An Electrochemical Signal-Amplification Strategy for

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An Electrochemical Signal-Amplification Strategy for Electrochemiluminescent Immunoassay with g-CN as Tags 3

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Yuchen Jin, Qi Kang, Xinli Guo, Bin Zhang, Dazhong Shen, and Guizheng Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03554 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 6, 2018

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Analytical Chemistry

An Electrochemical Signal-Amplification Strategy for Electrochemiluminescent Immunoassay with g-C3N4 as Tags Yuchen Jin, † Qi Kang, †,* Xinli Guo, † Bin Zhang,‡ Dazhong Shen , † Guizheng Zou‡,* †

College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, P. R. China

‡

College of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China

Corresponding author email: [email protected]; [email protected]

ABSTRACT: Signal amplification for electrochemiluminescence (ECL) was conventionally achieved by employing effective matrix, which could accelerate the electrochemical redox processes and/or carry more electrochemiluminophores. Herein, a convenient signal-amplification strategy was proposed for ECL immunoassay with carboxylated g-C3N4 nanosheets (NSs) as tags and carcinoembryonic antigen (CEA) as model target via electrochemically pretreating the substrate, i.e. glassy carbon electrode (GCE) modified with polymerized 2-aminoterephthalic acid (ATA) film (GCE/ATA). Bio-conjugates of g-C3N4 NSs and signal CEA antibody (Ab2), i.e. g-C3N4 NSs-Ab2 were immobilized on GCE/ATA via sandwich immunoreaction to form GCE/ATA-Ab1-Ag-Ab2NSs. Electrochemical impedance spectroscopy and potential-resolved ECL characterization proved that GCE/ATA play an important role on electron-transfer-resistance (Ret) of GCE/ATA-Ab1-Ag-Ab2-NSs for ECL, successively scanning GCE/ATA-Ab1Ag-Ab2-NSs in K2S2O8+H2O2 containing medium from 0 to -1.6 V could reduce the Ret and bring out 3.3 times enhanced ECL at the tenth scan cycle than the first scan cycle, which was about 10.2 times to the ECL of GCE/ATA-Ab1-Ag-Ab2-NSs in medium merely containing K2S2O8. Inspired by this, directly and successive scanning GCE/ATA in K2S2O8+H2O2 containing medium was employed in fabricating procedure, which could dramatically reduce Ret of GCE/ATA-Ab1-Ag-Ab2-NSs, and bring out obviously enhanced ECL response for selectively determine CEA from 0.1 pg/mL~1 ng/mL with a detection limit of 3 fg/mL.

Electrochemiluminescence (ECL) is a special form of chemiluminescence phenomenon, in which the excited state of luminophor is produced via electrochemical redox.1,2 The absence of unselective interference from exciting-light-resource makes ECL more attractive than fluorescence, and ECL assays have demonstrated excellent reproducibility, sensitivity.3-5 A microbead-based ECL system along with Ru(bpy)32+/tri-npropylamine based ECL reagent kits has been commercialized by IGEN9 (technology later acquired by Roche Diagnostics Corp.) for a number of assays with a particular focus on immunoassays.6,7 As the footstone of ECL, electrochemiluminophores play a crucial role on ECL assays.8 To overcome the shortcomings of dyestuff-kind electrochemiluminophores, such as Ruthenium complex9 and luminol,10 ECL methods based on various semiconductor nanocrystals (NCs) and/or nanoparticle have been explored for sensing.11-17 Graphite-like carbon nitride (g-C3N4) is a kind of two-dimensional semiconductor nanomaterial, and has several promising advantages over II-VI NCs, such as low energy band (~ 2.7 eV), high chemical stability and toxicelement-free nature.18,19 ECL of g-C3N4 has aroused much attention. 20-24 Xu et al also demonstrated that ECL from gC3N4 nanosheets (NSs) embedded with g-C3N4 quantum dots was stronger than that from bare g-C3N4 NSs, and designed a signal-on aptasensing strategy with the enhanced ECL.25 Up to now, understanding on the way to enhance ECL from g-C3N4 NSs is still very limited for their sensing application.

