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Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted PB@PANI Core-Shell Nanocubes as a Signal Probe

Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted... Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 7009624, 9 pages https://doi.org/10.1155/2023/7009624 Research Article Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted PB@PANI Core-Shell Nanocubes as a Signal Probe 1,2 2 1,2 1 Dexiang Feng , Lingzhi Chen , Ke Zhang , Shuangshuang Zhu , 2 2 2 1,2 2 Meichen Ying , Peng Jiang , Menglan Fu , Yan Wei , and Lihua Li Department of Chemistry, Wannan Medical College, Wuhu 241002, China Institute of Synthesis and Application of Medical Materials, Department of Pharmacy, Wannan Medical College, Wuhu 241002, China Correspondence should be addressed to Yan Wei; yanwei@wnmc.edu.cn and Lihua Li; llh05530226@126.com Received 10 September 2022; Revised 1 November 2022; Accepted 23 March 2023; Published 7 April 2023 Academic Editor: Jose Vicente Ros Lis Copyright © 2023 Dexiang Feng et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Herein, a method was developed for the sensitive monitoring of carcinoembryonic antigen (CEA) by gold nanoparticles dotted prussian blue@polyaniline core-shell nanocubes (Au NPs/PB@PANI). First, a facile low-temperature method was used to prepare the uniform PB@PANI core-shell nanocubes with the assistance of PVP, where PB acted as the electron transfer mediator to provide electrochemical signals, and the PANI with excellent conductivity and desirable chemical stability not only played the role of a protective layer to prevent etching of PB in basic media but also efectively improved electron transfer. Importantly, to further enhance the electrical conductivity and biocompatibility of PB@PANI and to further enhance the electrochemical signal and capture a large amount of Ab , Au NPs were doped on the surface of PB@PANI to form Au NPs/PB@PANI nanocomposites. Subsequently, benefting from the advantages of core-shell structure nanoprobes and gold-platinum bimetallic nanofower (AuPt NF), a sandwich-type electrochemical immunosensor for CEA detection was constructed, which provided a wide linear detection −1 −1 −1 range from 1.0 pg·mL to 100.0 ng·mL and a low detection limit of 0.35 pg·mL via DPV (at 3σ). Moreover, it displayed a satisfactory result when the core-shell structure nanoprobe-based immunosensor was applied to determine CEA in real human serum samples. energy consumption and trace detection, and more suitable 1. Introduction for the detection of biomarkers in low concentration [5, 6]. As we all know, carcinoembryonic antigen (CEA) is a tumor More recently, lots of electrochemical immunosensors have marker for colon cancer, breast cancer, ovarian cancer, and been constructed for quantitation of CEA. For example, other cancers, which can provide reliable information for the Wang et al. Jozghorbani et al. and Wang et al. designed three early diagnosis and treatment of tumor patients [1–3]. kinds of label-free CEA immunosensors with detection −1 Consequently, the highly sensitive determination of CEA is limits of 0.005, 0.05, and 0.0429 ng·mL , respectively [7–9]. a pressing need by virtue of accurate and efcient analytical Especially, a large number of reports have focused on the techniques. Immunosensors based on antibody-antigen sandwich-type electrochemical immunosensor for CEA interaction are one of the most widely used analytical detection [10–13], thanks to their distinct advantages such as techniques in the quantitative detection of biomarkers [4]. lower background noise, higher sensitivity, and importance, Among them, electrochemical immunosensors have and they have higher selectivity for analytes through two attracted much attention due to their characteristics of high specifc reactions, which are superior to label-free coun- specifcity, good sensitivity, short time consumption, low terparts [14–16]. 2 Journal of Analytical Methods in Chemistry Prussian blue (PB) with a face-centered cubic lattice (H PtCl 6H O), bovine serum albumin (BSA), polyvinyl 2 6 2 structure is popular as a kind of electrochemical redox- pyrrolidone (PVP), L-cysteine (L-Cys), aniline, ammonium active species in electrochemical biosensors because of its persulfate, and so on were purchased from Aladdin Reagent high electrochemical/electrocatalytic properties and low Co., Ltd. (Shanghai, China). redox potential [17, 18]. Unfortunately, the poor stability and low conductivity of PB limit its further applications 2.2. Devices. Electrochemical measurements were made at in biosensors [19]. To minimize these problems, some the CHI 660E electrochemical workstation (Shanghai Co., conducting materials can be introduced to meet the Ltd., China). Te morphology of nanomaterials was ob- requirements mentioned above. Polyaniline (PANI) as tained by a scanning electron microscopy (SEM, JSM-7100F, one of the most desired materials with good chemical Japan) and a transmission electron microscopy (TEM, JEM- stability, low cost, and good electrical conductivity can 6700F, Japan). not only provide a conductive substrate but also can form an efective coating layer on “the core” such as carbon nanotubes (CNTs) or PB [20, 21]. Te combination of PB 2.3. Synthesis of PB Nanocubes. PB nanocubes were syn- and PANI (PB@PANI) can play a signifcant synergistic thesized according to a previous work despite a minor −1 efect and improve the electrocatalytic performance, modifcation [27]. 