Ultrasensitive Homogeneous Electrochemical Detection of

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Ultrasensitive Homogeneous Electrochemical Detection of Transcription Factor by Coupled Isothermal Cleavage Reaction and Cycling Amplification Based on Exonuclease III Lihua Lu, Huijuan Su, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01538 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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Analytical Chemistry

Ultrasensitive Homogeneous Electrochemical Detection of Transcription Factor by Coupled Isothermal Cleavage Reaction and Cycling Amplification Based on Exonuclease III Lihua Lu, Huijuan Su, and Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China * Corresponding author. Tel/Fax: 86-532-86080855 E-mail: [email protected]

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ABSTRACT The assay and monitoring of transcription factors (TFs) has attracted extensive attention, due to their important roles in regulation of gene expressions. Herein, a simple, low cost, rapid, and highly sensitive homogeneous electrochemical method utilizing the coupled isothermal cleavage reaction and cycling amplification based on exonuclease III (Exo III) was explored for the analysis of transcription factor NF-κB p50 in aqueous solution. In the assay, a 3′-methylene blue (MB)-labeled hairpin probe is designed, which can be opened up by the single stranded DNA (ssDNA) protected by NF-κB p50 from the Exo III cleavage, to trigger the subsequent Exo III-assisted digestion, thus a large amount of MB-labeled mononucleotides are liberated to result in the greatly amplified electrochemical signal. By virtue of this Exo III-assisted target recycling, the present assay allows the detection of NF-κB p50 at the picomolar level, which is an exciting level for TFs detection. Furthermore, this detection possesses excellent selectivity, demonstrating high application potential in biological system and convenient TFs’ inhibitors screening. Comparing with the other reported strategies for TFs detection, this Exo III-assisted homogeneous electrochemical detection platform was just composed of one kind of enzyme and two DNA probes, offered a really simple and low-cost electrochemical detection for TFs assay.

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INTRODUCTION Transcription factors (TFs) are a type of functional proteins, which can specifically bind to a certain DNA sequence (usually, 6–12 base pairs) and function in a complex manner to dominate the transcription of a target gene.1,2 Regular transcription plays pivotal roles in the regulation of gene expression, especially to the cell development, differentiation and growth in biological system. However, the improper activation or inhibition of TFs can cause various diseases including congenital heart disease, developmental disorders, diabetes and cancers.3,4 Therefore, in order to effectively prevent or diagnose the TFs-related human diseases, rapid and ultrasensitive assays of TFs are eagerly desired. Traditionally, DNA footprinting,5 Western blotting,6 electrophoretic mobility shift assay (EMSA)7 and enzyme-linked immunosorbent assay (ELISA)8 have been employed to assay TFs. Although these techniques were widely used, they are generally tedious, discontinuous and expensive for the routine detection of TFs, and necessitate the utility of stringent safety techniques to prevent radiographic exposure or the use of unstable antibodies.9 Recently, luminescent,10-13 surface enhanced resonance Raman scattering (SERRS),14 colorimetric15 and electrochemical methods16,17 have been used for the detection of TFs based on the DNA-binding property and/or the exonuclease III (Exo III) resistance property of TFs (TF and its recognition DNA sequence combines into a stable TF/double-stranded DNA (dsDNA) complex, which sterically resist Exo III digestion, because Exo III can only specifically cleave the 3′-terminus of a dsDNA). Among these strategies, electrochemical strategies have attracted great attention due to their promising advantages of high sensitivity, low cost and easy-operation.17-19 For example, Plaxco and colleagues developed an E-DNA-like biosensor for TATA-binding protein (TBP) detection.16 Li and co-workers presented an electrochemical nanosensor for NF-κB p50 detection by employing triplex DNA and gold nanoparticles (AuNPs).19 Wang and colleagues constructed an electrochemical sensing template for NF-κB 3