The signal amplification for common ECL assays is mainly achieved via three conventional strategies.26 One is to employ effective matrix for the indirect signal amplification, such as carbon nanotube,27 carbon nanofiber,28 and noble metal nanostructures.29 Another is to accelerate electrochemical redox processes via chemocatalysis and/or enzyme catalysis of coreactants.30,31 The last one employs some materials as good carriers or bridges to improve the loading number of electrochemiluminophores, such as silica microspheres32 and magnetic beads.33,34 Herein, we proposed a convenient electrochemical signalamplification strategy to achieve sandwich-typed and highsensitive “signal on” ECL immunoassay with the carboxylated g-C3N4 NSs as tags and carcinoembryonic antigen (CEA) as model analyte (Scheme 1). The g-C3N4 NSs were immobilized onto glass carbon electrode (GCE) via forming sandwichtyped immune-complexes and linking them to GCE with poly(2-amino-terephthalic acid) (ATA). For the first time, we demonstrated that the electrochemically activating the substrate of GCE/ATA with K2S2O8 and H2O2 in fabricating procedure could reduce impedance of the finally obtained ECL sensor and bring out greatly enhanced reductive-oxidation ECL from surface-confined g-C3N4 NSs in the immunecomplexes, as the impedance of substrate GCE/ATA plays a crucial role on performance of ECL sensor. EXPERIMENTAL SECTION

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Materials and reagents. All reagents were of analytical grade or better. All aqueous solutions including the phosphate buffer (PB) were prepared with ultrapure water (specific resistance of 18 MΩ.cm). Melamine, nitric acid, potassium ferricyanide, potassium ferrocyanide, n-hydroxysuccinimide (NHS), and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA), 2-aminoterephthalic acid, potassium persulfate, ethylenediamine (En), and hydrogen peroxide were obtained from Aladdin Industrial Corporation (Shanghai, China); CEA (Ag), corresponding capture antibody (Ab1) and signal antibody (Ab2) were purchased from Beijing Keyuezhongkai Biology &Technology Co. Ltd. (Beijing, China). Alpha-fetal protein (AFP) was purchased from Biocell Biotechnology Co. Ltd. (Zhengzhou, China). Carbohydrate antigen 15-3 (CA15-3) were obtained from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). Human IgG was purchased from Beijing Dingguo Biotechnology Development Center (Beijing, China). Apparatus. ECL profiles were recorded on MPI-E ECL analyzer (Xi'an Remex Analytical Instrument Co., Ltd. China) with three-electrode system including an Ag/AgCl (sat. KCl) as reference electrode, a platinum wire counter electrode and a modified GCE working electrode. The electrochemical impedance spectroscopy (EIS) was conducted on CHI 660E electrochemical workstation (Chenhua Instrument Company, Shanghai, China). Absorbance, FTIR and fluorescence spectra were measured with UV-1700 spectrophotometer (Shimadzu, Japan), FT-IR 470 spectrometer (Thermo Fisher Scientific Co., U.S.A.) and F-7000 spectrofluorimeter (Hitachi, Japan), respectively. ECL spectrum was recorded on a homemade ECL spectrum analyzer, including multichannel optical analyzer (SpectraPro300i, Acton Research Co., Acton, MA, USA) and a CHI 832 analyzer (Shanghai CHI Instruments, China).35,36 Transmission electron microscopy (TEM) was conducted with a HT7000 microscope (Hitachi, Japan). The X-ray powder diffraction (XRD) patterns were measured on a Bruker D8 Advance diffractometer (Bruker, Germany). Preparation of carboxylated g-C3N4 NSs and g-C3N4 NSs-labeled Ab2 conjugates. Carboxylated g-C3N4 NSs were synthesized according to literatures with slight modification.37,38 In brief, 5.0 g of melamine powder was placed into a semi-closed ceramic crucible and heated at 550°C for 4 h in a muffle furnace under open air condition. The obtained yellow powder was ground carefully, then 1.0 g of the bulk g-C3N4 powder was dispersed in 50 mL ultrapure water and ultrasonicated for 1 h. The suspension was introduced into 50 mL 10 M HNO3 and refluxed for 24 h, the resulting product was centrifuged and washed with ultrapure water to neutral pH. The final product was dried in vacuum oven for further use. To prepare NSs-Ab2 conjugate (Scheme 1A), 1.0 mg g-C3N4 NSs was dispersed in 1.0 mL 0.01 M PB (pH 7.4) and sonicated for 10 min, then activated with 20 µL EDC (100 mg/mL) and NHS (100 mg/mL). 50 µL 100 µg/mL Ab2 was added in and stirred for 12 h at 4℃. The mixture was centrifuged to remove residual Ab2, and re-dispersed in 200 µL PB containing 0.1% BSA for 30 min to block any nonspecific binding sites. After further centrifugation, the purified g-C3N4-Ab2 conjugates was re-dispersed in 1.0 mL of PB and stored at 4 °C for further use.

