Bobcat339 | Prep Signal

Assay for TET1 activity and its inhibitors screening with signal ampliﬁcation by both nanoparticles and Ru(III) redox recycling
Ying Chenga,b, Jing Tanga, Xue Chena, Fang Wanga,b,∗, Zilin Chena,b,∗
a Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China
b State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing, 100080, China

A R T I C L E I N F O A B S T R A C T

Article history:
Received 22 February 2021
Received in revised form 7 June 2021 Accepted 19 June 2021
Available online 24 June 2021

Keywords:
Electrochemical biosensor
Ten-eleven translocation protein 1 Demethylation
Nanoparticles Inhibitors screening

Ten-eleven translocation protein 1 (TET1) is one member of TET proteins family which plays a key role in dynamic DNA methylation-demethylation process. Herein, a novel biosensor was constructed for TET1 detection and its inhibitors screening utilizing restriction digestion of endonuclease enzyme MspI. Half- methylated oligonucleotide (5mC DNA) was used as target and Ru(NH3)63+ as electrochemical signal probe. After the treatment by TET1 and T4 β-glucosyltransferase (T4 β-GT), target oligonucleotide would not be recognized and digested. If there was no TET1, the target would be digested and the response of biosensor decreased greatly. The current difference of biosensor with and without the incubation with TET1 was therefore dependent on the concentration of TET1. To increase sensitivity of the biosensor, nanostructured ﬁlm at electrode surface and nanoparticles modiﬁed oligonucleotides were employed as signal ampliﬁcation elements for Ru(NH3)63+ recycling. Finally, this biosensor showed high performance with a wide linear range of TET1 concentration from 3.5–21 ng/µL and a low detection limit of 0.33 ng/µL, which is superior to other existing methods. The inhibition effects of Bobcat339 on TET1 was successfully proved by our biosensor with an IC50 of 38 µM. Not only that, but the feasibility of the biosensor for inhibitors screening was evaluated and further conﬁrmed by other compounds including two anticancer drugs and three active ingredients of traditional Chinese medicine.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction

As an important epigenetic marker, DNA methylation occurs at the C5 position of cytosine which is critical for gene expres- sion and transcription [1,2]. Abnormal level of DNA methylation in human cells leads to various diseases, such as epilepsy, childhood asthma and carcinogenesis progression [3–5]. Ten-eleven translo- cation (TET) proteins family (TET1, TET2 and TET3) mediates active DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC), and further to 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC) [6]. 5hmC plays an important role in demethylation. It can either be passively depleted through DNA replication or actively reverted to cytosine through itera- tive oxidation and thymine DNA glycosylase (TDG)-mediated base excision repair [7]. A great deal of research indicate that abnor-

∗ Corresponding authors at: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China.
E-mail addresses: [email protected] (F. Wang), [email protected] (Z. Chen).

mal expression of TET dioxygenases is associated with cancer [8–10]. Hence, a sensitive and speciﬁc method for TET detection and inhibitors screening is urgent to be developed considering scarce and expensive commercial kit.
Many methods have been proposed for evaluation of DNA methylation, but only a few are related to detection of TET1 including ﬂuorescent assays, enzyme linked immunosorbent assay (ELISA) and 2D-TCL [11–13]. For the most part, indirect method is employed by LC–MS through quantitative analysis of 5mC and 5hmC [14]. These methods suffer from poor sensitivity, hazardous radiation, multi-step or large sample consumption, preventing their extensive application in practical TET assay. More impor- tantly, the abundance of 5hmC, 5fC and 5caC is much lower than 5mC, which increases the difﬁculty of TET1 detection. At this time, electrochemical sensors with high sensitivity and low cost have been concerned as a strong competitor [15–17]. An N-heterocyclic carbine-based electrochemical biosensor was recently reported for the rapid detection of measles virus with a low detection limit of 6 µg/mL, providing a platform technology for COVID-19 detec- tion [18]. Of course, sensitive detection of DNA demethylation and demethylase have also been allowed by electrochemical biosensor

