Lu
Li
,
Yaqing
Weng
,
Chenglong
Sun
,
Yueyi
Peng
* and
Qingji
Xie
*
Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China. E-mail: pengyueyi@hunnu.edu.cn; xieqj@hunnu.edu.cn
First published on 30th June 2023
We report the sensitive photoelectrochemical (PEC) sandwich-type immunosensing of alpha-fetoprotein (AFP) by using SnO2/In2S3 as a novel heterojunction material and horseradish peroxidase (HRP)–CeO2 as an efficient biolabel. A SnO2/In2S3/fluorine-doped tin oxide (FTO) photoanode was fabricated by hydrothermally synthesizing SnO2/In2S3 and cast-coating it on a FTO slice. A HRP–CeO2–secondary antibody–bovine serum albumin complex was immunologically immobilized on the photoanode, and the PEC immunoassay of AFP was realized by the HRP and mimetic enzyme CeO2 catalyzed oxidation of 3-amino-9-ethylcarbazole by H2O2 to generate a precipitate with a steric hindrance effect. Under optimized conditions, the value of photocurrent decrease was linear with the common logarithm of AFP concentration from 500 fg mL−1 to 50 ng mL−1, and the limit of detection (S/N = 3) is 0.15 pg mL−1.
Photoelectrochemical (PEC) analysis has the advantages of low background and high sensitivity due to separation of the excitation source and output signal.9–11 PEC analysis performance relies heavily on the PEC materials, which can be used both as light-harvesting substances to generate PEC signals and as substrates to immobilize biomolecules. Many metal oxides, metal sulfides, quantum dots, and metal–organic frameworks have been widely used due to their excellent PEC properties. Among them, SnO2 (Eg ∼ 3.6 eV) is a typical metal oxide semiconductor exhibiting high ultraviolet absorption, high stability and environmental friendliness.12,13 The wide bandgap of SnO2 limits its ability to absorb only ultraviolet light and it has a high electron–hole recombination degree, but the PEC performance can be improved by heterojunction formation, doping, sensitization, and noble metal loading.14 In2S3 (Eg ∼ 2.0 eV) is a narrow band gap n-type semiconductor with high photoactivity and is promising for photocatalysis, CO2 reduction and solar cell applications.15–17 As far as we know, construction and application of SnO2/In2S3 heterojunctions in the PEC field have not been reported.
Signal output and amplification strategies also play a key role in determining the sensitivity and selectivity of a PEC biosensor.18 To date, steric hindrance effects, energy transfer principles, p–n semiconductor quenching effects and enzymatic reactions have been widely used to output and amplify PEC signals.19,20 The catalyzed precipitation (CP) reaction can be used to quench the photocurrent signal by generating insoluble precipitates with a steric hindrance effect on the electrode surface and thus give obvious response signals and high sensitivity.21 CP based on natural enzymes such as horseradish peroxidase (HRP) and alkaline phosphate (ALP) has been reported.19,22 On the other hand, nanozymes (nanomaterials with enzyme-like properties) have also been widely used because of their good stability and low cost. CeO2 has received extensive attention in the fields of photocatalysis and biosensor due to its favorable electrical, optical and peroxidase-like properties.23 Here, CeO2 of a uniform nanocube structure is employed as a secondary antibody (Ab2) label, because it can act as both an excellent carrier for loading biomolecules and a peroxidase-like nanozyme to catalyze precipitation together with the natural enzyme HRP. As far as we know, Ab2 labeling with a CeO2 nanozyme and HRP natural enzyme to catalyze the production of an insoluble precipitate on the photoelectrode surface for signaling in a PEC AFP immunoassay has not been reported to date.