Scheme 1. Schematic illustration on the fabricating procedures of the proposed ECL immunosensor.

Fabricating procedures of the ECL Immunosensor. GCE was polished with 0.3 and 0.05 µm alumina slurry, washed with ultrapure water and dried with a stream of high-purity nitrogen. The fabricating procedure was similar to that of previously reported sandwich-immunosensor (Scheme 1B).35 The fresh GCE was immersed in 10 mL PB containing 10 mM ATA and scanned from 0.2 to 1.2 V at 50 mV/s for 4 cycles to electrochemically polymerize ATA on GCE surface with carboxyl groups outside,35 as the one-electron oxidation of the amino group via electrochemical redox could form a carbonnitrogen linkage at the GCE surface.39 According to Scheme 1, two routes were employed in fabricating procedure to immobilize ECL tags onto the obtained GCE/ATA via immunoreaction. In the route 1, carboxyl groups of GCE/ATA were activated with 20 µL EDC (100 mg/mL) and NHS (100 mg/mL) for 30 min, and was further linked with Ab1 to form GCE/ATA-Ab1 via incubating with 20 µL 10 µg/mL Ab1 for 2 h. After the obtained GCE/ATA-Ab1 was rinsed with PB to eliminate physically absorbed Ab1 and immersed in BSA solution (1% in PB) for 30 min to block any remaining activated carboxyl sites, 20 µL CEA of different concentration was coated its surface and incubated for 80 min at 37°C to obtain GCE/ATA-Ab1-Ag, which was then incubated with g-C3N4 NSs-Ab2 conjugates for 1 h to form GCE/ATA-Ab1-Ag-Ab2NSs. In the route 2, GCE/ATA was first activated electrochemically by potential scanning from 0 to −1.6 V for 4 cycles


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(except special explanation) in PB containing 100 mM K2S2O8 and 60 mM H2O2 to reduce significantly the impedance of GCE/ATA, i.e. GCE/ATA(Ω↓). Then, the Ab1, Ag, and gC3N4 NSs-Ab2 conjugates were immobilized onto GCE surface in a similar way to route 1, and finally achieved GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs for ECL immnoassay. In a control experiment, g-C3N4 NSs were directly linked to GCE/ATA with En to form GCE/ATA-En-NSs upon the assistance of EDC and NHS.39 1.6

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states.36 The Ret value of GCE/ATA also demonstrated increased tendency from to 0.7 kΩ to 2.6 kΩ via successively scanning GCE in 10 mM ATA solution from one to four cycles (curves b & c), indicating the poor conductivity of theATA film. GCE/ATA-Ab1 displayed a Ret value around 1.4 kΩ, as immobilizing Ab1 onto GCE/ATA would reduce negativecharge-density in pH7.4 PB (curve d). Similar to previously reported sandwich-type ECL sensor with NCs as tags,35 immobilizing the target antigen and Ab2-NSs conjugates in the followed steps brought out enlarged Ret value around 1.6 kΩ and 1.8 kΩ respectively, because the formation of the immune-complex with a very large formula weight could also block electron transfer of the redox probe. The EIS data indicate that the Ab2 preserved its biological activity in Ab2-NSs conjugates, and the NSs could be immobilized onto GCE surface and formed CEA target-NSs pairs via the proposed immunoassay strategy. As illustrated in Scheme 1, the charge transfer between GCE and electrochemiluminophore, i.e. carboxylated g-C3N4 NSs should overcome the impedance related to both GCE/ATA (R1) and sandwich immunocomplexes (R2). Figure 1 indicated that the states of GCE/ATA played a dominant role on the total Ret of GCE/ATA-Ab1-Ag-Ab2-NSs. The way to reduce impedance of GCE/ATA (R1) would be promising for both enhanced electrochemical redox and ECL of g-C3N4 in GCE/ATA-Ab1-Ag-Ab2-NSs. 16k