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p50 assay by virtue of nicking endonuclease-assisted and gold nanoparticle-aided amplification reaction.18 However, these reported detections are heterogeneous assays. In order to avoid relatively low binding efficiency in tedious and time-consuming immobilization procedures, homogenous electrochemical biosensing strategies have been highlighted as the best alternative to the heterogeneous electrochemical methods.20-23 Compared with heterogeneous assays, homogeneous electrochemistry avoid immobilization and all of the combination between the DNA probes and target molecules as well as the enzymatic reactions occur in the solution phase instead of on the electrode surface, since the signal of a homogeneous electrochemistry comes from the different diffusivity between electroactive reporter labeled DNA and mononucleotides to a charged electrode surface. A typical homogeneous electrochemistry usually consists of three necessary components, an electrode with conductive film (such as a negatively charged indium tin oxide (ITO) electrode), an electroactive agent (such as methylene blue or other electroactive agents) labeled hairpin or dsDNA molecular beacon and one kind of exonuclease (such as Exo III or other exonucleases) to release the electroactive agent into the electrode surface.20,24-27 By virtue of this simply designed homogeneous electrochemistry strategy, various targets, such as metal ions, DNA and small biological molecules, could be sensitively detected.22,28-33 In order to sensitively detect trace amount of TFs in biological systems, several DNA or enzyme-assisted amplification approaches have been demonstrated, including the isothermally exponential amplification-based chemiluminescence or colorimetric assay,34,35 solid-phase rolling circle amplification (RCA)-based near-infrared fluorescent detection,15 the target-converted helicase-dependent amplification assay and a hairpin DNA cascade reaction signal amplifier.36 Despite the fact that these methods can dramatically increase the detection sensitivity of TFs, several kinds of enzymes and multiple DNA probes were used simultaneously in one assay, which leads to rather complicated operation procedures and 4


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increased cost. By contrast, Exo III-assisted target recycling amplification have been widely used in colorimetric, fluorescent, electrochemical detections, especially in homogeneous electrochemistry assays, due to its robustness, simplicity and low cost.37-40 Inspired by these concepts, in this study, we developed an ultrasensitive homogeneous electrochemical strategy for transcription factor NF-κB p50 assay by using Exo III-assisted cleavage and cycling amplification. In the strategy, there were just one kind of enzyme and two DNA probes involved in the sensitive homogeneous electrochemistry detection, presenting a really simple and low-cost homogeneous electrochemical strategy for TFs detection. To the best of our knowledge, no homogeneous electrochemical-based assay for the analysis of TFs has yet been published in literatures.

EXPERIMENTAL SECTION Reagents. MgCl2, KCl, HCl, tris(hydroxymethyl)aminomethane (Tris) and other agents with analytical grade were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further treatment. Exo III was purchased from New England Biolabs Inc. (Beverly, MA, USA). The Milli Q water (Millipore Corp., Bedford, MA, USA) was used in all experiments. NF-κB p50 was bought from Cayman Chemical (Ann Arbor, MI, USA). Thrombin (TB), bovine serum albumin (BSA) and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA) for the selectivity experiment. Oridonin was bought from Solarbio (Beijing, China). All oligonucleotides (including MB-labeled DNA) were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) and was dissolved in Tris-HCl buffer (10 mM, pH = 7.4) to get the stock solution with the concentration of 50 µM. All DNA sequences used in this work were listed in Table 1.

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Table 1. DNA sequences used in this study a Name ON1

5′-AGATG2A3GTC3T2ATACGTACACTC3TA-3′

ON2

5′-TAG3AGTGTACGTATA2G3ACT3C2ATCT-3′

ON4-MB

5′- GTGTACGCT2ATACGTACACTC3TA-MB-3′

ON4

a

Sequence (from 5′ to 3′)