Herein, we hydrothermally synthesize SnO2 and SnO2/In2S3, and the SnO2/In2S3 heterojunction material is cast-coated on a fluorine-doped tin oxide (FTO) electrode to form a SnO2/In2S3/FTO photoanode. CeO2 nanocubes are synthesized by a hydrothermal method and calcination. The CeO2 surfaces are aminated by 3-aminopropyltriethoxysilane (APTES) treatment, and then HRP and Ab2 are tethered through amide bonds after 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation. An HRP–CeO2–Ab2–bovine serum albumin (BSA) immune-complex is prepared after blocking the nonspecific sites of HRP–CeO2–Ab2 with BSA. The first antibody (Ab1) is immobilized on the SnO2/In2S3/FTO photoanode after chitosan (CS) coating and the Schiff base reactions of glutaraldehyde (GLD) with CS and Ab1. After blocking the nonspecific sites with BSA, Ag (i.e. AFP) is immunologically immobilized, and then the HRP–CeO2–Ab2–BSA immune-complex is also immunologically linked to Ag. The labeled HRP and CeO2 can catalyze the oxidation of 3-amino-9-ethylcarbazole (AEC) by H2O2 to generate a precipitate on the photoanode surface and thus attenuate the photocurrent, and thus, the sensitive PEC detection of AFP is achieved. The photocurrent linearly decreases with the increase in the logarithm of AFP concentration, with a limit of detection (LOD, S/N = 3) of 0.15 pg mL−1.
Second, the SnO2/In2S3 heterojunction material was synthesized by a facile one-pot method, as shown in Scheme 1A. 0.1000 g of the synthesized SnO2 nanoparticles were sonicated and dispersed in 70 mL of ultrapure water, followed by adding 0.1408 g of InCl3·4H2O and 0.1163 g of L-cysteine (L-Cys). The dispersion was transferred to a 100 mL high-pressure reactor and reacted in an oven at 180 °C for 12 h. After cooling to room temperature, earthy yellow SnO2/In2S3 was collected by centrifugation and washed alternately with ultrapure water and ethanol several times, and then dried overnight in an oven at 60 °C. In addition, different SnO2/In2S3 mass ratios were examined by changing the mass of SnO2 (optimized to be 0.1 g SnO2) while other synthesis conditions remained constant. The dispersant is ultrapure water.
Scheme 1 Schematic for preparing the SnO2/In2S3 heterojunction (A), the HRP–CeO2–Ab2–BSA complex (B), and the immune-photoelectrode (C). |
Finally, the SnO2/In2S3/FTO electrode was prepared by casting 20.0 μL of 2.00 mg mL−1 SnO2/In2S3 suspension on the surface of a FTO electrode with an effective area of 0.250 cm2, and then dried in a 60 °C oven.
Amino-functionalized CeO2 (NH2–CeO2) was synthesized. 200 μL APTES was dispersed in 20 mL ethanol, and the pH was adjusted to 7.0 with 3 M HCl. Afterwards, 0.5 g CeO2 was added and the dispersion was continuously stirred for 14 h. After centrifuging and washing with anhydrous ethanol three times and drying, NH2–CeO2 was obtained.
The HRP–CeO2–Ab2–BSA complex was prepared as shown in Scheme 1B. 10 mg EDC and 5 mg NHS were added in 1 mL of 100 μg mL−1 Ab1 containing 2 mg mL−1 HRP. After gently shaking at 4 °C for 2 h, the carboxyl groups on HRP and Ab1 were activated. Afterwards, 2 mg mL−1 well-dispersed NH2–CeO2 was added into the mixture, and the amidation reaction occurred for 2 h. Then, the HRP–CeO2–Ab2 complex was obtained after centrifugation and washing with 0.01 M phosphate buffer solution (PBS) three times. To hinder nonspecific adsorption, HRP–CeO2–Ab2 was dispersed in 1 mL of 0.01 M PBS containing 300 μL 1% BSA. After gently shaking at 4 °C for 1 h, centrifuging, washing three times with 0.01 M PBS and then redispersing in 1 mL of 0.01 M PBS, the HRP–CeO2–Ab2–BSA complex was prepared. The dispersion was stored at 4 °C before use.