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Figure 1. EIS of (a) bare GCE, (b) GCE scanned in ATA containing PB for one cycle, (c) GCE scanned in ATA containing PB for four cycles, (d) GCE/ATA-Ab1, (e) GCE/ATA-Ab1-Ag, and (f) GCE/ATA-Ab1-Ag-Ab2-NSs. Samples containing 1 ng/mL CEA were employed in the fabricating procedures.

RESULTS AND DISCUSSION Characterization of the carboxylated g-C3N4 NSs. The carboxylated g-C3N4 NSs displayed obvious sheet structure (Figure S1) with typical XRD diffraction and infrared absorption spectrum (Figure S2).38 The infrared absorption spectrum (Figure S2B) exhibited characteristic absorption of carboxyl groups around 1700 cm-1, indicating that g-C3N4 NSs were carboxylated.37 The drastically improved solubility of g-C3N4 NSs in the aqueous medium than common g-C3N4 NSs also confirms the carboxylation of g-C3N4 NSs. The carboxylated g-C3N4 exhibit obviously absorption around 310 nm, and strong fluorescence peak around 458 nm (Figure S3), which is in accordance with the bandgap of g-C3N4 (~ 2.7 eV). Electrochemical characterization on fabricating procedure of the proposed ECL immunoassay. Impedance of the modified electrode plays an important role on the chargetransfer processes occurred at electrode surface,40 and has significant impacts on the performance of ECL sensors.39 The diameter of the semicircle in the high frequency range of the Niquist plot equals to the electron transfer resistance (Ret) at the electrode interface.26 Consequently, EIS was conducted in 0.10 M pH 7.4 PB with Fe(CN)63−/Fe(CN)64− redox couple as probes to record the changed surface state of GCE in each fabricating step of GCE/ATA-Ab1-Ag-Ab2-NSs (Figure 1). Compared with bare GCE, the formation of ATA film on GCE via electrochemical polymerization blocked the electrontransfer occurred at GCE surface, as the carboxyl group of ATA would lose protons and form negative-charged surface

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Figure 2. (A) ECL-potential profiles of GCE/ATA-Ab1-Ag-Ab2NSs in 0.1 M PB (pH7.4) containing 100 mM K2S2O8 + 60 mM H2O2 (black line) by continuous scanning potential from 0 to -1.6 V at the (a) first, (b) third, (c) fifth, and (d) tenth cycle and that in