5′- GTGTACGCT2ATACGTACACTC3TA-3′

ON1-mut1

5′-AGATC2G3TCA3T2ATACGTACACTC3TA-3′

ON2-mut1

5′-TAG3AGTGTACGTATA2T3GAC3G2ATCT-3′

ON1-mut2

5′-AGATG2A3GTC3C2GAT2GACTGCAGT3TA-3′

ON2-mut2

5′-TAA3CTGCAGTCA2TCG2G3ACT3C2ATCT-3′

In ON1 and ON2, the letters in boldface represent the recognition sequences for NF-κB p50. In ON4-MB

and ON4, the italic and underlined letters represent the sequences forming the stems of the hairpin probes, respectively. For ON1-MUT1, ON2-MUT1, ON1-MUT2 and ON2-MUT2, the boldface and underlined letters represent the mutant bases in these sequences.

Exo III-assisted homogeneous electrochemical NF-κB p50 detection. The single-stranded DNA (ssDNA) ON1 (50 µM) and ON2 (50 µM) were mixed in Tris-HCl buffer (10 mM, 100 mM NaCl, pH = 7.5) and were heated to 95 oC for 5 min, cooled to room temperature at 0.1 oC/s, then kept at room temperature for 1 h to ensure the production of completely hybridized double-stranded DNA (dsDNA) ON1/ON2 (25 µM). The prepared duplex substrate ON1/ON2 was stored at 20 oC for later use. The newly formed dsDNA ON1/ON2 contains a NF-κB p50 recognition site near one of its ends. The methylene blue (MB)-labeled DNA (50 µM) ON4-MB or non-labeled DNA ON4 (used in gel electrophoresis experiment) was treated to form a stable hairpin structure by using the same procedures as that for the ON1/ON2 dsDNA, excluding the mixing step. For the assay of NF-κB p50, certain amount of NF-κB p50 was added into 20 µL NF-κB p50 binding buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, pH = 7.5) containing ascertained amount of as-prepared dsDNA ON1/ON2. 6


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The mixture was incubated at room temperature for 30 min to allow NF-κB p50 protein binding with its recognition DNA sequences. Subsequently, the digestion reaction was started at 37 oC upon the addition of the indicated amount of Exo III. After 30-min incubation, the hairpin DNA ON4-MB was put into the reaction system to actuate the signal amplification procedure. At the same time, certain volume of the reaction buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.1 mM EDTA, pH = 7.5) was added to the system to get a final volume of 60 µL. Then, the mixture was further incubated at 37 oC for about one hour to let the Exo III-aided cycle happen before the electrochemical test. The control experiment was carried out under the same condition in the absence of NF-κB p50. All experiments were repeated three times. Detection of NF-κB p50 in cell lysate. 1 µM of as-prepared dsDNA was added in NF-κB p50 binding buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, pH = 7.5) containing 6 µL SK-BR-3 cell lysate and certain amount of NF-κB p50. It means that the cell lysate was diluted 10 times in this detection since the final volume of the mixture was 60 µL. Then, the following procedures were the same as the detection procedures in buffered solution.

Screening of NF-κB p50 inhibitor. Certain amount of NF-κB p50 were added in 20 µL NF-κB p50 binding buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, pH = 7.5) containing the prepared dsDNA ON1/ON2 and the indicated concentration of oridonin (3 µM). Then, the following procedures were the same as the NF-κB p50 assay in buffered solution.

Gel Electrophoresis. In order to avoid the effect of MB molecules, in gel electrophoresis the non-labeled hairpin DNA ON4, possessed the same sequences as ON4-MB, displaced MB-labeled ON4-MB. Then, the different products in the Exo III-assisted homogeneous electrochemical NF-κB p50 7



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assay were loaded on a 12% nondenaturing polyacrylamide gel and the gel was run at room temperature in TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) at 110 V for 50 min. The SYBR Gold stained gel was imaged by the Gel Doc XR+ Imaging System (BIO-RAD, America).