PEC detection was done in 0.1 M PBS (pH 7.40) containing 0.05 M ascorbic acid (AA) at a bias of 0.05 V vs. SCE. The PEC detection was performed with a 100 mW cm−2 visible light source, with the light source switched on and off every 10 s.
Second, the FTO-supported SnO2, In2S3 and SnO2/In2S3 were characterized by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), as shown in Fig. 1. The SnO2 consists of irregular particles, while the In2S3 shows a flower-like microsphere structure composed of a large number of nanosheets. The SEM image of the SnO2/In2S3 heterojunction displays the deposition of particulate SnO2 on the In2S3 surface. The EDS pattern of SnO2/In2S3/FTO and the revealed quantitative percentages of O, Sn, In, and S (inset in panel D) demonstrate that the SnO2/In2S3 material was successfully synthesized by the one-pot method.
Fig. 1 SEM images of (A) SnO2/FTO, (B) In2S3/FTO and (C) SnO2/In2S3/FTO. (D) EDS spectrum of SnO2/In2S3/FTO. |
Third, bare FTO, SnO2/FTO, In2S3/FTO and SnO2/In2S3/FTO electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 0.1 M PBS (pH 7.40) containing 2.0 mM K4[Fe(CN)6] and 0.1 M Na2SO4, as shown in Fig. S2A and B.† The cleaned FTO electrode shows a pair of redox peaks with a peak-to-peak separation (ΔEp) of 106 mV and a charge transfer resistance (Rct) of 0.115 kΩ, verifying the well reversible electrode process of Fe(CN)63−/Fe(CN)64− on the bare FTO electrode. The ΔEp values obey the order 126 mV (In2S3/FTO) < 138 mV (SnO2/FTO) < 151 mV (SnO2/In2S3/FTO), and the Rct values follow the order 0.303 kΩ (In2S3/FTO) < 0.594 kΩ (SnO2/FTO) < 0.952 kΩ (SnO2/In2S3/FTO). These results reasonably indicate the sequentially increased blocking of the electron transfer between solution-state Fe(CN)63−/Fe(CN)64− and the electrode surface by the electrode-surface modification. The PEC performance of the FTO, SnO2/FTO, In2S3/FTO and SnO2/In2S3/FTO electrodes was characterized at 0.05 V in 0.1 M PBS (pH 7.40) containing 0.05 M AA, as shown in Fig. S2C.† The SnO2/In2S3/FTO electrode shows a photocurrent notably larger than those of In2S3/FTO and SnO2/FTO, indicating that the formation of a SnO2/In2S3 heterojunction can improve the separation and transfer of photogenerated charges. As shown in Fig. S2D,† the photocurrent of the SnO2/In2S3/FTO photoanode barely attenuated after switching on/off the light for ten cycles, indicating that the electrode has good stability.
