Introduction

The detection of tumor markers plays an indispensable role in early discovery and diagnosis for malignant tumor [1]. Immunoassays for tumor markers depend on various analytical techniques, such as ELISA [2], chemiluminescent [3], photoelectrochemical [4], electrochemical [5], fluorescence [6], colorimetric [7] and so on. Photoelectrochemical immunoassay as a powerful and promising technology has gained more and more attention, concerning with the interaction of light and electrochemical systems [8, 9]. The high sensitivity is obtained for photoelectrochemical immunoassay due to the mutual independence of excitation signal (light) and detection signal (electricity) [10].

The photoelectrochemical conversion requires a semiconductor to absorb light and generate electron-hole pairs, which play an important role for constructing photoelectrochemical platform. Photocatalyst semiconductor can accelerate the photoreaction and enhance the kinetics of electron transfer. Varieties of photocatalysts are developed including TiO2, CdS, C3N4, BiVO4, Ta3N5, Bi2S3, WS2 and so on [11,12,13,14,15,16]. Different kinds of photocatalysts are applied into the field of water splitting, hydrogen production, biosensing, solar cell and so on [17]. For wide band gap semiconductor, high energy source (particularly ultraviolet light) is usually required to excite them to generate photocurrent. To make full use of the sunlight, more research has focused on visible-light photocatalyst such as CdS, MoS2, C3N4, BiOCl and so on [18,19,20,21]. Among graphitic carbon nitride (g-C3N4) as metal-free photocatalyst has obtained more and more attention because of its narrow band gap and feasible preparation [22]. However, the photocatalytic performances of g-C3N4 are limited due to its rapid recombination rate of photo-induced carriers.

To overcome this limitation and improve the photocatalytic activity, different modification methods for g-C3N4 including doping of metal or nonmetal elemental, combination of narrow band or wide band semiconductor and introduction of co-catalysts have been exploited. And heterojunctions structure of C3N4 with other semiconductor such as NiTiO3, Bi4NbO8Cl, Cu3P, MnO2 and so on can accelerate the separation of photo-induced carriers due to mutual contaction of two semiconductors [23,24,25,26]. For example, Zhong et al. constructed CdSe quantum dots/g-C3N4, which showed remarkably intensive photocatalytic activity for visible-light-induced H2 evolution because of its excellent visible absorption and high charge separation efficiency [27]. Zhang et al. reported SnO2/SnS2/C3N4 nanocomposites exhibited intense PEC signal responses compared with each single component, which were utilized for building PEC immunoassay [28]. And sacrificial donor such as ethanol, H2O2, ascorbic acid, cysteine also can enhanced photocatalytic activity through oxidation of sacrificial donor. For example, Manwar et al. used ethanol as a sacrificial donor to enhance photocatalytic hydrogen evolution [29].

Herein, C3N4-BiOCl semiconductor was prepared to construct photoelectrochemical sensing by photovoltaic conversion. And we explored L-cysteine as sacrificial donor to assist photoelectrochemical reaction and enhanced the photocurrent response. Sandwich-type immunoassay was constructed on 96-well microtiter plate based on CuO nanoparticles as labels. The released Cu2+ ions from CuO nanoparticles can weaken the photocurrents of C3N4-BiOCl by chelate with cysteine and as the electron receptors. And the low-abundant CEA biomarker (as a model) was quantitatively detected based on this new photoelectrochemical immunoassay system.

Experimental

Chemicals

CEA antigen with various concentrations (D620003–0100), 96-well microtiter plate and polyclonal CEA antibody (D120003–0025, Ab2, 0.01 mg mL−1) were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China, http://www.sangon.com/). L-cysteine (L-Cys), bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), melamine, copper acetate (Cu(AC)2), bovine serum albumin (BSA) and polydiallyldimethylammonium chloride (PDDA) were purchased from Aladdin Reagent Company (Shanghai, China, http://www.aladdin-e.com/). The 0.2 M phosphate buffer at various pH values were prepared by mixing the stock solutions of 0.2 M NaH2PO4, 0.2 M Na2HPO4 and 0.2 M KCl with different proportion. All the other chemicals were of analytical reagents grade and used without further purification. Clinical patient’s serum samples were made available by first hospital of Shanxi medical university.