https://doi.org/10.1016/j.jpba.2021.114228

such as the ferrocene acetic acid (FcA)-labeled DNA sensor [19]. To our knowledge, one biosensor was reported for quantitative detec- tion of 5mC-DNA and TET1 using electrochemiluminescence (ECL). However, it mainly aimed at the quantiﬁcation of 5hmC and under- estimated the activity of TET1, while the secondary and tertiary products (5fC, 5caC) of 5mC are not detected [20].
Previous work has developed a cost-effective electrochemical method for sensitive determination of TET1. This method shows a good linear range from 0.0042 µg/µL to 0.0210 µg/µL and a low limit of detection 0.00098 µg/µL [21].To improve sensitiv- ity of biosensors, endeavors have been put into the development of ampliﬁcation strategies. Here, we proposed a new electro- chemical strategy to develop a biosensor for TET1 detection. This strategy was based on restriction digestion of endonuclease enzyme MspI. Because gold nanoparticles (AuNPs) possess electri- cal conductivity and high surface area-volume ratio, AuNPs ﬁlm at electrode surface and AuNPs modiﬁed oligonucleotides (AuNPs- DNA) were employed as signal ampliﬁcation elements [22–25]. The electrochemical signal was obtained by loading of redox-active indicator, Ru(NH3)63+ (RuHex), which can bind to anionic phos- phate backbone of DNA strands via electrostatic interaction [26,27]. In addition, Ru(NH3)63+ can be electrically reduced on the electrode and the resulting Ru(NH3)62+ can be cycled by a chemical reductant
(Fe(CN)63−). With signal ampliﬁcation strategies of AuNPs-DNA
complexes, endonuclease enzyme MspI and Ru(III) redox recycling, TET1 is able to be monitored sensitively and its inhibitors can be evaluated accurately, thus offering a novel platform for the discov- ery of anticancer drugs. In addition, all three TET proteins (TET1, TET2, TET3) belong to a family of dioxygenase enzymes. They share a common core catalytic domain at their C termini and identical catalytic activity to successively oxidize the methyl group of 5mC, yielding three distinct forms of oxidized methylcytosines. So, this method might also be suitable for evaluation the activity of TET2 and TET3.

2. Experimental section

2.1. Reagents and apparatus

Recombinant TET1 protein was purchased from Epigentek (Cat.No. E12002, USA) and stored at 80 ◦C. Endonucleases MspI enzyme (Cat.No. R0106 L) and 10 Cutsmart buffer (Cat.No. B7204S) were supplied by New England Biolabs (Ipswich, MA). T4 phage β-glucosyltransferase (T4 β-GT) and 10 uridine diphosphate glucose (UDP-glucose) was purchased from Thermo scientiﬁc company. Bobcat339 hydrochloride was purchased from Med Chem Express (Cat.No. HY-111558A). Hexaammineruthe- nium(III) chloride ([Ru(NH3)6]3+, RuHex), mercaptohexanol (MCH) and 5-ﬂuorouracil were purchased from Sigma–Aldrich (MO, USA). Hydrogen tetrachloroaurate trihydrate (HAuCl4 3H2O), tri (2- carboxyethyl) phosphine hydrochloride (TCEP), DL-dithiothreitol (DTT), α-ketoglutaric acid (α-KG), ferrous alum and resvera- trol were purchased from Aladdin (Shanghai, China). ATP was sourced from Biosharp (Guangzhou, China). Sodium phosphates, Tris HCl, sodium chloride (NaCl), potassium chloride (KCl), bovine serum albumin (BSA), ethylenediaminetetraacetic acid disodium salt (EDTA), concentrated hydrochloric acid, sodium hydroxide, chloroauric acid, HEPES (N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid), ascorbic acid, potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6) and other chem- icals were purchased from Sinopharm Group (Shanghai, China). Luteolin and gallic acid were purchased from Herbpurify (Chengdu, China). All oligonucleotides were synthesized by Takara Bio- technology Co., Ltd. (Dalian, China) (Table S1).

Various buffer solutions were used in this assay, including DNA immobilization buffer: 10 mM Tris HCl, 1 mM EDTA, 1 M NaCl, 1 mM TCEP, pH 7.4. TET1 stock buffer: 40 mM Tris HCl, 110 mM NaCl, 2.2 mM KCl, 0.04 % Tween-20, 20 % glycerol, pH 8.0. TET1 reaction buffer: 50 mM HEPES, 75 µM Fe(NH4)2(SO4)2, 2 mM ascor- bic acid, 1 mM α-KG, 2.5 mM DTT, 100 mM NaCl, pH 7.9. Washing buffer: 10 mM Tris HCl, 1 mM EDTA and 100 mM NaCl (pH = 7.4). All solutions were prepared with Milli-Q water (18.2 M▲ cm).

2.2. Instrumentation

All electrochemical experiments were carried out using a CHI660D electrochemical workstation (Shanghai Chenhua Instru- ments, China). A three-electrode system was employed all through the experiment, consisting of the AuNPs-modiﬁed working elec- trode, a platinum auxiliary electrode, and a saturated calomel electrode (SCE). All electrochemical measurements were carried out at room temperature in 1 mM PBS (pH 7.4) containing 4 mM Fe(CN)63− and 50 mM Ru(NH3)63+. UV–vis absorption spectroscopy (UV-2600, Shimadzu, Japan) and transmission electron microscopy (JEM-2100, JEOL, Japan) were used to investigate the morphology of AuNPs and AuNPs-DNA complexes. Currents were measured by differential pulse voltammetry (DPV) from 0.5 to +0.1 V. Error bars shown on individual ﬁgures correspond to variabilities among three independent trials of each experiment.