8.0 mmoL·L Na Fe(CN) .8H O, 4.0 mL 4 6 2 conductivity, and stability [21]. Furthermore, to further hydrochloric acid (37%), and 2.0 g polyvinyl pyrrolidone enhance the conductivity and biocompatibility of PB@ (M ∼40000) were added into deionized water (400 mL) and PANI, gold nanoparticles (Au NPs) were introduced over ° stirred for 30 min, and then were refuxed at 60 C for 6 h. the surface of PB@PANI by Au-N bonds between Au NPs After that, the blue products were washed with deionized and -NH from PANI to form Au NPs/PB@PANI ° water and dried in vacuum at 100 C for 24 h. nanocomposites [15]. To the best of our knowledge, the preparation and application of Au NPs/PB@PANI 2.4. Preparation of PB@PANI Core-Shell Nanocubes. nanocomposites in electrochemical immunosensors have PB@PANI core-shell nanocubes were synthesized with not been reported. Hence, the PB@PANI loaded with Au reference to previous work with slight variations [21]. NPs (Au NPs/PB@PANI) would be a promising Briefy, 0.6 g·PB, 0.5 g polyvinyl pyrrolidone (M ∼40000) nanoprobe. w and 100 μL aniline monomer were dissolved in 100 mL For sandwich-type electrochemical immunosensors, to −1 hydrochloric acid (1 moL·L ) with sonication for 1 h. Ten, achieve signal amplifcation and high sensitivity, it is a more −1 ammonium persulfate solution (1.6 mmoL·L ) was added important key to efectively immobilize the primary anti- dropwise into the above solution and stirred for 18 h in an body (Ab ) [22, 23]. Gold and platinum nanoparticles are the ice bath. Finally, the PANI-coated PB (core-shell structure most promising bimetallic materials for various applications PB@PANI) was collected by centrifugation and washed with due to their superior biocompatibility, higher specifc sur- deionized water and ethanol for several times. face area, and superior electrocatalytic properties towards the reduction of H O [24–26]. Tey can be decorated onto 2 2 the surface of the L-cysteine (L-Cys)-modifed electrode to 2.5. Preparation of Ab -Au NPs/PB@PANI Bioconjugates. provide an available microenvironment for loading amounts 15 mg PB@PANI nanocubes were added into the concen- of Ab , thus greatly improving the sensitivity of the −1 trated Au NPs colloidal solution (5.0 mL, 1.0 mg·mL ) and immunosensor. reacted 30 min under shaking. Au NPs were assembled on Tis work was developed by electrodeposition of AuPt the surface of PB@PANI to form the Au NPs/PB@PANI bimetallic nanofower (AuPt NFs) on the GCE modifed with nanocomposites by the chemical bond of Au-NH [15]. L-Cys as a sensing platform and Au NPs/PB@PANI as Followed by centrifugation and washing twice with deion- a novel label prepared by a facile low-temperature method. ized water, the obtained precipitate was redispersed into PBS Te existence of AuPt NFs not only fxed the primary an- solution (pH 7.0). Subsequently, 500 μL CEA-Ab tibody but also accelerated the electron transfer. Further- −1 (2.0 mg·mL ) was added into the Au NPs/PB@PANI sus- more, the as-developed Au NPs/PB@PANI nanoprobes were pension (2 mL) and stirred for 12 h at 4 C. To get rid of used as a tracer for the generation and amplifcation of nonspecifc adsorption, 100 μL of 1% BSA was mixed with electrochemical signals and easily captured second anti- the obtained conjugates for 6 h at 4 C. Finally, the bio- bodies (Ab ) via Au-N bond. As expected, a sandwich-type conjugates were centrifuged and redispersed in PBS (pH 7.0, electrochemical immunosensor capable of achieving large −1 10 mmoL·L ), and then stored at 4 C. signal amplifcation was developed by connecting Ab -AuPt NFs/L-Cys with Ab -Au NPs/PB@PANI. 2.6. Fabrication of the Immunosensor. For polymer nano- composite flm deposition, polished glass carbon electrode 2. Materials and Methods −1 (GCE) was frst immersed in 0.5 mmoL·L cysteine con- 2.1. Materials and Chemicals. Carcinoembryonic antigen taining PBS (pH 7.0) and was scanned in the potential range (CEA) and its antibodies, α-fetoprotein (AFP), were pur- of −0.0 to 1.7 V at 50 mV/s for 3 cycles. Te obtained −1 chased from Biocell Biotech. Co., Ltd. (Zhengzhou, China). electrode was dried in air, then dipped into 0.2 moL·L −1 Chloroauric acid (HAuCl ·4H O), chloroplatinic acid H SO and the solution containing 0.5 mmoL·L HAuCl 4 2 2 4 4 BSA Journal of Analytical Methods in Chemistry 3 −1 and 0.5 mmoL·L H PtCl , and electrodeposited with 2 6 (b) chronoamperometric method at −0.2 V vs. Ag/AgCl for Au NPs Ab PANI PB 600 s at room temperature. After rinsing, the AuPt NFs/L- Cys/GCE-modifed electrode was immersed in primary Red-PB antibody (CEA-Ab ) solution at 4 C overnight, and Ab was L-Cys 1 1 (a) connected to the modifed electrode. Next, to remove excess AuPt NFs Ox-PB E (V) binding sites, the modifed electrode was immersed in 1 wt% BSA solution at room temperature for 40 min. Finally, the obtained electrode was washed three times with PBS and Ag Ab BSA GCE stored at 4 C. Scheme 1(a) displays the schematic of the designed immunosensor, and Scheme 1(b) shows the Scheme 1: (a) Te construction process of electrochemical preparation procedure of Ab -Au NPs/PB@PANI immunosensor. (b) Assembly diagram of immunoprobes. bioconjugates. 3.2. Characterization of the Constructed Immunosensor. 