Synthesis of the C3N4-BiOCl semiconductor

Initially, carboxylated g-C3N4 was synthesized completely according to our previously reported method [30]. First, melamine powder (5.0 g) was calcined at 550 °C for 4 h in air. And the yellow g-C3N4 powder was collected by grinding at room temperature. To get the carboxyl group, the prepared g-C3N4 powder was refluxed in HNO3 (100 mL, 5 M) for 24 h at 125 °C. Cooling to room temperature (RT, 25 ± 0.5 °C), the product was adjusted to pH 7.0 by centrifugation and cleanse with double distilled water. The final product was dried at 60 °C in a vacuum drying oven for 12 h to obtain carboxylate g-C3N4.

After, 0.30 g of g-C3N4 powder dispersed into water (25 mL) by ultrasound for 30 min to form suspension. Subsequently, bismuth nitrate pentahydrate (0.49 g), polyvinyl pyrrolidone (0.40 g) and glycerol (25 mL) were added into above solution and the mixed solution stirred vigorously for 1 h at RT. Then, 5 mL of saturated NaCl solution was dropwise added into this mixture solution and stirred for other 1 h. Then the hydrothermal reaction was carried out at 160 °C for 6 h in high pressure reactor. Finally, the product was collected by centrifugation and wash with double distilled water, which designed as C3N4-BiOCl.

Synthesis of CuO nanoparticles and bioconjugates

50 mL of ethanol mixture containing Cu(Ac)2 (1 mM) and HAc (2 mM) was refluxed at 78 °C. Then, 5 mL of NaOH solution (4 mM) was dropwise added into the solution under vigorous stirring and reacted for 1 h. And CuO NPs formed by the alcohothermal method [31]. Then the suspension was centrifuged and washed three times with deionized water. The CuO powder was collected by drying at 60 °C. To get CuO-antibody conjugates, 1 mg of CuO NPs dispersed into 1 mL of phosphate buffer (0.2 M, pH 7.4) and sonicated for 10 min. Then, 500 μL of CEA polyclonal antibody was added into the CuO NPs solution. The mixture was transferred into refrigerator and incubated overnight at 4 °C. After centrifugation and washing, the CuO-Ab2 conjugates were collected and re-dispersed in 1 mL of phosphate buffer (pH 7.4) for further use. To block nonspecific bonding sites, the CuO-Ab2 dispersion incubated with BSA (0.5%) for 30 min at room temperature. Finally, the CuO-Ab2 conjugates were collected by centrifugation and washing.

Immunoassay protocol

The sandwich-type immunocomplex was constructed on 96-well microtiter plate. First, a 96-well plate was coated with 100 μL of CEA (10 μg mL−1) in phosphate buffer (pH = 7.4) and incubated overnight at 4 °C. After washing with phosphate buffer three times, 200 μL of BSA (10%) blocking phosphate buffer was added into each well and incubated for 2 h at RT. Finally, the 96-well plate stored at 4 °C for further use after washing three times with phosphate buffer. CEA antigen (50 μL) were added into the 96-well plate and incubated for 70 min at RT. Afterwards, the 96-well plate was washed three times with ultrapure water to remove dissociative CEA. Then, 50 μL of CuO-Ab2 bioconjugates was added into the microplate and incubated for another 70 min. After washing three times, 200 μL of HNO3 (0.1 M) was added into the 96-well plate to release Cu2+ from the CuO nanolabels. Subsequently, the Cu2+ solution was transferred into PEC test cell. The preparation process and principle of the immunoassay was described in Scheme 1a.