RESULTS AND DISCUSSION ITO electrode pretreatment and homogeneous electrochemical measurement. The ITO electrode, with an active surface area of 0.12 cm2, was carefully pretreated according to the reported method.26 Specifically, ITO electrode was sonicated in an Alconox aqueous solution, propan-2-ol, acetone, and Milli Q water for 15 min, in turn. Then, the treated ITO electrode was dipped into NaOH solution (1 mM) for about 4 h at room temperature and sonicated in Milli Q water for 15 min. After these treatment steps, the negatively charged electrode surface was achieved and was ready for later use.41 Figure S1 showed the differential pulse voltammetric (DPV) peak current of MB with the treated ITO electrode and with the un-treated ITO electrode. The results showed that the treated ITO electrode exhibits a well-formed DPV peak with a slight left-shift when compared with the untreated one. The electrochemical measurements were implemented on an Autolab electrochemical workstation (Metrohm, Netherland). Before the homogenous electrochemical measurements, we tested the DPV peaks of MB with potential scanning from −0.5 to 0 V or with potential from 0 to −0.5 V, to get oxidation current or reduction current signal, respectively. The results showed that the two signals are similar at DPV peak position and size (Figure S2). So, the DPV peak with oxidation current was then used in DVP measurements in this work. Furthermore, the pH-dependent experiments of MB were carried out to investigate the influence of pH to this assay. The DPV peak current of MB exhibited the highest value at pH = 7.5, and the peaks shifted left as the pH value increased from 5.5 to 9.5 (Figure S3). So in all of the rest experiments, we fixed the pH value at 7.5. Other parameters for electrochemical measurements were set as follows: modulation time = 50 8


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ms, step potential = 5 mV, interval time = 0.5 s, modulation amplitude = 25 mV, scan rate = 10 mV/s. The common three-electrode system was used to perform all of the electrochemical testing, in which, ITO electrode, Ag/AgCl and platinum wire were used as the working electrode, reference electrode and auxiliary electrode, respectively.

Mechanism of Homogeneous Electrochemical-based NF-κB p50 Assay. A simple homogeneous electrochemical method was designed and the two-part formed mechanism of this assay was outlined in Figure 1. The homogeneous electrochemical detection was based on the different diffusivity between MB tagged by mononucleotides and a hairpin-structured DNA or dsDNA toward the negatively charged ITO electrode surface, since the MB-tagged mononucleotides have much stronger diffusivity to the negatively charged ITO electrode attributed to its smaller size and less negative charge, demonstrating an obvious electrochemical signal enhancement. In the signal converter part, ssDNA ON1 and ON2 initially formed into a complete hybridized dsDNA, which contained the binding sequence (purple double lines in Scheme 1) of NF-κB p50 at one of its ends. After being incubated with NF-κB p50 and treated by Exo III, the ssDNA ON3 attached with the Exo III’s digestion product NF-κB p50/dsDNA complex, was released from the original dsDNA ON1/ON2 due to the protection of NF-κB p50 against the digestion of the 3′-end of ON2 by Exo III. So, the signal converter part played an important role in transducing the NF-κB p50 signal into the ssDNA signal. Subsequently, the carefully designed hairpin DNA ON4-MB was introduced into the reaction system. Importantly, the ON4-MB had an MB molecule attached to its protruded 3′-end, which could effectively resist the Exo III digestion, and the attached MB molecule offered an electrochemical signal for the assay. Then, the nascent ssDNA part of ON3 was able to open up ON4-MB hairpin DNA to trigger the subsequent digestion in the presence of the already existed Exo III. This digestion not only liberated the free ssDNA part of ON3, which then opened up new ON4-MB DNA to 9



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trigger the next recycling digestion procedures, but also led to the quantity growing of MB-tagged mononucleotides and brought about the significantly increased electrochemical signal. In the absence of NF-κB p50, the duplex substrate ON1/ON2 will be completely digested and cannot produce the promoter DNA ON3. Therefore, the MB will be still attached to ON4-MB, and will lose the ability to generate the increased electrochemical signal because of the electrostatic repulsive force between the negatively charged ON4-MB probe and ITO electrode. Importantly, both of the two parts were isothermal processes for this Exo III-aided homogeneous electrochemical strategy for NF-κB p50 assay.