2.7. Electrochemical Detection. Te prepared immuno- CV and EIS measurements were utilized to monitor the sensors were incubated in the solutions of CEA antigen stepwise assembly process of the immunosensors [30]. As with diferent concentrations for 40 min and then con- shown in Figure 2(a), the peak current of L-Cys flm tinued incubated in Ab -Au NPs/PB@PANI solution at modifed GCE electrode by one-step electro- 37 C for the same time. Subsequently, diferential pulse polymerization was decreased clearly (curve b) com- voltammetry (DPV) and electrochemical impedance pared with the bare GCE electrode (curve a), indicating spectroscopy (EIS) were performed in PBS (pH 6.5) and L-Cys flm hindered electron transfer. In addition, −1 3-/4- 5 mmoL·L [Fe (CN) ] , respectively. modifcation of AuPt NFs onto the flm of L-Cys caused an increase in peak current benefting from excellent conductivity of the AuPt NFs (curve c). After Ab being 3. Results and Discussion 1 adsorbed on the modifed electrode (curve d), blocking 3.1. Characterization of the Nanomaterials. SEM images of with BSA (curve e), and modifying with Ag (curve f), the PB, the PB@PANI, and the Au NPs/PB@PANI are a further decrease in the peak currents was seen, due to displayed in Figures 1(a)–1(c). Te as-prepared PB the efect of biomacromolecules hindering electron nanocomposites were made up of cubes with side lengths transport. However, the peak currents increased sig- in the range of 300–320 nm (Figure 1(a)), which had nifcantly after the modifed electrodes were combined a rough surface to provide abundant nucleation sites for with Ab -Au NPs/PB@PANI bioconjugates (curve g), the uniform growth of PANI shell [28]. It was clearly seen indicating that Ab -Au NPs/PB@PANI bioconjugates that the nanocubes of PB were completely covered by the can greatly enhance electron transfer. Moreover, EIS PANI shell, and the thickness of the PANI was about results of each step of electrode modifcation were in 10 nm after the reaction time of 18 h (Figure 1(b)). When accordance with those of CV. Au NPs with diameters of about 15 nm were attached to the surface of PB@PANI, we clearly observed that Au NPs with uniform globular morphology were absorbed 3.3. Optimization of Experimental Conditions. To improve on the surface of PB@PANI core-shell nanocubes the sensitivity and analysis efciency of the proposed (Figure 1(c)), which can also immobilize CEA-Ab immunosensor, the experimental conditions were opti- through Au-N bonds. mized. As shown in Figure 3(a), when incubation time was Figures 1(d) and 1(e) show the morphology of the 20 min–80 min, the DPV response increased gradually and electrode. As exhibited in Figure 1(d), the surface of the then reached a plateau after 40 min because the combination bare GCE was not smooth. By electrodeposition, the L- of the antigen with Ab reached equilibrium in approxi- Cys flm was deposited on the surface of GCE mately 40 min. (Figure 1(e)), which maybe infuence on the size and Te pH value of bufer solution has a signifcant infu- crystalline structure of AuPt bimetallic nanofowers ence on the stability and activity of electrochemical bio- (AuPt NFs) according to previous reports [29]. At the sensors. As can be seen from Figure 3(b), with the change of same time, the presence of L-cysteine helped to bind the pH, the signal of DPV response also changed. In the range of AuPt NFs on the surface of GCE. Quasi-spherical fower- pH 5.0–8.0, DPV value frst increased and then slightly like AuPt NFs with diameters of about 50 nm were ob- decreased. Au NPs/PB@PANI nanoprobes possessed opti- served after the electrodeposition on L-Cys/GCE mal activity and stability in approximately neutral or acid (Figure 1(f)). environment due to an efective coating layer of conducting Meanwhile, Au, Pt, C, N, and S elements were found in PANI. Terefore, we chose PBS solution with a pH of 6.5 as the energy dispersive spectroscopy (EDS) of AuPt NFs/L- the appropriate electrolyte. Cys/GCE (Figure S1A). EDS can further prove that Au NPs/ In the meantime, the deposition time of AuPt NFs had PB@PANI nanocomposites were composed of Au, Pt, K, N, a signifcant efect on DPV response as well. As displayed in C, O, and Fe elements (as shown in Figure S1B). Figure 3(c), as the deposition time of AuPt NFs increased I (μA) 4 Journal of Analytical Methods in Chemistry 100 nm 100 nm 100 nm (a) (b) (c) 500 nm 1 μm 2 μm (d) (e) (f) Figure 1: (a) SEM images of PB, (b) PB@PANI, (c) Au NPs-PB@PANI, (d) GCE, (e) L-Cys/GCE, and (f) AuPt NFc/L-Cys/GCE. A D F 600 -15 -30 -45 0 -0.6 -0.4 -0.2 0.0 0.2 0.4 0 500 1000 1500 2000 Potential (V) Z´ (Ω) (a) (b) Figure 2: (a) CVs and (b) EIS of (A) bare GCE, (B) L-Cys/GCE, (C) AuPt NFs/L-Cys/GCE, (D) Ab1/AuPt NFs/L-Cys/GCE, (E) BSA/Ab1/ 3-/4- AuPt NFs/L-Cys/GCE, (F) Ag/BSA/Ab1/AuPt NFs/L-Cys/GCE, (G) Bioconjugates/Ag/BSA/Ab1/AuPt NFs/L-Cys/GCE in [Fe(CN) ] . from 200 to 1000 s, the current signal increased gradually 3.4. Quantitative Detection of CEA. Under optimal experi- and then reached a plateau at 600 s. Terefore, the deposition mental conditions, CEA was quantifed by using the de- time of AuPt NFs was selected as 600 s for the preparation of veloped immunosensor. As shown in Figure 4, the DPV the immunosensor. signal increased gradually as the CEA concentration in- Similar to reported biosensor, the performance of creased. Tere was a good linear relationship between the immunosensor was highly afected by the concentration of current intensity and the logarithm of CEA concentration in −1 Au NPs/PB@PANI. As shown in Figure 3(d), with the in- the range of 0.001–100 ng·mL . Te regression equation was −1 −1 2 −1 creasing concentration from 0.5 mg·mL to 2.0 mg·mL , I (μA) � 4.6652 + 0.7570x (R � 0.9981) with 0.35 pg·mL the current response increased rapidly, but when the con- detection limit. In comparison with other nanoprobe-based −1 centration was higher than 2.0 mg·mL , the current re- immunosensors [31–37], the detection limit of the prepared −1 sponse reached a plateau. Terefore, 2.0 mg·mL became immunosensor was lower (Table 1). Te reasons for the ex- the optimum concentration for this study. cellent analytical performance of the designed immunosensor Current (μA) -Z˝ (Ω) Journal of Analytical Methods in Chemistry 5 5.6 5.6 5.2 5.2 4.8 4.8 4.4 4.4 4.0 4.0 3.6 3.6 5.0 5.5 6.0 6.5 7.0 7.5 8.0 20 30 40 50 60 70 80 pH Time (min) (a) (b) 5.5 5.4 5.0 5.1 4.5 4.8 4.0 4.5 3.5 4.2 0 1 2 34 200 400 600 800 1000 -1 Conc. (mg mL ) Time (s) (c) (d) Figure 3: Infuence of (a) incubation time, (b) pH, (c) deposition time of AuPt NFs, and (d) the concentration Au NPs/PB@PANI on the −1 