Scheme 1
scheme 1

a Schematic illustration of photoelectrochemical immunoassay; b Principle of photoelectric conversion of C3N4-BiOCl; and (c) Mechanism of Cu2+-quenched photocurrent of C3N4-BiOCl containing L-cys

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Photoelectrochemical measurements

The photoelectrochemical measurements were carried out in CHI660E (Chenhua, Shanghai, China) with xenon lamp. First, the bare ITO electrodes were thoroughly sonicated in ethanol/NaOH, acetone and distilled water for 10 min respectively, and dried at RT. Thereafter, 20 μL of C3N4-BiOCl (2 mg mL−1) was carefully cast to electrode surface after 1% of PDDA (20 μL) formed in the surface of ITO (designed as C3N4-BiOCl/PDDA/ITO). For PEC detection, the C3N4-BiOCl/PDDA/ITO was inserted into phosphate buffer containing L-Cys and copper ions. And photocurrents were obtained in the three-electrode system with working voltage of 0 V.

Results and discussion

Characterizations of C3N4-BiOCl and CuO

Transmission electron microscope (TEM), X-ray powder diffraction (XRD), FTIR spectra, UV-vis diffuse reflectance spectra (DRS) of C3N4-BiOCl and transmission electron microscope (TEM), UV-vis absorbance spectra of CuO NPs are described in detail in ESM (Fig. S1 and Fig. S2).

Mechanism of photoelectrochemical assay

C3N4-BiOCl semiconductor showed excellent photoelectric conversion ability. To highlight this advantage, the photocurrent responses of different semiconductor were measured under visible light irradiation. As plotted in Fig. 1a, C3N4-BiOCl (curve ‘d’) shows highest photocurrent intensity compared with that of C3N4 (curve ‘b’) and BiOCl (curve ‘c’). The result attributed to the interfacial transition of photoexcited charge-carrier, which was proved by photoluminescence (PL) spectrometry. As shown in Fig. 1b, the PL spectrum was obtained at an excitation wavelength of 370 nm. C3N4-BiOCl (curve ‘b’) shows weak PL emission intensity compared with that of pure g-C3N4 sample (curve ‘a’). Those results showed C3N4-BiOCl hybrids can greatly suppress the recombination of photoexcited charge-carrier.

Fig. 1
figure 1

a Photocurrents of (a) bare ITO electrode, (b) BiOCl/ITO, (c) C3N4/ITO, (d) C3N4-BiOCl/ITO in phosphate buffer; b PL of (a) C3N4, (b) C3N4-BiOCl from 350 nm to 600 nm; c Photocurrent responses of C3N4-BiOCl/ITO electrode for L-cys at different concentration in phosphate buffer; and (d) Photocurrent responses of C3N4-BiOCl/ITO electrode in phosphate buffer containing L-cys for different concentrations of Cu2+

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The photoelectric response capacity of C3N4-BiOCl modified ITO electrode for L-cys and Cu2+ is then inspected. Figure 1c manifests the typical photocurrents of C3N4-BiOCl/PDDA/ITO toward different L-cys concentration. The photocurrents suggest the charge excitation, separation, and transfer in the C3N4-BiOCland the response to L-cys. It can be seen in all photocurrent curves, the anodic photocurrent increased with the increase of L-cys levels and the growth tended to balance after 1 mM, indicating the near saturation of the target C3N4-BiOCl reaction [32]. In this reaction, L-cys as electron donors was liable to PEC oxidation at current conditions, which can restrain the recombination of e− and h+. However, when copper ions are present, L-cysteine as bio-thiols is a type of chelators, which can capture and coordinate with Cu2+ [33]. As shown in Fig. 1d, the anodic photocurrent decreased with the increase of Cu2+ concentration. More interestingly, the photocurrent is lower than the blank current of C3N4-BiOCl (~60 nA, without L-cys) when the concentration of Cu2+ was greater than or equal to 1. The results ascribed to the transfer of photoinduced electrons from the conduction band of the C3N4 and BiOCl nanosheets to Cu2+ [34]. The mechanisms of PEC toward L-cys and Cu2+ are graphically described in Scheme 1b, c. Hence, the L-cys assisted PEC system is sensitive for Cu2+.

