key: cord-0960096-hpaysnqy
authors: Liu, Jinhua; Ruan, Guotong; Ma, Wenlin; Sun, Yujie; Yu, Haidong; Xu, Zhihui; Yu, Changmin; Li, Hai; Zhang, Cheng-wu; Li, Lin
title: Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum
date: 2021-11-21
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2021.113823
sha: 9e85d6280e3ef2a23482ded9457add889cc16800
doc_id: 960096
cord_uid: hpaysnqy

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5–180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0–120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.

Owing to its excellent accuracy, practicality, low cost and high-throughput, 2 enzyme-linked immunosorbent assay (ELISA) has been extensively employed in food Unfortunately, ALP-labeled enzymes can only hydrolyze product with phosphate 9 groups and need to be in the strict alkaline environment, which limit its application 10 (Chen et al., 2018; Chen et al., 2020) . Alternative enzyme, HRP, gets relatively more 11 common application in diagnosis, biosensing due to its high specific activity, stability, (electron-donating groups and electron-withdrawing groups) into the PPD structure. 8 Additionally, we speculated that the strong fluorescence original of FCNPs was also 9 attributed to PEI with many cationic reactive primary amino groups and high charge 10 density. The in situ fluorogenic reaction platform could not only reduce the 11 interference of background signal, but also exhibit outstanding features such as 12 stability, wavelength-tunability, highly fluorescence quantum yield. Noteworthy, using 13 the cTnI and SARS-CoV-2 nucleocapsid protein (N protein) as the models, the 14 proposed fluorescent ELISA displayed high sensitivity for detecting cTnI and 15 SARS-CoV-2 N protein with a low limit of detection (LOD) of 0.19 ng/mL and 0.33 3 Briefly, horseradish peroxidase (120 mU/mL) and hydrogen peroxide (500 μM) 4 were dissolved in Tris-HCl buffer (10 mM, pH=7.4) . After incubating at 25 ° C for 15 5 minutes on a shaker, Polyethyleneimine (5 mg/mL, MW = 70000) and 6 p-phenylenediamine (60 μM) were added in it. The polymeric nanocluster was 7 obtained by mixture at room temperature within minutes. The relative quantum yield 8 of FCNPs was calculated by the following equation:

Where Φ is the fluorescence quantum yield, F is the integrated area of emitted 11 fluorescence spectra, and A is the absorbance at the excitation wavelength. The 12 subscript P and D implied product of PEI-PPD and quinine sulfate, respectively. 13 

All analysis performance processes have been listed in Supporting Information. chromogenic reaction using PPD and PEI as the substrates (Scheme 1A). 10 The catalytic activity of HRP toward PEI and PPD in the presence of H2O2 was To obtain better sensing performance, we optimized the pH and incubation time in the subsequent experiments. 5 As the emission spectral of reaction could be modulated by introducing various 6 substitutions, we administrated electron donating groups (EDGs) and electron 7 withdrawing groups (EWGs) to achieve wavelength tunability of FCNPs. The 3.2. Characterization of FCNPs. 5 The FCNPs were synthesized in the presence of HRP and H2O2. The optical 6 properties of FCNPs were characterized by absorption and fluorescence spectra. As These results illustrated that ascorbic acid and glutathione had a good capacity as a 1 terminating agent and could make fluorescence intensity of the system leveled off for 2 a long time (Fig. S7B ). 3 To further study the effect of different precursors on the formation of FCNPs, we The FL intensity of FCNPs gradually enhanced as the molecular weights increasing 2 (Fig. 3D) , implying that long-chain PEI precursors were more liable to achieve 3 folding and warping. To eliminate the possibility of alkali-induced fluorophores 4 formation, we introduced NaOH and other bases to replace PEI in the system (Fig.   5   3E) . The results showed that only PEI and EDA could react with PPD in the presence 6 of HRP and H2O2 to form a blue and green fluorescent copolymer, respectively. PEI, 7 as a polymer, had more cationic reactive primary amino groups and higher charge 8 density than EDA. So we speculated that the formation of the green fluorescent 9 copolymer was associated with the structure of PEI itself. 10 

After optimization, we use the assay to analyze HRP activity. The relationship 12 between HRP concentration and FL intensity at 510 nm was investigated in detail. As 13 presented in Fig. 4A , when HRP increased from 0 to 150 mU/mL, FL intensity of the 14 system at around 510 nm augmented gradually, and it displayed a two-stage good 15 linear relationship (Fig. 4B) color changes could be easily read under ultraviolet light (Fig. 4C) . The selectivity of 20 our proposed assay was investigated by comparing with other common biomolecules, 21 such as ALP, ACP, Ppase, BSA, Pepsin, Cyt-c, Tyrosinase, Trypsin and GOx. All the tests were performed in 1.5 mL centrifuge tubes. As shown in Fig. 4D , those 1 biomolecules, except HRP, with concentration of 120 mU/mL did not lead to 2 noticeable fluorescence change. These results indicated that our detection system 3 exhibited the excellent selectivity and selectivity toward HRP. 4 3.5. Fluorescent Immunoassay for cTnI and SARS-CoV-2 N protein. 5 Inspired by comprehensive application of HRP in ELISA, we initiated to explore respectively. Fig. 5A vividly displayed the strategy of HRP-labeled immunoassay for 13 the detection of cTnI. The specific antibody was pre-immobilized on a 96-well plate 14 to capture the target cTnI. Subsequently, through the specific recognition of the 15 antigen and antibody, the goat anti-cTnI antibody and HRP secondary antibody labels 1 previously reported assay for cTnI detection (Table S3) (Table S4 ). To 7 validate the sensing ability and specificity of our sensor toward target antigen cTnI, 8 nonspecific proteins, including alpha fetoprotein, lysozyme, pepsin and trypsin were 9 administrated, and some co-existing reducing species, such as AA, GSH, Cys, as well 10 as metal ions (Fe 3+ , Cu 2+ ) had been added the system for target sensing. None of those 11 substances induced obvious increase of fluorescence signal ( Fig. 5D and Fig. S11 ), 12 suggesting excellent selectivity of our assay for detecting cTnI. 13 In order to further verify the versatility of the proposed platform, we used the (Table S5) . Noteworthy, the cTnI concentration determined with our system was in 17 good agreement with the results obtained by commercial TMB-based standard ELISA 18 kit. Additionally, we listed the cost of our methods and commercial kit tests (Table S6,   19   Table S7 and Table S8 ). Compared with the traditional ELISA for testing a cTnI Table S9 , diluted human serum samples with 6 different concentration of N protein (0-120 ng/mL) were applied to the system. The other proteins are both 120 mU/mL. 

Microchimica Acta. 186

 13 The authors acknowledge the National Science Foundation of China (Grant nos.14 22174065) and financial support from the National Key R&D Program of China