A Colorimetric Immunoassay Based on g-C3N4@Fe3O4 Nanocomposite for Detection of Carcinoembryonic Antigen

Zhao, Yanling; Wen, Yanfei; Hu, Xing; Zhang, Bing

doi:https://doi.org/10.1155/2022/6966470

Journal of Analytical Methods in Chemistry

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Abstract Introduction Experimental Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 6966470 | https://doi.org/10.1155/2022/6966470

A Colorimetric Immunoassay Based on g-C₃N₄@Fe₃O₄ Nanocomposite for Detection of Carcinoembryonic Antigen

Yanling Zhao,¹Yanfei Wen,²Xing Hu,²and Bing Zhang²

Academic Editor: Larisa Lvova

Received01 Nov 2021

Revised06 Dec 2021

Accepted15 Jan 2022

Published28 Jan 2022

Abstract

We proposed a colorimetric immunosensor based on g-C₃N₄@Fe₃O₄ nanocomposite-mediated transformation strategy for sensitive detection of carcinoembryonic antigen (CEA). The g-C₃N₄@Fe₃O₄ nanocomposite was synthesized and characterized by the scanning electron microscope (SEM), energy dispersive X-ray spectra (EDX), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Fe³⁺ derived from g-C₃N₄@Fe₃O₄ nanocomposite could combine with sodium salicylate to form purple complex products. Based on this color development, the sandwich colorimetric immunoassay was built by utilizing g-C₃N₄@Fe₃O₄ nanocomposite as nanolabels on the microplate. With the increasement of CEA concentration, the purple color showed a gradient change. Under optimal conditions, the linearity range is 0.001–50 ng/mL with the detection limit of 0.35 pg/mL for CEA. More importantly, the colorimetric immunoassay has good selectivity, specificity, repeatability, and stability.

1. Introduction

The level of carcinoembryonic antigen (CEA) is related to many diseases such as breast cancer, lung cancer, colon cancer, rectal cancer, and so on [1]. CEA as broad-spectrum tumor marker is important for early diagnosis and treatment of cancer [2, 3], and the methods for CEA detection are of great concern [4–6]. In recent years, great efforts have been made to develop various immunoassay methods, such as photoelectrochemical [7], fluorescence [8], chemiluminescence [9], surface-enhanced Raman scattering [10], enzyme-linked immunosorbent assay (ELISA) [11], colorimetry [12], and so on. In these methods, colorimetric immunoassay has attracted much attention due to its simplicity and conveniency [13, 14].

A key challenge for the development of colorimetric immunoassay is to transform the detection event into color change. Various strategies have been developed for this purpose, such as aggregation-based colorimetric immunoassay, lateral-flow colorimetric immunoassay, enzyme-mediated colorimetric immunoassay, and light-enabled colorimetric immunoassay [15–18]. Aggregation-based colorimetric immunoassay and colorimetric lateral-flow immunoassay are user-friendly, fast, and cost-effective, which are usually employed for fast on-site analysis. Enzyme-mediated colorimetric immunoassay and light-enabled colorimetric immunoassay need the participation of bioenzyme and laser, which is expensive and complex to operate. Hence, it is meaningful to develop the novel colorimetric method to enhance practicability. Various chemical color reactions have been developed and utilized in the field of pharmaceutical analysis and environmental analysis [19–21]. The chromogenic reaction between the phenolic hydroxyl group and Fe³⁺ is often used to identify the presence of phenols, which forms a purple complex [22].

In order to improve the sensitivity of chromogenic reaction and its application in immunoassay, nanomaterials with a specific interfacial effect and small-size effect have been employed [23–25]. Carbon nitrogen (C₃N₄), as a kind of metal-free material, has attracted much attention in biosensing filed due to its easy preparation, good biocompatibility, and high specific surface area. A series of C₃N₄-based composite nanomaterials including WO₃/g-C₃N₄/MnO₂, Ni-doped SnO₂/g-C₃N₄, g-C₃N₄-COOH/ZnSe, and so on are synthesized and used in analytical applications [26–29]. Zhang et al. synthesized graphitic carbon nitride nanosheets-supported palladium nanosheets composite (Pd/g-C₃N₄) with oxidase-like activity for acetylcholinesterase (AChE) activity detection [30]. Ding et al. realized tumor marker detection using ternary GO-C₃N₄-AgBr heterojunction nanophotocatalyst [31].