peak currents to C � 10.0 ng·mL . Error bars represent standard deviations from fve repeated measurements. CEA 7 7 5 5 4 4 y=4.6652+0.7570x 3 3 R =0.9981 2 2 -0.4 -0.2 0.0 0.2 0.4 -3 -2 -1 0 1 2 Potential (V) lgC (ng/mL) (a) (b) Figure 4: (a) DPV responses of the as-prepared immunosensor after incubation with CEA concentrations (From a–g: 0.001, 0.01, 0.1, 1.0, −1 10.0, 50.0, and 100 ng·mL ) in 0.1 M PBS (pH 6.5). (b) Calibration curves of the immunosensor. Error bars represent standard deviations from fve repeated measurements. may be summarized as follows: frst, PANI, as the conductive conduction efciency of signal molecules, and can be used as shell of PB@PANI core-shell structure, maintained the a signal amplifcation system to improve the sensitivity of de- structural stability of PB and efectively improved the electron tection. Finally, AuPt NFs with multifunctionality were used as transfer from PANI to PB due to the intimate adhesion. an ideal sensing platform, not only can capture more Ab and Second, a large amount of Au NPs distributed on the PB@ improve conductivity but also constituted a dual signal am- PANI surface provided a large specifc surface area for the plifcation system together with Au NPs/PB@PANI nanoprobes. binding of antibodies, which enhanced the immobilization of CEA-Ab . In addition, the Au NPs/PB@PANI nanoprobes 3.5. Specifcity, Stability, and Reproducibility of the with the unique structure and synergetic contributions Immunosensor. In order to investigate the specifcity of the −1 exhibited good conductivity and high stability, increased the constructed immunosensor targeting CEA, 100.0ng·mL of Current (μA) Current (μA) Current (μA) Current (μA) Current (μA) Current (μA) 6 Journal of Analytical Methods in Chemistry Table 1: Comparison of analytical performance of CEA immunosensors with diferent nanoprobes. −1 −1 Nanoprobes Linear range (ng·mL ) Detection limit (ng·mL ) Reference −4 Ag-BSA-Pt NPs 0.005–100.00 7.60 ×10 [31] −3 CPS@PANI@Au 0.006–12.00 1.56 ×10 [32] −4 PT-Au 0.3–30.00 1.47 ×10 [33] −3 3D-rGO-MWCNTs/Ag-Au Ps 0.001–80.00 3.00 ×10 [34] −3 AuNPs-PAN@CNTs 0.002–80.00 8.00 ×10 [35] −3 CNSs@Au NPs 0.002–80.00 3.00 ×10 [36] −4 Ce-MoF@HA/Ag-HRP 0.001–80.00 2.00 ×10 [37] −4 AuNPs-PB@PANI 0.001–100.00 3.50 ×10 Tis work Figure 5: Te specifcity of the CEA immunosensor. Error bars � SD (n � 5). 6 6 5 5 4 4 3 3 2 2 1 1 0 0 5 10 15 20 25 30 14 2 35 6 Time (days) Samples (a) (b) Figure 6: (a) Te stability and (b) reproducibility of the CEA immunosensor. Error bars � SD (n � 5). four interferences including alpha fetoprotein (AFP), ascorbic protection of PB in the PANI coating made the developed acid (AA), glucose (Glu), and BSA was added to CEA immunosensor have high stability. −1 (1.0ng·mL ) solution, respectively. As displayed in Figure 5, To further verify the reproducibility of the immuno- −1 these interferences made a neglectful infuence to the target, sensor, six immunosensors were incubated in 10.0 ng·mL suggesting the proposed biosensor presented high specifcity for of CEA for DPV determination. As shown in Figure 6(b), the the specifc recognition of CEA. RSD was 3.1% after measurement, demonstrating the re- Te stability experiments of the immunosensor were producibility of the immunosensor was acceptable. carried out by storing several immunosensors at 4 C for 30 days, which were tested for CEA every 5 days 3.6. Determination of Real Serum Sample. To certify ana- (Figure 6(a)). After 30 days, the DPV value retained lytical reliability and application potential of the developed 88.6% of its initial response. Te efective fxation of Ab immunosensor, ten clinical serum specimens containing on the Au NPs/PB@PANI nanoprobe and the efcient CEA were analyzed. As displayed in Table 2, the relative Current (μA) Current (μA) CEA Blank AA AFP Arg Current (μA) BSA CEA+AA CEA+AFP CEA+Arg CEA+BSA Journal of Analytical Methods in Chemistry 7 Table 2: Comparison with ELISA for CEA. −1 a −1 Serum sample no. Tis method (ng·mL ) ELISA (ng·mL ) RSD (%) 1 0.06± 0.01 0.058± 0.02 +3.45 2 0.54± 0.12 0.57± 0.08 −5.26 3 1.88± 0.14 1.85± 0.05 +1.62 4 5.00± 0.22 5.14± 0.13 −2.72 5 13.26± 0.42 13.76± 0.57 −3.63 6 18.78± 1.23 17.56± 1.65 +6.94 7 23.55± 1.09 24.06± 0.79 −2.12 8 37.67± 0.78 36.87± 1.52 +2.17 9 50.33± 1.77 52.26± 0.67 −3.69 10 78.38± 1.39 75.97± 1.83 +3.17 Mean value± SD of fve measurements. error between the two methods was in the range from (202010368027), the National Natural Science Foundation of −5.26% to 6.94%, and these data revealed that the developed China (21645006), and 2022 Anhui Provincial College immunosensor had good practicability for clinical serum Students Innovation and Entrepreneurship Training Pro- analysis. gram, College of Pharmacy 013. 4. Conclusions Supplementary Materials In conclusion, an electrochemical immunosensor based on gold For the characterization of the signal probe material, we nanoparticles functionalized PB@PANI core-shell nanocubes as added the energy dispersive spectroscopy (EDS) as sup- a signal amplifcation strategy was fabricated for CEA detection. plementary material, provided with the manuscript. Figure Te high sensitivity of the immunosensor was attributed to the S1. EDX spectra of (A) AuPt NFs/L-Cys/GCE and (B) Au reasons as follows. First, the abundant N-species in PANI can be NPs/PB@PANI. (Supplementary Materials) coupled with PB to efectively protect the structural degradation of PB in basic media. Second, Au NPs/PB@PANI nanocubes References containing abundant electron mediators provided a large signal of DPV. Finally, AuPt NFs were dotted on the L-Cys/GCE [1] D. L. Zheng, J. Y. Yang, Z. Y. 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Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted PB@PANI Core-Shell Nanocubes as a Signal Probe