Scheme 1. The mechanism of Exo III-aided homogeneous electrochemical strategy for NF-κB p50 assay.

Feasibility of Homogeneous Electrochemical-based NF-κB p50 Assay. To verify the implement of the designed strategy, we tested the differential pulse voltammograms (DPV) responses of the reaction system under different conditions. As shown in Figure 2A, there is no DPV signal when just dsDNA ON1/ON2 and NF-κB p50 were added into the buffer (curve a). When MB-labeled hairpin DNA ON4-MB was put into the system, the system gave a weak DPV signal (curve b) due to the electrostatic repulsive force between the negatively charged ITO electrode and the MB-tagged ON4 probe. After incubating ON4-MB probe with Exo III at 37 °C for 30 min, a slightly increased electrochemical signal

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was obtained (curve c), which might be ascribed to the nonspecific digestion of ON4-MB probes by Exo III. However, when ON4-MB probe was put into the reaction system after the duplex DNA ON1/ON2 was incubated with NF-κB p50 and treated by Exo III afterwards, the electrochemical signal showed a significant enhancement (curve d). This demonstrates that ON4-MB can be opened up by the trigger probe ON3 and then be cleaved by Exo III, releasing MB-labeled mononucleotides and free ON3 again. The hybridization of ON3 and excessive ON4-MB probes followed by the digestion of Exo III, resulted in a significantly increased electrochemical signal. The current peaks exhibited by curve b and c were relatively weak and can be ignored comparing with the obviously bigger signal in curve d. To further investigate this assay, the gel electrophoresis experiment was carried out (Figure 1B). In order to avoid the interference of MB molecules, the non-labeled hairpin DNA ON4, possessed the same sequences as ON4-MB, displaced MB-labeled ON4-MB in the gel electrophoresis experiment. The ssDNA ON2 was used as the reference band (lane 1). Hairpin DNA ON4 only (lane 2) showed a similar band as that demonstrated by ON4 + Exo III system (lane 3), showing that Exo III cannot digest the protruding 3′-end of hairpin DNA ON4. The dsDNA ON1/ON2 was incubated with NF-κB p50, exhibiting a slower moving band (lane 4) than the reference band of ON2. The comparative gel electrophoresis experiments between ON1/ON2 and ON1/ON2 + NF-κB p50 were presented in Figure S4, and the results showed no obvious difference between ON1/ON2 (lane 3) and ON1/ON2 + NF-κB p50 (lane 4). We inferred that the binding of NF-κB p50 with the dsDNA was destroyed during the running of non-denaturing PAGE. While the duplex ON1/ON2 treated by Exo III in the absence of NF-κB p50, demonstrated no signal (lane 5) and revealed the complete digestion of ON1/ON2 duplex by Exo III. The system containing ON1/ON2 + hairpin DNA ON4 + Exo III, just showed the band of the hairpin DNA ON4 in the absence of NF-κB p50 (lane 6), indicating that the ON1/ON2 duplex is also digested and the trigger DNA ON3 is not generated. The system 11



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containing ON1/ON2 + NF-κB p50 + Exo III and the system containing ON1/ON2 + NF-κB p50 + Exo III + hairpin DNA ON4, demonstrated the same new bands in lane 7 and lane 8, respectively. According to the detection principle, ON1/ON2 in lane 7 was completely digested by Exo III, and the DNA triggers ON3 protected by NF-κB p50 was remained. While, the added hairpin DNA ON4 in lane 8 was continually opened by ON3 and then was cleaved by Exo III, so what left was also DNA trigger ON3. ON3 possesses a shorter ssDNA sequence than ON2 because of the cleavage by Exo III, which led to the faster motion of ON3 than the reference DNA ON2. So the experimental result agrees well with the proposed assay principle. Two mutant experiments were carried out to further confirm the feasibility of this detection. One mutant dsDNA is ON1-mut1/ON2-mut1, which does not contain the binding site of NF-κB p50, and the other one is dsDNA ON1-mut2/ON2-mut2, which contains the DNA sequences not complementary to the hairpin DNA ON4-MB. As expected, for these two mutant DNA substrates, the reaction system only demonstrated minimal change in the DPV peaks of MB, indicating that the feasibility of this two part-formed detection platform (Figure S5). Taken together, these results confirmed the detection mechanism shown in Scheme 1, suggesting that the Exo III-assisted amplification assay can sensitively detect transcription factors.

