we prepared the optimized immunosensor and stored at 4 � C before use. Fig. S4C showed that the detection current response was decreased to 86% of its initial response after 4 weeks, which implied that the immunosensor had good stability.
4. Conclusions In this work, a sandwich-type electrochemical immunosensor for the detection of CEA was successfully developed by using MoS2/CuS-Au as sensing platform and Au@PtPd MPs as signal amplifiers. Under optimal experimental conditions, the designed immunosensor showed wide linear range (50 fg/mL to 100 ng/mL), low detection limit (16.7 fg/mL), good reproducibility, selectivity and stability. Compared with ELISA methods, the immunosensor exhibited good accuracy for CEA detection, which had a promising way for clinical diagnosis. However, there are still some areas where work restrictions need to be improved, such as simplifying the preparation of immunosensors, miniaturizing equipment and portability, which is the direction of further research.
3.8. Real sample analysis To certify the reliability and the applicability of the designed immunosensor in real sample analysis, recovery experiments were conducted by study the different concentrations of CEA in human serum sample. The human serum sample were provided from the school
Table 1 Detection of CEA in human serum samples with the proposed immunosensor and ELISA methods. Initial CEA concentration in sample (ng/mL)a
2.50
a b
Added CEA concentration (ng/mL)
1.50 3.00 4.50
The detection Content (ng/ mL)b
RSD (%, n ¼ 5)
Immunosensor
ELISA
Immunosensor
ELISA
Immunosensor
ELISA
3.98 � 0.24 5.63 � 0.19 6.86 � 0.24
3.97 � 0.18 5.60 � 0.19 6.87 � 0.23
3.89 2.35 2.41
3.82 2.61 2.36
99.20 104.3 96.9
98.10 103.47 97.3
: The human serum samples were provided from the school infirmary. : Each value is the average of five measurements. 6
Recovery (%, n ¼ 5)
Relative deviation (%)
4.03 4.28 2.33
Biosensors and Bioelectronics 149 (2020) 111842
Y. Jia et al.
Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Yilei Jia: Conceptualization, Methodology, Software, Data curation, Writing - original draft. Yueyun Li: Visualization, Investigation, Vali dation. Shuan Zhang: Software, Validation. Ping Wang: Supervision, Investigation. Qing Liu: Supervision, Investigation. Yunhui Dong: Re sources, Supervision. Acknowledgements This study was supported by the Key Research and Development Program of Shandong Province (No.2018GSF120001), National Natural Science Foundation of China (No. 21575079). All of the authors express their deep thanks. We also thank the instrument testing support pro vided by the analysis and testing center of Shandong University of technology. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2019.111842. References Amani, J., Maleki, M., Khoshroo, A., Sobhani-Nasab, A., Rahimi-Nasrabadi, M., 2018. An electrochemical immunosensor based on poly p-phenylenediamine and graphene nanocomposite for detection of neuron-specific enolase via electrochemically amplified detection. Anal. Biochem. 548, 53–59. Bard, A.J., Faulkner, L.R., Leddy, J., Zoski, C.G., 1980. Electrochemical Methods: Fundamentals and Applications. wiley, New York. Barton, M.K., 2014. Early outpatient referral to palliative care services improves end-oflife care. Ca - Cancer J. Clin. 64 (4), 223. Barua, S., Dutta, H.S., Gogoi, S., Devi, R., Khan, R., 2017. Nanostructured MoS2 based advanced biosensors: a review. ACS Appl. Nano Mater. acsanm.7b00157. Bu, L., Shao, Q., B, E., Guo, J., Yao, J., Huang, X., 2017. PtPb/PtNi intermetallic core/ atomic layer shell octahedra for efficient oxygen reduction electrocatalysis. J. Am. Chem. Soc. 139 (28) jacs.7b03510. Chaoliang, T., Hua, Z., 2015. Two-dimensional transition metal dichalcogenide nanosheet-based composites. Chem. Soc. Rev. 44 (9), 2713–2731. Chen, D., Li, C., Liu, H., Ye, F., Yang, J., 2015. Core-shell Au@Pd nanoparticles with enhanced catalytic activity for oxygen reduction reaction via core-shell Au@Ag/Pd constructions. Sci. Rep. 5 (JUL), 11949. Chen, Y., Fu, G., Li, Y., Gu, Q., Lin, X., Sun, D., Tang, Y., 2016. L-Glutamic acid derived PtPd@Pt core/satellite nanoassemblies as an effectively cathodic electrocatalyst. J. Mater. Chem. 5 (8), 10.1039.C1036TA09451A. Chinen, A.B., Guan, C.M., Ferrer, J.R., Barnaby, S.N., Merkel, T.J., Mirkin, C.A., 2015. Nanoparticle probes for the detection of cancer biomarkers, cells, and tissues by fluorescence. Chem. Rev. 115 (19), 150827151416009. Du, D., Zou, Z., Shin, Y., Wang, J., Wu, H., Engelhard, M.H., Liu, J., Aksay, I.A., Lin, Y., 2010. Sensitive immunosensor for cancer biomarker based on dual signal amplification strategy of graphene sheets and multi-enzyme functionalized carbon nanospheres. Anal. Chem. 82 (7), 2989–2995. Gangemi, A., Salehi, P., Hatipoglu, B., Martellotto, J., Barbaro, B., Kuechle, J.B., Qi, M., Wang, Y., Pallan, P., Owens, C., 2010. Islet transplantation for brittle type 1 diabetes: the UIC protocol. Am. J. Transplant. 8 (6), 1250–1261. Garrett, B.R., Polen, S.M., Click, K.A., He, M., Huang, Z., Hadad, C.M., Wu, Y., 2016. Tunable molecular MoS2 edge-site mimics for catalytic hydrogen production. Inorg. Chem. 55 (8), 3960. Garzon, R., Liu, S., Fabbri, M., Liu, Z., Heaphy, C.E., Callegari, E., Schwind, S., Pang, J., Yu, J., Muthusamy, N., 2009. MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood 113 (25), 6411–6418. Ho, M.Y., D’Souza, N., Migliorato, P., 2012. Electrochemical aptamer-based sandwich assays for the detection of explosives. Anal. Chem. 84 (84), 4245–4247. Hui, W., Erkang, W., 2013. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 42 (14), 6060–6093. Jia, H., Gao, P., Ma, H., Wu, D., Du, B., Wei, Q., 2015. Preparation of Au-Pt nanostructures by combining top-down with bottom-up strategies and application in
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