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Hindawi Publishing Corporation
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10.1155/2023/7009624
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Abstract

Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 7009624, 9 pages https://doi.org/10.1155/2023/7009624 Research Article Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted PB@PANI Core-Shell Nanocubes as a Signal Probe 1,2 2 1,2 1 Dexiang Feng , Lingzhi Chen , Ke Zhang , Shuangshuang Zhu , 2 2 2 1,2 2 Meichen Ying , Peng Jiang , Menglan Fu , Yan Wei , and Lihua Li Department of Chemistry, Wannan Medical College, Wuhu 241002, China Institute of Synthesis and Application of Medical Materials, Department of Pharmacy, Wannan Medical College, Wuhu 241002, China Correspondence should be addressed to Yan Wei; yanwei@wnmc.edu.cn and Lihua Li; llh05530226@126.com Received 10 September 2022; Revised 1 November 2022; Accepted 23 March 2023; Published 7 April 2023 Academic Editor: Jose Vicente Ros Lis Copyright © 2023 Dexiang Feng et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Herein, a method was developed for the sensitive monitoring of carcinoembryonic antigen (CEA) by gold nanoparticles dotted prussian blue@polyaniline core-shell nanocubes (Au NPs/PB@PANI). First, a facile low-temperature method was used to prepare the uniform PB@PANI core-shell nanocubes with the assistance of PVP, where PB acted as the electron transfer mediator to provide electrochemical signals, and the PANI with excellent conductivity and desirable chemical stability not only played the role of a protective layer to prevent etching of PB in basic media but also efectively improved electron transfer. Importantly, to further enhance the electrical conductivity and biocompatibility of PB@PANI and to further enhance the electrochemical signal and capture a large amount of Ab , Au NPs were doped on the surface of PB@PANI to form Au NPs/PB@PANI nanocomposites. Subsequently, benefting from the advantages of core-shell structure nanoprobes and gold-platinum bimetallic nanofower (AuPt NF), a sandwich-type electrochemical immunosensor for CEA detection was constructed, which provided a wide linear detection −1 −1 −1 range from 1.0 pg·mL to 100.0 ng·mL and a low detection limit of 0.35 pg·mL via DPV (at 3σ). Moreover, it displayed a satisfactory result when the core-shell structure nanoprobe-based immunosensor was applied to determine CEA in real human serum samples. energy consumption and trace detection, and more suitable 1. Introduction for the detection of biomarkers in low concentration [5, 6]. As we all know, carcinoembryonic antigen (CEA) is a tumor More recently, lots of electrochemical immunosensors have marker for colon cancer, breast cancer, ovarian cancer, and been constructed for quantitation of CEA. For example, other cancers, which can provide reliable information for the Wang et al. Jozghorbani et al. and Wang et al. designed three early diagnosis and treatment of tumor patients [1–3]. kinds of label-free CEA immunosensors with detection −1 Consequently, the highly sensitive determination of CEA is limits of 0.005, 0.05, and 0.0429 ng·mL , respectively [7–9]. a pressing need by virtue of accurate and efcient analytical Especially, a large number of reports have focused on the techniques. Immunosensors based on antibody-antigen sandwich-type electrochemical immunosensor for CEA interaction are one of the most widely used analytical detection [10–13], thanks to their distinct advantages such as techniques in the quantitative detection of biomarkers [4]. lower background noise, higher sensitivity, and importance, Among them, electrochemical immunosensors have and they have higher selectivity for analytes through two attracted much attention due to their characteristics of high specifc reactions, which are superior to label-free coun- specifcity, good sensitivity, short time consumption, low terparts [14–16]. 2 Journal of Analytical Methods in Chemistry Prussian blue (PB) with a face-centered cubic lattice (H PtCl 6H O), bovine serum albumin (BSA), polyvinyl 2 6 2 structure is popular as a kind of electrochemical redox- pyrrolidone (PVP), L-cysteine (L-Cys), aniline, ammonium active species in electrochemical biosensors because of its persulfate, and so on were purchased from Aladdin Reagent high electrochemical/electrocatalytic properties and low Co., Ltd. (Shanghai, China). redox potential [17, 18]. Unfortunately, the poor stability and low conductivity of PB limit its further applications 2.2. Devices. Electrochemical measurements were made at in biosensors [19]. To minimize these problems, some the CHI 660E electrochemical workstation (Shanghai Co., conducting materials can be introduced to meet the Ltd., China). Te morphology of nanomaterials was ob- requirements mentioned above. Polyaniline (PANI) as tained by a scanning electron microscopy (SEM, JSM-7100F, one of the most desired materials with good chemical Japan) and a transmission electron microscopy (TEM, JEM- stability, low cost, and good electrical conductivity can 6700F, Japan). not only provide a conductive substrate but also can form an efective coating layer on “the core” such as carbon nanotubes (CNTs) or PB [20, 21]. Te combination of PB 2.3. Synthesis of PB Nanocubes. PB nanocubes were syn- and PANI (PB@PANI) can play a signifcant synergistic thesized according to a previous work despite a minor −1 efect and improve the electrocatalytic performance, modifcation [27]. 