Finally, as shown in Fig. S3,† UV-vis DRS and Mott–Schottky experiments were carried out to estimate the energy band structure of the material and discuss the possible charge transfer pathways of the SnO2/In2S3 heterojunction (Scheme 2). Fig. S3A† shows the ultraviolet absorption edge of SnO2 at 358 nm32 and that of In2S3 at 650 nm.33 Notably, after the formation of the SnO2/In2S3 heterojunction, the absorption band is obviously red-shifted compared with those of the individual materials. Meanwhile, the enhanced visible light absorption is beneficial to improve the optoelectronic performance of the heterojunction. Both SnO2 and In2S3 are direct bandgap semiconductors.34,35 Therefore, in Fig. S3B and C,† according to the plots of (αhν)2versus photon energy (hν), the band gaps of SnO2 and In2S3 are estimated to be 3.7 eV and 2.1 eV, respectively, which are consistent with literature values.36,37 The Mott–Schottky curves of both SnO2 and In2S3 exhibit positive slopes, indicating that SnO2 and In2S3 exhibit n-type semiconductor characteristics. In Mott–Schottky plots, the flat band potential (Ef) of n-type semiconductors is equivalent to the conduction band level (ECB).38 Therefore, the ECB of SnO2 and In2S3 is −0.63 V (vs. SCE) and −0.75 V (vs. SCE), respectively (Fig. S3D and E†). Then, according to the conversion relationship Eg = EVB − ECB, the EVB of SnO2 and In2S3 is estimated as 3.07 V (vs. SCE) and 1.35 V (vs. SCE), respectively. Finally, according to ERHE = ESCE + 0.0591 pH + 0.2415, the ECB of SnO2 and In2S3 is 0.05 V (vs. RHE) and −0.07 V (vs. RHE), and the EVB of SnO2 and In2S3 is 3.75 V (vs. RHE) and 2.03 V (vs. RHE), respectively, which agree well with literature results.27,39
Scheme 2 Schematic of the possible charge transfer pathways of the SnO2/In2S3 heterojunction and the PEC immune-sensing principle. |
Due to the matching of the energy band structures of SnO2 and In2S3, the combination of SnO2 and In2S3 can form a heterojunction that is beneficial to the separation and transfer of photogenerated charges and greatly improves the PEC performance. The possible charge transfer mechanism of this system is shown in Scheme 2. When the energy provided by the external light source with a visible light filter (400–700 nm) slightly excites SnO2 and notably excites In2S3, electron–hole pair separation occurs in/on SnO2 and especially In2S3. Subsequently, at the SnO2/In2S3/FTO electrode, the photogenerated electrons are transferred from the relatively negative CB of In2S3 to the CB of SnO2, and the holes are somewhat transferred from the relatively positive VB of SnO2 to the VB of In2S3. Anyway, the electron transfer from In2S3 to SnO2 should be obvious in the SnO2/In2S3 heterojunction, though SnO2 itself can only be slightly photoexcited by the filtered visible light to give a minor photocurrent and thus the hole transfer from SnO2 to In2S3 is insignificant, probably leading to the formation of a special type-II heterojunction with notably unequal electron and hole flows. In fact, a conventional type-II heterojunction usually has comparable electron and hole flows (but not definitely strictly symmetric electron and hole flows in our opinion, due to the two different employed semiconductors with different photoelectric conversion efficiencies and with different CB-to-CB and VB-to-VB spacing). The successful construction of the heterojunction reduces the recombination degree of electron–hole pairs and improves the photocurrent response. When CP occurs on the SnO2/In2S3/FTO electrode to prevent AA from approaching the FTO and In2S3 surfaces, the photocurrent will be decreased for signaling, as discussed in detail later. In summary, the above characterizations with XRD, XPS, SEM, EDS, CV, EIS, PEC detection, UV-vis DRS and Mott–Schottky curves have verified the involved materials and electrodes and the construction of the SnO2/In2S3 heterojunction.
Ultraviolet-visible (UV-vis) absorption spectra were used to verify that HRP and Ab2 were successfully modified on NH2–CeO2, as shown in Fig. S4E.† The characteristic absorption peak of NH2–CeO2 is found at ca. 300 nm. Ab2 exhibits its characteristic absorption peak near 280 nm.41 HRP as a protein has a typical protein absorption peak at ca. 270 nm. The absorption peak near 270–280 nm in HRP–CeO2–Ab2 becomes larger. These results validate the successful preparation of HRP–CeO2–Ab2.
