key: cord-0694207-8qebgi3h
authors: Zhang, Kai; Fan, Zhenqiang; Yao, Bo; Zhang, Tingting; Ding, Yuedi; Zhu, Sha; Xie, Minhao
title: Entropy-Driven Electrochemiluminescence Ultra-Sensitive Detection Strategy of NF-κB p50 as the Regulator of Cytokine Storm
date: 2020-12-30
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112942
sha: f3a15682699b5b5dee6b324afc9c1e78ad66bd7a
doc_id: 694207
cord_uid: 8qebgi3h

2019 novel coronavirus (2019-nCoV) with strong contagion in the crowd, has ravaged worldwide and severely impacts the human health and epidemic prevention system, by producing a series of significant stress reactions in the body to induce further cytokine storm. Transcription factors (TFs) served as essential DNA binding proteins play an integral role in regulating cytokine storm, and the detection of it in the human coronavirus environment provides especially valuable approaches to diagnosis and treatment of 2019-nCoV and development of antiviral drugs. In this work, an entropy-driven electrochemiluminescence (ECL) biosensor was constructed for ultra-sensitive bioassay of NF-κB p50. The strategy primarily capitalizing the splendid double-stranded DNA (dsDNA) binding properties of transcription factors, employing GOAu-Ru composite material as ECL emitter, utilizing entropy-driven reactions for signal amplification method, offered a repeatable proposal for TFs detection. In the absence of TFs, the released DNA1 further went in the entropy-driven reaction, contributing to an “ECL off” state. However, in the presence of TFs, the dsDNA avoided being digested, which blocked DNA1 for participating in the entropy-driven reaction, and the system exhibited an “ECL on” state. Most importantly, the ECL bioanalytical method denoted broad application prospects for NF-κB p50 detection with a lower detection limit (9.1 pM).

). In addition, ECL analysis also avoids the problem that some 9 chemiluminescent reagents are laborious to deposit or unstable to use under specific 10 circumstance. For example, gold nanoparticles participated colorimetric reaction, 11 exists a situation where gold nanoparticles tend to aggregate which makes it difficult 12 to reuse and extremely reduces the reproducibility of the assays(Chen et al. 2014). In 13 various ECL biosensing systems, tris (2,2'-bipyridyl)-ruthenium (II) (Ru(bpy) 3 2+ ) and 14 its derivatives were commonly used for ECL emitters (Zhang et al. 2017 ). For the fact 15 that the Ru(bpy) 3 2+ ECL system consumes more emitting materials in the 16 homogeneous solution, therefore, the stability of the system is affected, which greatly 17 cuts down the detection efficiency and highly increases the cost. The oligonucleotides purified by high-performance liquid chromatography (HPLC) 9 used in our detection strategy were obtained from Genscript Biotech. Co., Ltd.