8.0 mmoL·L Na Fe(CN) .8H O, 4.0 mL 4 6 2 conductivity, and stability [21]. Furthermore, to further hydrochloric acid (37%), and 2.0 g polyvinyl pyrrolidone enhance the conductivity and biocompatibility of PB@ (M ∼40000) were added into deionized water (400 mL) and PANI, gold nanoparticles (Au NPs) were introduced over ° stirred for 30 min, and then were refuxed at 60 C for 6 h. the surface of PB@PANI by Au-N bonds between Au NPs After that, the blue products were washed with deionized and -NH from PANI to form Au NPs/PB@PANI ° water and dried in vacuum at 100 C for 24 h. nanocomposites [15]. To the best of our knowledge, the preparation and application of Au NPs/PB@PANI 2.4. Preparation of PB@PANI Core-Shell Nanocubes. nanocomposites in electrochemical immunosensors have PB@PANI core-shell nanocubes were synthesized with not been reported. Hence, the PB@PANI loaded with Au reference to previous work with slight variations [21]. NPs (Au NPs/PB@PANI) would be a promising Briefy, 0.6 g·PB, 0.5 g polyvinyl pyrrolidone (M ∼40000) nanoprobe. w and 100 μL aniline monomer were dissolved in 100 mL For sandwich-type electrochemical immunosensors, to −1 hydrochloric acid (1 moL·L ) with sonication for 1 h. Ten, achieve signal amplifcation and high sensitivity, it is a more −1 ammonium persulfate solution (1.6 mmoL·L ) was added important key to efectively immobilize the primary anti- dropwise into the above solution and stirred for 18 h in an body (Ab ) [22, 23]. Gold and platinum nanoparticles are the ice bath. Finally, the PANI-coated PB (core-shell structure most promising bimetallic materials for various applications PB@PANI) was collected by centrifugation and washed with due to their superior biocompatibility, higher specifc sur- deionized water and ethanol for several times. face area, and superior electrocatalytic properties towards the reduction of H O [24–26]. Tey can be decorated onto 2 2 the surface of the L-cysteine (L-Cys)-modifed electrode to 2.5. Preparation of Ab -Au NPs/PB@PANI Bioconjugates. provide an available microenvironment for loading amounts 15 mg PB@PANI nanocubes were added into the concen- of Ab , thus greatly improving the sensitivity of the −1 trated Au NPs colloidal solution (5.0 mL, 1.0 mg·mL ) and immunosensor. reacted 30 min under shaking. Au NPs were assembled on Tis work was developed by electrodeposition of AuPt the surface of PB@PANI to form the Au NPs/PB@PANI bimetallic nanofower (AuPt NFs) on the GCE modifed with nanocomposites by the chemical bond of Au-NH [15]. L-Cys as a sensing platform and Au NPs/PB@PANI as Followed by centrifugation and washing twice with deion- a novel label prepared by a facile low-temperature method. ized water, the obtained precipitate was redispersed into PBS Te existence of AuPt NFs not only fxed the primary an- solution (pH 7.0). Subsequently, 500 μL CEA-Ab tibody but also accelerated the electron transfer. Further- −1 (2.0 mg·mL ) was added into the Au NPs/PB@PANI sus- more, the as-developed Au NPs/PB@PANI nanoprobes were pension (2 mL) and stirred for 12 h at 4 C. To get rid of used as a tracer for the generation and amplifcation of nonspecifc adsorption, 100 μL of 1% BSA was mixed with electrochemical signals and easily captured second anti- the obtained conjugates for 6 h at 4 C. Finally, the bio- bodies (Ab ) via Au-N bond. As expected, a sandwich-type conjugates were centrifuged and redispersed in PBS (pH 7.0, electrochemical immunosensor capable of achieving large −1 10 mmoL·L ), and then stored at 4 C. signal amplifcation was developed by connecting Ab -AuPt NFs/L-Cys with Ab -Au NPs/PB@PANI. 2.6. Fabrication of the Immunosensor. For polymer nano- composite flm deposition, polished glass carbon electrode 2. Materials and Methods −1 (GCE) was frst immersed in 0.5 mmoL·L cysteine con- 2.1. Materials and Chemicals. Carcinoembryonic antigen taining PBS (pH 7.0) and was scanned in the potential range (CEA) and its antibodies, α-fetoprotein (AFP), were pur- of −0.0 to 1.7 V at 50 mV/s for 3 cycles. Te obtained −1 chased from Biocell Biotech. Co., Ltd. (Zhengzhou, China). electrode was dried in air, then dipped into 0.2 moL·L −1 Chloroauric acid (HAuCl ·4H O), chloroplatinic acid H SO and the solution containing 0.5 mmoL·L HAuCl 4 2 2 4 4 BSA Journal of Analytical Methods in Chemistry 3 −1 and 0.5 mmoL·L H PtCl , and electrodeposited with 2 6 (b) chronoamperometric method at −0.2 V vs. Ag/AgCl for Au NPs Ab PANI PB 600 s at room temperature. After rinsing, the AuPt NFs/L- Cys/GCE-modifed electrode was immersed in primary Red-PB antibody (CEA-Ab ) solution at 4 C overnight, and Ab was L-Cys 1 1 (a) connected to the modifed electrode. Next, to remove excess AuPt NFs Ox-PB E (V) binding sites, the modifed electrode was immersed in 1 wt% BSA solution at room temperature for 40 min. Finally, the obtained electrode was washed three times with PBS and Ag Ab BSA GCE stored at 4 C. Scheme 1(a) displays the schematic of the designed immunosensor, and Scheme 1(b) shows the Scheme 1: (a) Te construction process of electrochemical preparation procedure of Ab -Au NPs/PB@PANI immunosensor. (b) Assembly diagram of immunoprobes. bioconjugates. 3.2. Characterization of the Constructed Immunosensor. 