Scheme 1C illustrates the modification steps of the immune-sensing electrodes. First, CV and EIS were used to characterize each modification step of the immune-electrode in 0.1 M PBS (pH 7.40) containing 2.0 mM K4Fe(CN)6 and 0.1 M Na2SO4, as shown in Fig. 2A and B. With step-by-step modifications, the ΔEp and Rct values increase as follows: SnO2/In2S3/FTO (151 mV, 0.952 kΩ) < GLD/CS/SnO2/In2S3/FTO (172 mV, 1.38 kΩ) < Ab1/GLD/CS/SnO2/In2S3/FTO (187 mV, 1.58 kΩ) < BSA/Ab1/GLD/CS/SnO2/In2S3/FTO (194 mV, 1.92 kΩ) < Ag/BSA/Ab1/GLD/CS/SnO2/In2S3/FTO (211 mV, 2.23 kΩ) < HRP–CeO2–Ab2–BSA/Ag/BSA/Ab1/GLD/CS/SnO2/In2S3/FTO (229 mV, 2.84 kΩ) before CP < HRP–CeO2–Ab2–BSA/Ag/BSA/Ab1/GLD/CS/SnO2/In2S3/FTO after CP (252 mV, 3.83 kΩ). These results reasonably indicate the decrease in the electrode activity after modifying an electron-insulating material and confirm the successful stepwise construction of the immune-electrode.
Photocurrents of the immune-electrodes at different fabrication steps were measured at 0.05 V in 0.1 M PBS (pH 7.40) containing 0.05 M AA, as shown in Fig. 2C. The photocurrent values follow the order 70 μA (SnO2/In2S3/FTO) > 55 μA (GLD/CS/SnO2/In2S3/FTO) > 46 μA (Ab1/GLD/CS/SnO2/In2S3/FTO) > 42 μA (BSA/Ab1/GLD/CS/SnO2/In2S3/FTO) > 36 μA (Ag/BSA/Ab1/GLD/CS/SnO2/In2S3/FTO), because the surface modifications of GLD/CS, Ab1, BSA and Ag (AFP) on the electrode can hinder the mass transfer and electron transfer of the hole scavenger AA. After the HRP–CeO2–Ab2–BSA labeling of the captured AFP, the photocurrent of HRP–CeO2–Ab2–BSA/Ag/BSA/Ab1/GLD/CS/SnO2/In2S3/FTO further decreased to 30 μA, due to the further increased steric hindrance effect. CeO2 and HRP are capable of catalyzing the oxidation of AEC by H2O2 to produce a red precipitate, and the precipitation of the insoluble precipitate on the electrode and semiconductor surface can further quench the photocurrent (Scheme 2). As a result, after CP on the immune-electrode, the photocurrent of the immune-electrode decreased significantly to 12 μA because the insoluble precipitate notably prevents the transfer of electron donors to the electrode surface. In summary, both HRP–CeO2–Ab2–BSA and the generated red precipitate can decrease the photocurrent of the immune-electrode, enabling the construction of a signal-off immune-sensor for the sensitive PEC detection of AFP.
Under the optimal conditions, the photocurrent of the PEC immune-electrode decreases with the increase in AFP concentration, due to the CP effect (Fig. 3). The photocurrent has a linear relationship with the common logarithm of AFP concentration from 500 fg mL−1 to 50 ng mL−1, with a linear regression equation of I = −4.23lgcAg + 10.5 (R2 = 0.997) and a LOD of 0.15 pg mL−1 (S/N = 3). The analytical performance of the PEC immune-electrode is better than many reported results, as listed in Table S1.†
The stability, reproducibility and selectivity of the immune-electrode were investigated, as shown in Fig. S6.† The immune-electrode still has good stability after 200 s cycling. Five immune-electrodes were prepared in parallel to detect 500 pg mL−1 AFP, and the relative standard deviation (RSD) was 8.4%, indicating good reproducibility. In comparison with those to IgG, CEA, CYFRA21-1 and PCT (each at 50 ng mL−1, see ESI† for their full names), the photocurrent response to 500 pg mL−1 AFP was the most obvious. Hence, this immunosensor has good stability, reproducibility and selectivity.
We detected AFP in serum samples from healthy people using this immunosensor and the standard addition method. As listed in Table S2,† the recovery (92.0–106%) and RSD (3.3–7.6%) results are acceptable, suggesting that the PEC immunoassay has the potential to detect AFP in actual samples.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3sd00105a |
This journal is © The Royal Society of Chemistry 2023 |