Figure 1. (A) The DPV peak current of the system under different conditions: (a) dsDNA ON1/ON2 + NF-κB p50; (b) MB-labeled hairpin DNA ON4-MB; (c) MB-labeled hairpin DNA ON4-MB + Exo III; (d) ON1/ON2 + NF-κB p50 + Exo III + ON4-MB. The concentrations of ON1/ON2, NF-κB p50, ON4-MB 12


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and Exo III were 1 µM, 1 nM, 2 µM and 30 U/mL, respectively. (B) Nondenaturing PAGE imaging of Exo III-assisted homogeneous electrochemical NF-κB p50 products: lane 1, ON2; lane 2, Hairpin DNA ON4; lane 3, Hairpin DNA ON4 + Exo III; lane 4, ON1/ON2 + NF-κB p50; lane 5, ON1/ON2 + Exo III; lane 6, ON1/ON2 + Hairpin DNA + Exo III; lane 7, ON1/ON2 + NF-κB p50 + Exo III; lane 8, ON1/ON2 + NF-κB p50 + Exo III + Hairpin DNA. The concentrations of ON1/ON2, NF-κB p50, ON4-MB and Exo III were 1 µM, 30 nM, 1 µM and 30 U/mL, respectively.

Homogeneous electrochemical detection of NF-κB p50 in aqueous solution. As shown in Scheme 1, in the presence of NF-κB p50, the Exo III-assisted signal amplification was triggered in virtue of the protection of NF-κB p50. To obtain a higher sensitivity of this assay, appropriate amount of ON4-MB and Exo III are required in the reaction system to ensure the amplification part works properly. In the case of fixing the concentration of ON1/ON2, the optimization of ON4-MB concentration is achieved by testing the concentration ratio between dsDNA and hairpin DNA ON4-MB. As shown in Figure 2A, the DPV peak current change (the difference between the DPV peak current with 5 nM of NF-κB p50 and that without NF-κB p50) displayed the highest value when the concentration ratio between ON1/ON2 (1 µM) and ON4-MB was 1:2 in the reaction system. This is because insufficient ON4-MB will not supply plenty amplification signal, while overmuch ON4-MB will lead to a high background even in the absence of NF-κB p50. Therefore, 2 µM was chosen as the optimal concentration of ON4-MB. As shown in Figure 2B, the DPV peak current gradually increased with the amount of Exo III elevated from 10 to 30 U/mL, but the DPV peak current kept almost unchanged and leveled off with the amount of Exo III over 30 U/mL. Therefore, 30 U/mL was chosen as the optimal amount of Exo III in the subsequent experiments. In addition, in order to obtain the best amplification efficiency, the reaction time for Exo III-assisted NF-κB p50 assay was also studied. As shown in Figure 2C, the DPV peak current gradually reached its plateau when the reaction time was one hour, exhibiting a rapid detection performance of this assay. Thus, the reaction time was selected as one hour in the rest of the experiments. 13