2.7. Electrochemical Detection. Te prepared immuno- CV and EIS measurements were utilized to monitor the sensors were incubated in the solutions of CEA antigen stepwise assembly process of the immunosensors [30]. As with diferent concentrations for 40 min and then con- shown in Figure 2(a), the peak current of L-Cys flm tinued incubated in Ab -Au NPs/PB@PANI solution at modifed GCE electrode by one-step electro- 37 C for the same time. Subsequently, diferential pulse polymerization was decreased clearly (curve b) com- voltammetry (DPV) and electrochemical impedance pared with the bare GCE electrode (curve a), indicating spectroscopy (EIS) were performed in PBS (pH 6.5) and L-Cys flm hindered electron transfer. In addition, −1 3-/4- 5 mmoL·L [Fe (CN) ] , respectively. modifcation of AuPt NFs onto the flm of L-Cys caused an increase in peak current benefting from excellent conductivity of the AuPt NFs (curve c). After Ab being 3. Results and Discussion 1 adsorbed on the modifed electrode (curve d), blocking 3.1. Characterization of the Nanomaterials. SEM images of with BSA (curve e), and modifying with Ag (curve f), the PB, the PB@PANI, and the Au NPs/PB@PANI are a further decrease in the peak currents was seen, due to displayed in Figures 1(a)–1(c). Te as-prepared PB the efect of biomacromolecules hindering electron nanocomposites were made up of cubes with side lengths transport. However, the peak currents increased sig- in the range of 300–320 nm (Figure 1(a)), which had nifcantly after the modifed electrodes were combined a rough surface to provide abundant nucleation sites for with Ab -Au NPs/PB@PANI bioconjugates (curve g), the uniform growth of PANI shell [28]. It was clearly seen indicating that Ab -Au NPs/PB@PANI bioconjugates that the nanocubes of PB were completely covered by the can greatly enhance electron transfer. Moreover, EIS PANI shell, and the thickness of the PANI was about results of each step of electrode modifcation were in 10 nm after the reaction time of 18 h (Figure 1(b)). When accordance with those of CV. Au NPs with diameters of about 15 nm were attached to the surface of PB@PANI, we clearly observed that Au NPs with uniform globular morphology were absorbed 3.3. Optimization of Experimental Conditions. To improve on the surface of PB@PANI core-shell nanocubes the sensitivity and analysis efciency of the proposed (Figure 1(c)), which can also immobilize CEA-Ab immunosensor, the experimental conditions were opti- through Au-N bonds. mized. As shown in Figure 3(a), when incubation time was Figures 1(d) and 1(e) show the morphology of the 20 min–80 min, the DPV response increased gradually and electrode. As exhibited in Figure 1(d), the surface of the then reached a plateau after 40 min because the combination bare GCE was not smooth. By electrodeposition, the L- of the antigen with Ab reached equilibrium in approxi- Cys flm was deposited on the surface of GCE mately 40 min. (Figure 1(e)), which maybe infuence on the size and Te pH value of bufer solution has a signifcant infu- crystalline structure of AuPt bimetallic nanofowers ence on the stability and activity of electrochemical bio- (AuPt NFs) according to previous reports [29]. At the sensors. As can be seen from Figure 3(b), with the change of same time, the presence of L-cysteine helped to bind the pH, the signal of DPV response also changed. In the range of AuPt NFs on the surface of GCE. Quasi-spherical fower- pH 5.0–8.0, DPV value frst increased and then slightly like AuPt NFs with diameters of about 50 nm were ob- decreased. Au NPs/PB@PANI nanoprobes possessed opti- served after the electrodeposition on L-Cys/GCE mal activity and stability in approximately neutral or acid (Figure 1(f)). environment due to an efective coating layer of conducting Meanwhile, Au, Pt, C, N, and S elements were found in PANI. Terefore, we chose PBS solution with a pH of 6.5 as the energy dispersive spectroscopy (EDS) of AuPt NFs/L- the appropriate electrolyte. Cys/GCE (Figure S1A). EDS can further prove that Au NPs/ In the meantime, the deposition time of AuPt NFs had PB@PANI nanocomposites were composed of Au, Pt, K, N, a signifcant efect on DPV response as well. As displayed in C, O, and Fe elements (as shown in Figure S1B). Figure 3(c), as the deposition time of AuPt NFs increased I (μA) 4 Journal of Analytical Methods in Chemistry 100 nm 100 nm 100 nm (a) (b) (c) 500 nm 1 μm 2 μm (d) (e) (f) Figure 1: (a) SEM images of PB, (b) PB@PANI, (c) Au NPs-PB@PANI, (d) GCE, (e) L-Cys/GCE, and (f) AuPt NFc/L-Cys/GCE. A D F 600 -15 -30 -45 0 -0.6 -0.4 -0.2 0.0 0.2 0.4 0 500 1000 1500 2000 Potential (V) Z´ (Ω) (a) (b) Figure 2: (a) CVs and (b) EIS of (A) bare GCE, (B) L-Cys/GCE, (C) AuPt NFs/L-Cys/GCE, (D) Ab1/AuPt NFs/L-Cys/GCE, (E) BSA/Ab1/ 3-/4- AuPt NFs/L-Cys/GCE, (F) Ag/BSA/Ab1/AuPt NFs/L-Cys/GCE, (G) Bioconjugates/Ag/BSA/Ab1/AuPt NFs/L-Cys/GCE in [Fe(CN) ] . from 200 to 1000 s, the current signal increased gradually 3.4. Quantitative Detection of CEA. Under optimal experi- and then reached a plateau at 600 s. Terefore, the deposition mental conditions, CEA was quantifed by using the de- time of AuPt NFs was selected as 600 s for the preparation of veloped immunosensor. As shown in Figure 4, the DPV the immunosensor. signal increased gradually as the CEA concentration in- Similar to reported biosensor, the performance of creased. Tere was a good linear relationship between the immunosensor was highly afected by the concentration of current intensity and the logarithm of CEA concentration in −1 Au NPs/PB@PANI. As shown in Figure 3(d), with the in- the range of 0.001–100 ng·mL . Te regression equation was −1 −1 2 −1 creasing concentration from 0.5 mg·mL to 2.0 mg·mL , I (μA) � 4.6652 + 0.7570x (R � 0.9981) with 0.35 pg·mL the current response increased rapidly, but when the con- detection limit. In comparison with other nanoprobe-based −1 centration was higher than 2.0 mg·mL , the current re- immunosensors [31–37], the detection limit of the prepared −1 sponse reached a plateau. Terefore, 2.0 mg·mL became immunosensor was lower (Table 1). Te reasons for the ex- the optimum concentration for this study. cellent analytical performance of the designed immunosensor Current (μA) -Z˝ (Ω) Journal of Analytical Methods in Chemistry 5 5.6 5.6 5.2 5.2 4.8 4.8 4.4 4.4 4.0 4.0 3.6 3.6 5.0 5.5 6.0 6.5 7.0 7.5 8.0 20 30 40 50 60 70 80 pH Time (min) (a) (b) 5.5 5.4 5.0 5.1 4.5 4.8 4.0 4.5 3.5 4.2 0 1 2 34 200 400 600 800 1000 -1 Conc. (mg mL ) Time (s) (c) (d) Figure 3: Infuence of (a) incubation time, (b) pH, (c) deposition time of AuPt NFs, and (d) the concentration Au