Impedance correlated performance of ECL assay with g-C3N4 as electrochemiluminophores. Under optimized conditions, no ECL was detected with ether GCE/ATA-Ab1-Ag in PB containing K2S2O8 or GCE/ATA-Ab1-Ag-Ab2-NSs in coreactant-free PB (data not shown). The reductive-oxidation ECL of GCE/ATA-Ab1-Ag-Ab2-NSs in PB containing K2S2O8 further confirmed that g-C3N4 NSs were immobilized onto GCE/ATA via the designed procedure in Scheme 1 and were promising electrochemiluminophores for ECL immunoassay (Figure 2A, blue curve e). Although both the maximum ECL intensity and the potential for maximum ECL emission of GCE/ATA-Ab1-Ag -Ab2-NSs in PB merely containing K2S2O8 was stable along with successive potential scan for five cycles (blue curve, inset of Figure 2A), the ECL intensity of GCE/ATA-Ab1-Ag-Ab2-NSs in the K2S2O8 containing medium was relative lower. Enhanced ECL for GCE/ATA-Ab1-AgAb2-NSs were needed for their bioassay with desired performance. Recently, some researches presented a promising dualcoreactant strategy to enhance ECL of semiconductor nanoparticles,41,42 in which ECL of electrochemiluminophores with both H2O2 and K2S2O8 as dual-coreactants was much stronger than that single K2S2O8 coreactant. H2O2 was consequently introduced into the K2S2O8 containing ECL solution to enhance the reductive-oxidation ECL of g-C3N4 herein. As shown in Figure 2A, ECL response of GCE/ATA-Ab1-AgAb2-NSs in both K2S2O8 and H2O2 containing medium was about 3.1 times higher than that in merely K2S2O8 containing medium (curve a & e). Interestingly, ECL response of GCE/ATA-Ab1-Ag-Ab2-NSs in both K2S2O8 and H2O2 containing medium increased obviously with prolonged successive potential scan cycle (Figure 2A, curve a-d), their maximum ECL intensity eventually approached a constant value at the tenth potential scan cycle (Inset of Figure 2A), which was about 3.3 times higher than that at the first potential cycle, and about 10.2 times higher than the GCE/ATA-Ab1-Ag-Ab2-NSs in PB merely containing K2S2O8. ECL spectra of GCE/ATA-Ab1-Ag-Ab2-NSs in PB containing H2O2 and K2S2O8 demonstrated a solely peak around 464 nm (Figure S4), which was much closed to fluorescence spectrum of the carboxylated g-C3N4 NSs, and confirmed the ECL was originated from the carboxylated g-C3N4 NSs. Importantly, the potential for maximum ECL of GCE/ATA-Ab1-Ag-Ab2-NSs in both K2S2O8 and H2O2 containing PB also positively shifted along with the successive potential scan (Figure 2A and Figure S5). The maximum ECL emission potential was located around -1.21 V (vs. Ag/AgCl) for the tenth cycle (Figure 2A, black line e), which was shifted in the lowered electrochemical energy direction for 0.38 V when compared with GCE/ATA-Ab1-Ag-Ab2-NSs in PB merely containing K2S2O8 (-1.59 V). The enhanced ECL intensity and positively shifted potentials for the maximum ECL

emission of GCE/ATA-Ab1-Ag-Ab2-NSs in dual-coreactant containing medium were favorable for improved ECL response and less electrochemical interference. Importantly, as shown in Figure 2B, Ret of GCE/ATA-Ab1Ag-Ab2-NSs also demonstrated a decreased tendency via successively scanning them in H2O2 + K2S2O8 containing PB, indicating the enhanced ECL of GCE/ATA-Ab1-Ag-Ab2-NSs in both H2O2 and K2S2O8 containing medium via successive potential scan might due to enhanced electron-transfer ability, i.e. electrochemical redox, of GCE/ATA-Ab1-Ag-Ab2-NSs. Normalized ECL intensity

0.1 M PB (pH 7.4) merely containing 0.10 M K2S2O8 by scanning potential from 0 to -1.6 V for one cycle (curve e, blue line). (B) EIS of GCE/ATA-Ab1-Ag-Ab2-NSs 0.1 M PB (pH7.4) containing 5 mM K3Fe(CN)6 and 5.0 mM K4Fe(CN)6 (a) and corresponding EIS after they were continuous scanning in 0.1 M PB (pH 7.4) containing 100 mM K2S2O8 + 60 mM H2O2 for (a) one, (b) three, (c) five, and (d) ten cycles. Insert: ECL intensity-time profiles in 0.1 M PB (pH 7.4) containing 100 mM K2S2O8 (blue line) and 100 mM K2S2O8 + 60 mM H2O2 (black line) by continuous scanning potential from 0 to -1.6 V. GCE/ATA-Ab1-Ag-Ab2-NSs was fabricated with sample containing 1 ng/mL CEA.

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Figure 3. Effects of successive potential scan cycle on ECL intensity of GCE/ATA-En-NSs in (A) 100 mM K2S2O8 and (B) 100 mM K2S2O8 + 60 mM H2O2 containing 0.1 M pH7.4 PB. Effects of potential scan cycle on Ret of (a) GCE/ATA, (b) GCE/ATAEn-NSs, (c) GCE/ATA-Ab1-Ag-Ab2-NSs by successively scanning them in (C) 100 mM K2S2O8 and (D) 100 mM K2S2O8 + 60 mM H2O2 containing 0.1 M pH7.4 PB between 0 and -1.6 V at 50 mV/s. GCE/ATA-Ab1-Ag-Ab2-NSs was fabricated with sample containing 1 ng/mL CEA.