Performance of the photoelectrochemical immunoassay

The following parameters were optimized: (a) pH value; (b) incubation time; (c) temperature, and the description and figures are given in the ESM (Fig. S3). Under optimal experimental conditions, sandwich-type immunoassay was constructed on the 96-well microtiter plate for detection a variety of CEA. As shown in Fig. 2a, the photocurrents decreased with the increasing of CEA concentration (the inset). The photocurrents were linearly associated with CEA levels in the range of 0.1 pg mL−1 ~ 10 ng mL−1. The equation was y (nA) = −15.10 log C (ng mL−1) + 57.27 (R2 = 0.98, n = 27) with the detection limit (LOD) of 0.1 pg mL−1. On the one hand, since the threshold values in normal human serum is 3 ng mL−1 for CEA, this photoelectrochemical immunoassay can completely meet the requirements of clinical diagnosis. For another, an overview on recently reported methods for detection of CEA is listed in the Table 1. Compared with other methods, the immunoassay own wider linear range and lower detection limit. The excellent performance of photoelectrochemical immunoassay mainly attributed to enhanced photoelectric activity of C3N4-BiOCl and response capability toward Cu2+. However, the analysis procedures were complicated because the establishment process of immunoassay and detection system of photocurrent was mutually independent. The relationship was built by the function of Cu2+. Therefore, there has been an intense focus on integration in future.

Fig. 2
figure 2

a Calibration plot of the immunoassay toward different concentrations of CEA [the inset: photocurrent curves with different CEA concentration at an applied potential of 0 V]; b Specificity of photoelectrochemical immunoassay for PSA, AA, CA724, K+, Glu; c Stability of the immunoassay for 300 s; and (d) Five groups of immunoassays for 10 ng mL−1 CEA detection

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Table 1 An overview on recently reported nanomaterial-based methods for determination of CEA
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Selectivity, repeatability, and stability of the photoelectrochemical immunoassay

First, ascorbic acid (AA), prostate specific antigen (PSA), carbohydrate antigen 724 (CA724), glucose (Glu), and K+ were detected by this photoelectrochemical immunoassay. It can be seen in Fig. 2b the value for CEA was lower than that of blank, and values of other substances had no obvious difference with the blank value. As shown in Fig. 2c, the photocurrent of the C3N4-BiOCl modified platform for 1 pg mL−1 CEA is stability under the repeated light irradiation circles for 280 s. Five groups of immunoassays were built to detect 10 ng mL−1 CEA. The variation coefficients (CVs) were 2.58% (Fig. 2d). This immunocomplex was stored in fridge at 4 °C for three weeks, and the photocurrent value reserved 95% compared with initial value. Hence, this photoelectrochemical immunoassay displayed satisfactory selectivity, repeatability, and stability.

Analysis of real serum samples

To verify applicability of photoelectrochemical immunoassay for practical serum samples, t-test is employed by contrasting analysis results from photoelectrochemical method and commercial ELISA method. Before experiment, the collected serum samples were handled carefully with dilution by phosphate buffer (pH = 7.0). The experimental results and texp values are listed in the Table 2. The texp values in all samples were less than tcrit (tcrit = 4.30), which declare the immunoassay is believable and possess applicable value in further.

Table 2 Comparison of the assay results for human serum specimens by using the photoelectrochemical immunoassay and the referenced ELISA method
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Conclusion

A photoelectrochemical immunoassay for CEA detection was developed by utilizing CuO nanoparticles as labels and cysteine assisted C3N4-BiOCl photoelectrochemical system. The heterojunction nanostructure of C3N4-BiOC land sacrificial donor of cysteine doubly enhanced the photocurrent response of C3N4-BiOCl. The PEC system was sensitive to Cu2+ due to its chelation and electron receptors, which doubly decreased the photocurrent response. And Cu2+ was introduced from immunoassay format and related to CEA concentrations. Compared with conventional photoelectrochemical detection systems, highlights of this study can be concluded as follows: (i) the background signal of photocurrent was intensive, which was profitable for signal reduction method; (ii) the presence of cysteine improved sensitivity for Cu2+, which was beneficial to immunoassay. This strategy opens a new perspective for the application of photoelectrochemical bioanalysis in the future. Future works should focus on the detection of more biomolecule in serum.