NPs/PB@PANI on the −1 peak currents to C � 10.0 ng·mL . Error bars represent standard deviations from fve repeated measurements. CEA 7 7 5 5 4 4 y=4.6652+0.7570x 3 3 R =0.9981 2 2 -0.4 -0.2 0.0 0.2 0.4 -3 -2 -1 0 1 2 Potential (V) lgC (ng/mL) (a) (b) Figure 4: (a) DPV responses of the as-prepared immunosensor after incubation with CEA concentrations (From a–g: 0.001, 0.01, 0.1, 1.0, −1 10.0, 50.0, and 100 ng·mL ) in 0.1 M PBS (pH 6.5). (b) Calibration curves of the immunosensor. Error bars represent standard deviations from fve repeated measurements. may be summarized as follows: frst, PANI, as the conductive conduction efciency of signal molecules, and can be used as shell of PB@PANI core-shell structure, maintained the a signal amplifcation system to improve the sensitivity of de- structural stability of PB and efectively improved the electron tection. Finally, AuPt NFs with multifunctionality were used as transfer from PANI to PB due to the intimate adhesion. an ideal sensing platform, not only can capture more Ab and Second, a large amount of Au NPs distributed on the PB@ improve conductivity but also constituted a dual signal am- PANI surface provided a large specifc surface area for the plifcation system together with Au NPs/PB@PANI nanoprobes. binding of antibodies, which enhanced the immobilization of CEA-Ab . In addition, the Au NPs/PB@PANI nanoprobes 3.5. Specifcity, Stability, and Reproducibility of the with the unique structure and synergetic contributions Immunosensor. In order to investigate the specifcity of the −1 exhibited good conductivity and high stability, increased the constructed immunosensor targeting CEA, 100.0ng·mL of Current (μA) Current (μA) Current (μA) Current (μA) Current (μA) Current (μA) 6 Journal of Analytical Methods in Chemistry Table 1: Comparison of analytical performance of CEA immunosensors with diferent nanoprobes. −1 −1 Nanoprobes Linear range (ng·mL ) Detection limit (ng·mL ) Reference −4 Ag-BSA-Pt NPs 0.005–100.00 7.60 ×10 [31] −3 CPS@PANI@Au 0.006–12.00 1.56 ×10 [32] −4 PT-Au 0.3–30.00 1.47 ×10 [33] −3 3D-rGO-MWCNTs/Ag-Au Ps 0.001–80.00 3.00 ×10 [34] −3 AuNPs-PAN@CNTs 0.002–80.00 8.00 ×10 [35] −3 CNSs@Au NPs 0.002–80.00 3.00 ×10 [36] −4 Ce-MoF@HA/Ag-HRP 0.001–80.00 2.00 ×10 [37] −4 AuNPs-PB@PANI 0.001–100.00 3.50 ×10 Tis work Figure 5: Te specifcity of the CEA immunosensor. Error bars � SD (n � 5). 6 6 5 5 4 4 3 3 2 2 1 1 0 0 5 10 15 20 25 30 14 2 35 6 Time (days) Samples (a) (b) Figure 6: (a) Te stability and (b) reproducibility of the CEA immunosensor. Error bars � SD (n � 5). four interferences including alpha fetoprotein (AFP), ascorbic protection of PB in the PANI coating made the developed acid (AA), glucose (Glu), and BSA was added to CEA immunosensor have high stability. −1 (1.0ng·mL ) solution, respectively. As displayed in Figure 5, To further verify the reproducibility of the immuno- −1 these interferences made a neglectful infuence to the target, sensor, six immunosensors were incubated in 10.0 ng·mL suggesting the proposed biosensor presented high specifcity for of CEA for DPV determination. As shown in Figure 6(b), the the specifc recognition of CEA. RSD was 3.1% after measurement, demonstrating the re- Te stability experiments of the immunosensor were producibility of the immunosensor was acceptable. carried out by storing several immunosensors at 4 C for 30 days, which were tested for CEA every 5 days 3.6. Determination of Real Serum Sample. To certify ana- (Figure 6(a)). After 30 days, the DPV value retained lytical reliability and application potential of the developed 88.6% of its initial response. Te efective fxation of Ab immunosensor, ten clinical serum specimens containing on the Au NPs/PB@PANI nanoprobe and the efcient CEA were analyzed. As displayed in Table 2, the relative Current (μA) Current (μA) CEA Blank AA AFP Arg Current (μA) BSA CEA+AA CEA+AFP CEA+Arg CEA+BSA Journal of Analytical Methods in Chemistry 7 Table 2: Comparison with ELISA for CEA. −1 a −1 Serum sample no. Tis method (ng·mL ) ELISA (ng·mL ) RSD (%) 1 0.06± 0.01 0.058± 0.02 +3.45 2 0.54± 0.12 0.57± 0.08 −5.26 3 1.88± 0.14 1.85± 0.05 +1.62 4 5.00± 0.22 5.14± 0.13 −2.72 5 13.26± 0.42 13.76± 0.57 −3.63 6 18.78± 1.23 17.56± 1.65 +6.94 7 23.55± 1.09 24.06± 0.79 −2.12 8 37.67± 0.78 36.87± 1.52 +2.17 9 50.33± 1.77 52.26± 0.67 −3.69 10 78.38± 1.39 75.97± 1.83 +3.17 Mean value± SD of fve measurements. error between the two methods was in the range from (202010368027), the National Natural Science Foundation of −5.26% to 6.94%, and these data revealed that the developed China (21645006), and 2022 Anhui Provincial College immunosensor had good practicability for clinical serum Students Innovation and Entrepreneurship Training Pro- analysis. gram, College of Pharmacy 013. 4. Conclusions Supplementary Materials In conclusion, an electrochemical immunosensor based on gold For the characterization of the signal probe material, we nanoparticles functionalized PB@PANI core-shell nanocubes as added the energy dispersive spectroscopy (EDS) as sup- a signal amplifcation strategy was fabricated for CEA detection. plementary material, provided with the manuscript. Figure Te high sensitivity of the immunosensor was attributed to the S1. EDX spectra of (A) AuPt NFs/L-Cys/GCE and (B) Au reasons as follows. First, the abundant N-species in PANI can be NPs/PB@PANI. (Supplementary Materials) coupled with PB to efectively protect the structural degradation of PB in basic media. Second, Au NPs/PB@PANI nanocubes References containing abundant electron mediators provided a large signal of DPV. Finally, AuPt NFs were dotted on the L-Cys/GCE [1] D. L. Zheng, J. Y. Yang, Z. Y. 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Published: Apr 7, 2023

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