To verify the contribution of R1 and R2 on changed impedance of GCE/ATA-Ab1-Ag-Ab2-NSs, GCE/ATA-En-NSs was fabricated as control. As shown in Figure 3, GCE/ATA-EnNSs not only demonstrated stable ECL via successive potential scan in PB merely containing K2S2O8, but also displayed gradually enhanced ECL via successive potential scan in PB containing both H2O2 and K2S2O8. These results were similar to those of GCE/ATA-Ab1-Ag-Ab2-NSs, and indicated that the enhanced ECL of both GCE/ATA-Ab1-Ag-Ab2-NSs and GCE/ATA-En-NSs in both K2S2O8 and H2O2 containing medium via successive potential scan might be mainly related to the changed surface-states of GCE/ATA. According to Figure 3C, GCE/ATA, GCE/ATA-En-NSs and GCE/ATA-Ab1-Ag-Ab2-NSs displayed stable Ret around 2.6, 1.3 and 1.8 kΩ in K2S2O8 containing PB, respectively. Successive scanning in single coreactant of K2S2O8 had negli-


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gible effect on their Ret values. However, successive potential scanning them in both H2O2 and K2S2O8 containing PB would bring out drastically total Ret values to a constant value around 0.2 kΩ with the decreasing rate of GCE/ATA > GCE/ATAEn-NSs > GCE/ATA-Ab1-Ag-Ab2-NSs (Figure 3D). These results further proved that the enhanced ECL of GCE/ATAAb1-Ag-Ab2-NSs in both K2S2O8 and H2O2 containing medium via successive potential scan mainly due to the decreased Ret of the substrate GCE/ATA, i.e. R1, via electrochemically activating them with the dual-coreactants for enhanced electrochemical redox and/or electron transfer. Ret dependent ECL intensity have been observed in some ECL systems.43-45 Zhang developed an ECL biosensor for 8hydroxy-2′-deoxyguanosine (8-OHdG) via assembling hemin/Gquadruplex on carbon nitride nanosheets, and obtained decreased ECL response along with increased Ret.44 Lu designed an ECL aptasensor with CdSe NCs as electrochemiluminophores, their ECL response also decreased gradually with increased Ret.45 Inspired by these facts, a strategy to enhance ECL of GCE/ATA-Ab1-Ag-Ab2-NSs was proposed in fabricating procedure, which was conducted by directly treating GCE/ATA in both K2S2O8 and H2O2 containing medium via electrochemical method (route 2 of Scheme 1), as displayed in followed section.

rapidly reduced to a constant value in such solution (Figure 3D, curve a) and bring out reduced total Ret for enhanced electron transfer ability of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs. As shown in Figure 4, the obtained GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs not only displayed obvious enhanced ECL response in PB containing both single-coreactant K2S2O8 and dual-coreactant K2S2O8 + H2O2, but also displayed increased enhancement efficiency along with treating GCE/ATA in K2S2O8 + H2O2 containing medium with increased potential cycles (N). The enhancement efficiency of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs in PB containing dual-coreactant K2S2O8 + H2O2 was higher than that in PB merely containing single-coreactant K2S2O8. A high enhancement efficiency of 8.9 was achieved for ECL of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs with N=4 in PB containing both K2S2O8 and H2O2 to that of GCE/ATA-Ab1-Ag-Ab2-NSs in PB merely containing K2S2O8. These results indicated a promising nonenzymatic signal-amplification strategy for ECL assay. The ECL response of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs (N=4) in both K2S2O8 and H2O2 containing PB was consequently employed for the designed ECL immunoassay in the followed section. 20k 20k