In this study, g-C₃N₄@Fe₃O₄ nanocomposites were prepared as nanolabels to build colorimetric immunoassay for CEA detection. Under acidic conditions, g-C₃N₄@Fe₃O₄ generates a mass of Fe³⁺, which reacted with sodium salicylate and formed purple complex. Based on this colorimetric phenomenon, CEA concentration in the serum is analyzed by semiquantitative analysis by naked eye and quantitatively analyzed by UV-vis absorption.

2. Experimental

2.1. Materials and Reagents

Carcinoembryonic antigen (CEA), monoclonal CEA antibody (Ab₁, 0.1 mg/mL), and polyclonal CEA antibody (Ab₂, 0.1 mg/mL) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Sodium salicylate (C₇H₅NaO₃), N-hydroxysulfosuccinimide sodium salt (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), melamine, anhydrous ferric chloride (FeCl₃), ethylene glycol, nitric acid (HNO₃), hydrochloric acid, sodium acetate, Tween-20, and bovine serum albumin (BSA) were purchased from Aladdin Reagent Company (Shanghai, China). The phosphate buffer solution with various values was prepared with 0.1 M disodium phosphate.

2.2. Apparatus

Scanning electron microscopy (SEM) was carried out on a JSM-7100F scanning electron microscope (JEOL, Japan). X-ray powder diffraction (XRD) was tested on a Bruker D8 diffractometer (Germany) using Cu K radiation (40 kV, 40 mA) with a Ni filter. The ultraviolet-visible (UV-vis) absorption spectra were performed with a UV-3900 UV-vis spectrophotometer (Hitachi Co., Japan). Fourier transform infrared spectrum was recorded on FTIR Bruker alpha II (Germany).

2.3. Preparation of g-C₃N₄ Nanoparticles

Carboxyl-modified g-C₃N₄ nanosheets were prepared according to a previous report [28]. Briefly, 5 g of melamine was calcined at 550°C for 4 h in the muffle furnace. After cooling to room temperature, the yellow g-C₃N₄ product was ground into powder for further use. Then, 1 g of g-C₃N₄ powder was placed into a round-bottom flask with 100 mL of HNO₃ (5 M), and backwash was performed for 24 h at 125°C. Finally, the product of carboxylate g-C₃N₄ was obtained by cooling, centrifugation, and cleaning with deionized water to pH 7.0.

2.4. Preparation of g-C₃N₄@Fe₃O₄ Nanocomposites

g-C₃N₄@Fe₃O₄ nanocomposites were prepared according to a previous report with minor revision [29]. First, 0.40 g of above g-C₃N₄ nanosheets was added into 60 mL of ethylene glycol. Then, 0.65 g of FeCl₃ was added with ultrasound for 10 min. After 2.60 g of sodium acetate was added, the mixed solution was stirred vigorously for 20 min. Subsequently, the mixture was transferred to a Teflon-lined stainless-steel autoclave and reacted at 200°C for 8 h. After cooling to room temperature, the black product of g-C₃N₄@Fe₃O₄ was washed with ethanol several times and dried in vacuum at 60°C. To combine polyclone CEA antibody, g-C₃N₄@Fe₃O₄ nanocomposites were activated by EDC (0.0383 g) and NHS (0.0230 g) and shaked at room temperature for 30 min. Magnetic separation and washing were performed three times, and the conjugation of Ab₂-g-C₃N₄@Fe₃O₄ was collected and stored at 4°C for further use. For comparison, Fe₃O₄ nanoparticles and Ab₂-Fe₃O₄ conjugation were prepared according to the above steps.

2.5. Construction of Colorimetric Immunoassay

Scheme 1 displays the establishment process of colorimetric immunoassay for CEA detection. First, 100 μL of monoclonal CEA antibody was added into 96-microwell plate, and the plate was covered with plastic wrap and incubated at 4°C overnight. Then, surface solution was removed, and the wells were washed three times with phosphate buffer solution (0.01 M contains 0.05% Tween 20). Then, 100 μL of phosphate buffer solution (0.01 M contains 1% BSA) was added into each well and incubated for 45 min at 37°C to block the nonspecific adsorption sites. After cleaning steps, 100 μL of CEA standards with various concentrations were added into the wells and incubated at room temperature for 45 min. Next, 100 μL of Ab₂-g-C₃N₄@Fe₃O₄ was added and incubated at room temperature for 45 min, which designed as Ab₁/CEA/Ab₂-g-C₃N₄@Fe₃O₄. Subsequently, the colorimetric system was constructed by the reaction between sodium salicylate and Fe³⁺ [32]. Briefly, 100 μL of HCl solution (10 M) was added into each well of the above plates. The solution was transferred to a glass test tube containing sodium salicylate (6 mg/mL) to develop color changes. The absorption spectra in the range of 400–700 nm were monitored after reaction. For comparison, Ab₁/CEA/Ab₂-Fe₃O₄ also was designed according to the above steps.