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Figure 2. (A) The DPV peak current change (∆ip= ip − ip,0, in which ip is the DPV peak current in the presence of NF-κB p50, and ip,0 is the DPV peak current in the absence of NF-κB p50) versus the ratio of ON1/ON2 and ON4-MB. The concentrations of ON1/ON2, NF-κB p50 and Exo III were 1 µM, 5 nM and 30 U/mL, respectively; the reaction time was 1 hour. (B) The DPV peak current change versus the Exo III concentration. The concentrations of ON1/ON2, NF-κB p50 and ON4-MB were 1 µM, 5 nM and 2 µM, respectively; the reaction time was 1 hour. (C) The DPV peak current versus the reaction time. The concentrations of ON1/ON2, NF-κB p50, ON4-MB and Exo III were 1 µM, 5 nM, 2 µM and 30 U/mL, respectively. The error bars represent the standard deviation of three repetitive measurements. Under the optimal experimental conditions, the responses of the DPV peak current towards different concentrations of NF-κB p50 were investigated. As demonstrated by Figure 3A, the DPV peak current of the system increased with the concentration of NF-κB p50 increased from 0 to 50000 pM, which was consistent with the fact that higher concentration of NF-κB p50 would produce more ON3 triggers and more oligonucleotides tagged MB were released into the solution to increase the system’s diffusion current. The inset of Figure 3B showed a good linear relation between the value of DPV peak current and the 14


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logarithm of NF-κB p50 concentration ranging from 10 to 5000 pM. The directly measured detection limit for NF-κB p50 assay was 10 pM, which is comparable or superior to those of the previously reported strategies for TFs detection (Table S1). Furthermore, the repeatability of the homogenous electrochemical strategy was demonstrated through 7 successive detections in the presence of 0.5 nM NF-κB p50. The calculated relative standard deviation (RSD) was 3.52%, indicating an acceptable repeatability of the developed method for TFs assay (Figure S6). These results displayed that simple, fast and sensitive detection of NF-κB p50 was indeed achieved by this as-proposed homogenous electrochemical strategy.

Figure 3. (A) The responses of DPV peak current to the different concentrations of NF-κB p50: (a) 0, (b) 10, (c) 50, (d) 100, (e) 500, (f) 1000, (g) 5000, (h) 10000, (i) 30000 and (j) 50000 pM. (B) The relationship between DPV peak current and NF-κB p50 concentration in buffered solution. Inset: The linear relationship between the DPV peak current and the logarithm of NF-κB p50 concentration ranging from 10 to 5000 pM. The error bars represent the standard deviation of three repetitive testing.

Specificity of the homogeneous electrochemical detection of NF-κB p50. The specificity of the detection platform for NF-κB p50 assay was demonstrated by investigating the DPV peak current of the system by substituting NF-κB p50 with several kinds of typical nonspecific binding proteins or the mixture of the nonspecific binding protein with NF-κB p50, such as BSA, insulin, TB, BSA + NF-κB p50, TB + NF-κB p50, and insulin + NF-κB p50, respectively. The results showed that only NF-κB p50 significantly increased the DPV peak current of the assay (Figure 4), and no significant enhancement in DPV peak current was observed upon the addition of other nonspecific binding proteins. These results 15



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confirmed that the proposed strategy possesses excellent selectivity for NF-κB p50 over other nonspecific binding proteins. This excellent selectivity originates from the specific binding of NF-κB p50 with the ON1/ON2 probes at the recognition site and hence triggers the subsequent amplification part, resulting in the increase of electrochemical signal.

Figure 4. The DPV peak current corresponding to different proteins and their mixtures: (a) background, (b) NF-κB p50, (c) Insulin, (d) TB, (e) BSA, (f) TB + NF-κB p50, (g) Insulin + NF-κB p50 and (h) BSA + NF-κB p50. The concentration of NF-κB p50 was 1 nM, and the concentration of other kinds of protein was 10 nM. The error bars represent the standard deviation of three repetitive measurements.