(Nanjing, China) with the sequences, as shown in Table S1 . Silver nitrate (AgNO 3 ) 11 and sodium borohydride (NaBH 4 ) were bought from Boer Chemical Reagent Co. Ltd. China), and the ultrapure water was processed through Milli-Q purification system. For the exonuclease III (Exo III) cleavage process, the protein binding solution after 26 incubation was added 50 μL of Exo III (2 U μL -1 ) to react for 15 min at 37 °C. And 27 then, the mixture solution was slowly heated to 75 °C and held for 10 min to cease the 28 cleavage process. Therefore, the reaction solution was prepared, which was ready for the next assay procedure. After that, the already fabricated ECL biosensor was dipped The process of cell culture was briefly illustrated as followed. Firstly, HeLa cells were 20 implemented in cell cultural medium (RPMI-1640) with the addition of 10% fetal 21 bovine, 100 μg mL -1 penicillin, and streptomycin in moist air (containing 5% CO 2 ). The new strategy of NF-κB p50 detection we designed mainly includes the 3 exonuclease III (Exo III) assisted nicking enzyme reaction, and entropy-driven 4 reaction participated ECL assays. When NF-κB p50 is present in the system, the The TEM image showed that the Au nanoparticles (AuNPs) were dispersed in the reaction could be extremely triggered by DNA1 and then exposed toehold for next 20 hybridization of DNA6 to produce more DNA3/6 and expel more DNA5. Therefore, 21 we could infer that the PAGE images visually demonstrated the feasibility of the 22 entropy-driven reaction. We first obtained the fluorescence spectrum ( Figure 3B , black curve) the and the ECL 27 spectrum ( Figure 3B , red curve) through the spectrophotometer and optical filters, 28 separately. The maximum ECL emission wavelength of GOAu-Ru was at 622 nm, and the fluorescence emission spectrum had a maximum value at 637 nm. Compared to 1 the fluorescence spectrum of GOAu-Ru, the ECL spectrum assumes a hypsochromic 2 shift, presumably the reason that GO and Au nanoparticles with high electron transfer 3 efficiency, increase the energy of transition. Next, for the clarification of the ECL 4 mechanism of emitter composites, Figure 3C and Figure 3D acted as co-reactant for the emitting process, the ECL intensity was much higher than 12 that in the assay solution without co-reactant. The whole evidence showed that 13 GOAu-Ru is served as ECL emitter, and TEA was acted as a co-reactant to improve 14 ECL efficiency. According to the experimental phenomenon above and earlier work, Firstly, CV was used to characterize the progressively modified GCE as shown in Figure 4A . The GCE before modification presented a redox peak, while with the 1 modification of GOAu-Ru, the CV signal peak gone down noticeably. After the 2 hybrid DNA probes, MCH, DNA1 by degrees modified to the GOAu-Ru/GCE, the 3 redox peaks successively declined, mainly due to the increased impedance of charge 4 transfer. However, with the AgNC modified DNA6 immobilized to the electrode, the 5 redox peak was obviously raised, for that the AgNC increased the efficiency of 6 electron transfer. Next, electrochemical impedance spectroscopy (EIS) was further 7 employed to describe the surface impedance after each stepwise modification. In 8 Figure 4B , as the classical model circuit illustrated, the total impedances composed of 9 real Z′ and imaginary Z″, were reflected by circuit elements in the form of circuit 10 diagram, which mainly related to the resistance and capacitance of the 11 electrochemical system. The real Z′ chiefly included electrolyte's ohmic resistance 12 (Rs), the impedance of charge transfer (Rct), and Warburg diffusion impedance (W).

In this three-electrode system, Rs reflected the resistance between GCE and the 14 Ag/AgCl reference electrode, Rct corresponded the interfacial impedance of electrode, process were also discussed as depicted in Figure 4C . Apparently, the adsorption of Assay performance of the biosensor for testing various concentrations of NF-κB p50 8 was investigated, which was illustrated in Figure 5A . By calculating the degree of the 9 ECL signal increase, the concentrations of the target protein were analyzed. As Figure   10 5B given above depicted, the ΔECL intensity increased rapidly with the concentration 11 increase of the NF-κB p50 ranged from 0 pM to 1 nM. Besides, the relationship 12 between ΔECL intensity and concentration of target protein was also investigated 13 (inset in Figure 5B ), the linear equation was Y = 1.64 + 3.22X with R 2 = 0.998, in 14 which the Y represented the value difference of ECL between sample at a specific 15 concentration and the blank sample ranged from 0 to 300 pM, X represented the 16 concentrations of target proteins. What is more, the limit of the detection (LOD) was 17 9.1 pM, which was comparable to previous work shown in Table S3 in the past few 18 years. We also investigated the assay performance in diluted nuclear extracts to 19 evaluate the validity of the sensor. Table 1 showed that the recoveries in Tris-HCl 20 buffers with and without 10-fold diluted nuclear extracts ranged from 99. The specificity of the ECL system was verified by four selected non-specific proteins, 4 the concentration of which was 10-folded that of the target protein (100 pM), 5 consisting of bovine serum albumin (BSA), acid-binding immunoglobulin (Ig)-like 6 lectin 5 (Siglec-5), carcinoembryonic antigen (CEA), and interferon-γ (IFN-γ). As 7 depicted in Figure 5C , the quenching efficiency was much lower when BSA, Siglec-5, 8 CEA, IFN-γ existed, which was a similar quenching effect with the blank sample. In 9 contrast, a much higher quenching efficiency was noticed in the presence of NF-κB 10 p50, revealing the well-designed biosensor was with excellent selectivity towards 11 NF-κB p50. In addition, the stability of the ECL signal was explored by estimating 12 consequent potential canning of 8 cycles. The Figure 5D showed the ECL response 13 under different concentrations of target protein, the corresponding relative standard 14 deviations (RSD) calculated were within the acceptable range (5%), denoting the 15 sensing method was with excellent reproducibility and highly appliable value. 

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