3. Results and Discussion

3.1. Characterizations of g-C₃N₄@Fe₃O₄ Nanocomposites

The g-C₃N₄ nanosheets can load more Fe₃O₄ nanoparticles due to its large specific surface area. The morphology of g-C₃N₄@Fe₃O₄ nanocomposites was characterized by SEM. As shown in Figure 1(a), the Fe₃O₄ nanospheres with 200–300 nm is dispersed on the surface of g-C₃N₄ nanosheets. Energy dispersive X-ray (EDX) spectra state the elements of Fe, O, N, and C coexisting in g-C₃N₄@Fe₃O₄ nanocomposites (Figure 1(b)), which preliminarily indicated that the material is successfully synthesized. Furthermore, X-ray diffraction (XRD) is monitored to reveal crystalline structure of g-C₃N₄@Fe₃O₄ nanocomposite (Figure 1(c)). It can be clearly seen that the peaks at 2θ = 30.06°, 35.45°, 40.30°, 53.54°, and 57.16° were assigned to (220), (311), (400), (422), and (511) planes of Fe₃O₄, and the peak at 2θ = 27.49° was assigned to (002) plane of g-C₃N₄. Meanwhile, the function group of g-C₃N₄@Fe₃O₄ was proved by FTIR spectra. Figure 1(d) shows the FTIR spectra of Fe₃O₄, g-C₃N₄, and g-C₃N₄@Fe₃O₄ nanocomposite materials. Compared with the spectrum of a, b, and c, an apparent band at 3423 cm⁻¹ ascribed to O-H stretching vibrations, the band at 595 cm⁻¹ attributed to Fe-O vibrations of Fe₃O₄, and the band at 800–1600 cm⁻¹ attributed to characteristic peak of triazines. These characterizations complement each other and demonstrate the successful synthesis of g-C₃N₄@Fe₃O₄ nanomaterial.

(a)

(b)

(c)

(d)

3.2. Mechanism of the Colorimetric Assay

Ferric chloride reagent can react with the phenolic hydroxyl group for color development, which is a classical reaction and is often used to identify drugs, e.g., epinephrine. This reaction is exploited in our color-changing system. g-C₃N₄@Fe₃O₄ nanolabels can produce Fe³⁺ in acidic conditions, and Fe³⁺ reacts with sodium salicylate for generation of purple complex. This process can be represented by the following equation:

As shown in Figure 2(a), the sodium salicylate solution has no absorption peak (curve “a”), and the purple complex solution owns obvious absorption peaks at 530 nm (curve “b”). Whether or not Fe³⁺ has complexed with sodium salicylate, to verify this issue, EDTA is employed to chelate Fe³⁺. Obviously, the purple faded (the inset picture) and the absorption peak disappeared (curve “c”). The experimental results verified that the chromogenic mechanism is due to the influence of Fe³⁺.

(a)

(b)

To highlight the advantages of g-C₃N₄@Fe₃O₄, two types of colorimetric immunoassays were established based on g-C₃N₄@Fe₃O₄ and Fe₃O₄ nanolabels. At the same conditions, as shown in Figure 2(b), Ab₁/CEA/Ab₂-g-C₃N₄@Fe₃O₄ has a larger absorption value (curve “a”) compared with that of Ab₁/CEA/Ab₂-Fe₃O₄ (curve “b”) for 1 ng/mL CEA. This good performance is mainly attributed to the participation of g-C₃N₄, which has a large specific surface area and loads more Fe₃O₄ nanoparticles.