Detection of NF-κB p50 in diluted cell lysate and screening of inhibitors for NF-κB p50. To demonstrate the robustness of the detection platform, we investigated the performance of the developed assay for NF-κB p50 detection in the presence of 10% (v/v) SK-BR-3 cell lysate. Although 10% (v/v) of cell lysate would contain fewer interfering substrates compared with real undiluted samples, we found that the performance of our NF-κB p50 detection in diluted cell lysate was comparable to those recently reported protein assays, for example, Zhang and co-workers detected microphthalmia-associated transcription factor (MITF) in 10-fold diluted nuclear extracts,42 Ma and colleagues monitored various concentrations of protein tyrosine kinase-7 in 0.5% cell extract samples.43 In the diluted cell lysate, the assay still showed a linear growth in DPV peak current with the increase of NF-κB p50 concentration (Figure. 5A and 5B). Then the recovery test of NF-κB p50 in 10-fold diluted human serum was carried out. 16


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Table S2 showed the experimental results tested in 10-fold diluted serum samples spiked with NF-κB p50 with five concentrations (0, 0.5, 1.0, 3.0 and 5.0 nM), and the recoveries ranging from 94.9 to 107.5% were obtained.

Figure 5. (A) The responses of DPV peak current to the different concentrations of NF-κB p50: (a) 0, (b) 10, (c) 50, (d) 100, (e) 500, (f) 1000, (g) 5000, (h) 10000, (i) 30000 and (j) 50000 pM in the presence of 10% (v/v) SK-BR-3 cell lysate. (B) The relationship between DPV peak current and NF-κB p50 concentration in the presence of 10% (v/v) SK-BR-3 cell lysate. Inset: The linear relationship between the DPV peak current and the logarithm of NF-κB p50 concentration ranging from 10 to 5000 pM in the presence of 10% (v/v) SK-BR-3 cell lysate. The error bars represent the standard deviation of three repetitive testing. (C) The responses of DPV peak current to the different concentrations of oridonin: (a) 0, (b) 3, (c) 6, (d) 9, and (e) 12 µΜ in the presence of 5 nM NF-κB p50. Screening the compounds against NF-κB p50 through the proposed homogeneous electrochemical assay would be of great importance in exploring new treatment strategies for NF-κB p50-related diseases. Oridonin is a well-known inhibitor to the binding between NF-κB p50 and the recognition sites in dsDNA. As shown in Figure 5C, continuous reductions in the DPV peak current were observed when increasing 17



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amounts of oridonin were added into the detection system. This was because oridonin is able to inhibit the binding of NF-κB p50 with ON1/ON2 dsDNA and allow Exo III to completely cleave the dsDNA, leading to the decrease of electrochemical signal. The above result highlighted the feasibility of this strategy to the screening of certain molecules as inhibitors of NF-κB p50.

CONCLUSIONS We have developed an ultrasensitive homogeneous electrochemical strategy for transcription factor detection by using coupled isothermal cleavage reaction and Exo III-assisted cycling amplification. Taken advantage of its excellent sensitivity and selectivity to NF-κB p50 detection, the application of this NF-κB p50 assay in diluted cell lysate and even in the screening of the inhibitor of NF-κB p50 were carried out successfully. Furthermore, this strategy exhibits the advantages of simplicity and low cost, since just two DNA probes and one kind of enzyme were used in the assay and complicated probe immobilization processes were avoided. Therefore, the proposed homogeneous electrochemical TFs assay may become a promising strategy for simple and sensitive TFs detection and shows great potential to be applied in NF-κB p50-related clinical diagnostics and treatments.

ASSOCIATED CONTENT Supporting information

Cell lysate preparation, Supplementary Figure S1− −S6, Supplementary Table S1 and S2.

AUTHOR INFORMATION Corresponding Author

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*

E-mail: [email protected] (LF), Tel/Fax: 86-532-86080855

Author Contributions The manuscript includes contributions from all authors. All authors have approved the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China (Nos. 21375072 and 21575074), the Special Foundation for Distinguished Taishan Scholar of Shandong Province (No. ts201511052) and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 663-1116010).

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Ultrasensitive Homogeneous Electrochemical Detection of

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