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Figure 4. Effects of successive potential scan cycles (N) of treating GCE/ATA in 100 mM K2S2O8 + 60 mM H2O2 containing PB in the fabricating procedures on the maximum ECL intensity of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs by successively scanning the sensor in PB containing (A) merely 100 mM K2S2O8 and (B) 100 mM K2S2O8 +60 mM H2O2 from 0 to -1.6 V at 50 mV/s. The GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs were prepared with the electrochemically activated GCE/ATA and a sample containing 1 ng/mL CEA. Effects of directly activating GCE/ATA in fabricating procedures on the performance of ECL immunoassay. To lower the Ret of GCE/ATA-Ab1-Ag-Ab2-NSs, GCE/ATA was electrochemically activated in PB containing 100 mM K2S2O8 + 60 mM H2O2 via successive potential scan from 0 to -1.6 V at scan rate of 50 mV/s, as demonstrated in route 2 of Scheme 1, because the Ret of GCE/ATA, i.e. R1 in scheme 1, could be

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0.0

E vs. Ag/AgCl (V) Figure 5. ECL response of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs to samples containing CEA of different concentrations. Insert: corresponding calibration curve in semilogarithmic coordinates. The experiment was conducted by scanning GCE/ATA(Ω↓)-Ab1-AgAb2-NSs in 100 mM K2S2O8 + 60 mM H2O2 containing PB from 0 to -1.6 V at 50 mV/s. The concentration of CEA in curves a→k was shown in the inset section.

Performance of the designed ECL immunoassay with gC3N4 NSs as tags. As can be seen in Figure 5, ECL response of GCE/ATA(Ω↓)-Ab1-Ag-Ab2-NSs towards CEA samples of different concentrations displayed similar maximum-ECL-



emission-potential around -1.21 V, which was much closed to that of the GCE/ATA-Ab1-Ag-Ab2-NSs via successively scan in K2S2O8 + H2O2 containing PB for ten cycles (Figure 2A, black curve d). Importantly, the ECL peak intensity of GCE/ATA(Ω↓)Ab1-Ag-Ab2-NSs increased gradually towards sample with increasing CEA concentration. The maximum ECL intensity and logarithm of CEA concentration was linearly related in the range of 0.1 pg/mL ~ 1 ng/mL with the linear correlation coefficient of r2 = 0.992 and limit of detection of 3 fg/mL. The specificity of the proposed immunosensor was evaluated with three representative interfering proteins of high concentrations (Figure S6), such as AFP, CA15-3 and IgG. Compared with CEA blank samples, the existence of abundant interfering proteins in the samples, such as 10 pg/mL AFP, 10 pg/mL CA15-3, and 10 pg/mL IgG displayed negligible ECL response, respectively. The mixture containing 1 pg/mL CEA and all the three aforementioned interfering proteins with concentrations 10 times higher than that of CEA also displayed negligible effect on the ECL response of CEA too. All these results confirmed that electrochemically activating GCE/ATA with K2S2O8 + H2O2 in fabricating procedure would provide a promising nonenzymatic signal-amplification strategy to enhance ECL response of fabricated sensor with desired sensitivity.

CONCLUSIONS Reducing the Ret of ECL immunosensors was crucial to improve their sensing performance, because immobilizing electrochemiluminophores on electrode surface via immunereaction was the most frequently used strategy for ECL immunoassay. Herein, a promising signal-amplification strategy to reduce the impedance of ECL immunosensor for enhanced performance was proposed with carboxylated g-C3N4 NSs as electrochemiluminophores, effective signal-amplification was conveniently accomplished by electrochemically activating the substrate, i.e. the polymerized ATA film on GCE surface, in K2S2O8 + H2O2 containing PB before immobilizing biomolecules onto its surface. This electrochemical signalamplification strategy not only provided a promising alternative to the traditional enzymatic and nanoparticle based signalamplification strategy, but also proved that g-C3N4 NSs were promising toxic-element-free electrochemiluminophores with for bioassay.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI.

TEM, XRD, UV-Vis spectrum, infrared absorption spectrum, and PL spectrum of carboxylated g-C3N4 NSs; ECL spectrum of the GCE/ATA-Ag-Ab1-Ab2-NSs; effects of potential scan cycles on the potentials for maximum ECL emission of GCE/ATA-Ab1-Ag-Ab2-NSs; selectivity of the proposed immunoassay.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

Notes

ACKNOWLEDGMENT The authors gratefully acknowledge financial support of National Natural Science Foundation of China (21575080, 21427808), and the Fundamental Research Funds of Shandong University (2018JC017).

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