3.3. Performance Assessing of Colorimetric Immunoassay

For optimal performance of colorimetric immunoassay, experimental conditions related to bioactivity or biosensor sensitivity should be optimized. First, the construction conditions of immune structure including pH and incubation time were optimized. As shown in Figures 3(a) and 3(b), the absorbances have maximum at pH 7.0 and 45 min. Therefore, pH 7.0 and the incubation time of 45 min were used in immunoassay. Meanwhile, the concentration of HCl can resolve Fe₃O₄ for producing Fe³⁺, which is directly related to the chromogenic system. As shown in Figure 3(c), there was a maximum at concentration of 10 M, and then, the absorbance gradually declined with the increasing HCl concentration. Therefore, 10 M of HCl was chosen in the whole experiment. Under optimal conditions, CEA was tested by colorimetric immunoassay. As shown in Figure 3(d), the absorbance at 530 nm increased gradually with the increasing CEA concentration in the range of 0.001–50 ng/mL. The linear equation was y = 0.022 logC (ng/mL) + 0.120 (R² = 0.997, n = 27) with the detection limit (LOD) of 0.35 pg/mL (LOD = 3σ/s, where σ is the standard deviation of the blank and s is the slope of the calibration plot).

(a)

(b)

(c)

(d)

3.4. Selectivity, Repeatability, and Stability of the Colorimetric Immunoassay

In order to ensure the selectivity of colorimetric immunity, some interfering substances were selected for colorimetric detection such as ascorbic acid (AA), Ca²⁺, K⁺, and glucose (Glu) and prostate specific antigen (PSA). As shown in Figure 4(a), the absorbance value of the target CEA is the largest, while that of other interfering substances are smaller. In addition, there was no significant difference in the absorbance values of CEA in the presence of interfering substances (Figure 4(b)). Moreover, five groups of the colorimetric immunosensor were established to test reproducibility. As shown in Figure 4(c), the coefficient of variation (CVs) within the groups was 2.45%. Furthermore, the prepared colorimetric sensor was stored at 4°C for 4 weeks, and its absorbance was measured weekly. Compared with the original absorbance, the value retained 94% (Figure 4(d)). These results indicate that the developed colorimetric immunoassay has high selectivity, good repeatability, and stability.

(a)

(b)

(c)

(d)

3.5. Actual Serum Sample Analysis

In order to verify the practicability of the colorimetric system in the actual serum matrix, some clinical serum samples were obtained from the First Affiliated Hospital of Shanxi Medical University. Those samples were diluted by phosphate buffer solution (pH = 7.0) and detected by commercial ELISA. The results are given in Table 1, and the t test was calculated by the equation: (where x is the average value of three groups of experimental results; s is the pooled standard deviation of immunosensor and ELISA toward three groups of experimental results; n is the number of analysis (n = 3)). It can be seen that all t_exp values were smaller than t_crit (t_crit = 4.30). The result demonstrated that the colorimetric immunosensor is reliable for actual sample detection and own good clinical practical value in future.

4. Conclusion

In summary, a novel g-C₃N₄@Fe₃O₄ nanocomposite-mediated immunoassay was built based on colorimetric effects. Under acidic conditions, g-C₃N₄@Fe₃O₄ underwent dissociation to produce Fe³⁺, which combines with sodium salicylate to form purple complex. The complex products have a specific absorbance value in the UV-visible absorption spectrum. Thus, the quantitative detection of CEA could be realized by a UV-vis spectrophotometer. This strategy opens a new perspective for the application of colorimetric bioanalysis in the future. Future works should focus on the detection of more biomolecule in serum.

Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was supported by the Natural Science Foundation of Shanxi Province (3C392019035).

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Copyright © 2022 Yanling Zhao et al. This 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.

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Journal of Analytical Methods in Chemistry

A Colorimetric Immunoassay Based on g-C3N4@Fe3O4 Nanocomposite for Detection of Carcinoembryonic Antigen

Abstract

1. Introduction

2. Experimental

2.1. Materials and Reagents

2.2. Apparatus

2.3. Preparation of g-C3N4 Nanoparticles

2.4. Preparation of g-C3N4@Fe3O4 Nanocomposites

2.5. Construction of Colorimetric Immunoassay

3. Results and Discussion

3.1. Characterizations of g-C3N4@Fe3O4 Nanocomposites

3.2. Mechanism of the Colorimetric Assay

3.3. Performance Assessing of Colorimetric Immunoassay

3.4. Selectivity, Repeatability, and Stability of the Colorimetric Immunoassay

3.5. Actual Serum Sample Analysis

4. Conclusion

Data Availability

Conflicts of Interest

Acknowledgments

References

Copyright

A Colorimetric Immunoassay Based on g-C₃N₄@Fe₃O₄ Nanocomposite for Detection of Carcinoembryonic Antigen

2.3. Preparation of g-C₃N₄ Nanoparticles

2.4. Preparation of g-C₃N₄@Fe₃O₄ Nanocomposites

3.1. Characterizations of g-C₃N₄@Fe₃O₄ Nanocomposites