2.3. Preparation of AuNPs-DNA S3

AuNPs were prepared based on a standard citrate method [28]. First of all, the glassware was thoroughly washed with freshly pre- pared aqua regia (HCl: HNO3, 3:1) and ultrapure water successively. Next, 50.0 mL of HAuCl4 solution (0.01 %, m/v) was quickly brought to the boil under vigorously stirring, and 2.0 mL of trisodium citrate solution (1%, m/v) was added immediately into the boiling solution. The solution was allowed to react until its color changed from pale yellow to wine red, which was followed by continuing heating to reﬂux for 10 min. After stirring, cooled to room temperature, and stored the prepared wine-red AuNPs in a brown bottle at 4 ◦C for future use.
The thiolated DNA S3 was grafted onto the surface of AuNPs to prepare AuNPs-DNA complexes. 5.0 µL 100 µM DNA S3 and
200.0 µL AuNPs were mixed and incubated at 4 ◦C for 12 h. Then
5.0 µL 1% SDS was added to make AuNPs oscillate for 1 h at room temperature. Afterwards, 100.0 µL of 1 M PBS solution (pH 7.4) was slowly added to age the mixture for 12 h. Then, it was centrifuged three times at 12,000 rpm for 10 min and washed with 1 M PBS solution. Finally, the AuNPs-DNA complex (AuNPs-DNA-S3) was dissolved with 200.0 µL of 1 M PBS solution.

2.4. Gold nanoparticles deposition

The bare glassy carbon electrode (GCE, A = 0.071 cm2) was mechanically polished with polishing micro-cloth containing
0.05 µm Al2O3 slurry to a mirror ﬁnish, and then carefully cleaned in HNO3-H2O (v/v, 1:1) and ethanol, deionized water in turn via ultra-sonication each for 2 min. AuNPs were electrodeposited at -
0.20 V for 200 s in 1.0 mL of solution containing 3 mM HAuCl4 and
0.1 M KNO3. The prepared electrode is denoted as AuNPs/GCE.

2.5. Fabrication of the electrochemical biosensor

5.0 µL of DNA S1 solution (1 µM) was dropped on the surface of the AuNPs/GCE electrode for 13 h at 37 ◦C and then the obtained electrode was sealed with 10.0 µL MCH for 30 min. Hybridiza- tion was then performed by dropping 5.0 µL of DNA S2 solution (1 µM) for 2 h at 37 ◦C. Subsequently, 10.0 µL of AuNPs-DNA S3

Scheme 1. The principle of biosensor for TET1 detection.

were dropped onto the electrode surface for 2 h at 37 ◦C to hybridize the upper portion of DNA S1 (The obtained electrode is denoted as AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE). For demethy- lation, the electrode was incubated by adding 5.0 µL solution of TET1 reaction buffer containing different concentrations of TET 1 protein at 37 ◦C. After 1 h, 10.0 µL of T4 β-GT reaction buffer con- taining 50 unit/mL T4 β-GT and 40 µM UDP-glucose was dropped on the electrode and the electrode kept for 30 min at 37 ◦C. MspI cleavage was performed at 37 ◦C for 1 h in MspI buffer. After each step, the electrode was rinsed with Tris HCl buffer and distilled water.

2.6. Inﬂuence of inhibitors on TET1 activity

The inhibition effects of Bobcat339 on TET1 activity was quan- titatively analyzed using this method. All the drug inhibition experiments were performed under the same experimental con- ditions with TET1 assay, only with the addition of Bobcat339 in the oxidation step contained TET1 (21 ng/µL). Moreover, ﬁve com- pounds (100 µg/mL taxol, 5-ﬂuorouracil, resveratrol, gallic acid and luteolin) were also evaluated for the inhibition of TET1.

3. Results and discussion

3.1. Design of the electrochemical assay for TET 1 detection

The schematic diagram of this approach is shown in Scheme 1. The target DNA S1 (5mC DNA) modiﬁed with thiol was ﬁrstly self-assembled onto the surface of AuNPs modiﬁed GCE via Au- S bonding, followed by hybridization with DNA S2 to form double-stranded DNA containing a speciﬁc recognition sequence of 5r-CmCGG-3r for endonuclease enzyme MspI. AuNPs-DNA S3 ampliﬁcation unit was then successively brought onto the electrode surface through hybridization between DNA S3 and the upper por- tion of DNA S1. If there was no TET1, the target DNA S1 would be digested by MspI and the response of biosensor was weak. How- ever, after the treatment by TET1, 5mC was oxidized to 5hmC, 5fC or 5caC [29]. DNA S1 would not be recognized and digested by MspI because 5fC and 5caC could not be recognized by MspI as reported in the research. Besides, glycosylation of 5hmC residues by T4 β-GT can make CCGG residues insensitive to digestion by MspI [30–34]. As a DNA-binding and redox-active probe, Ru(NH3)63+

could bind to anionic phosphate backbone of DNA strands via electrostatic interaction. The oligonucleotide designed in biosen- sor has six methyl groups in the 5r as mentioned in Table S1 (ESI). So the phosphate keeps certain distance from the surface of the electrode which avoided the steric hindrance in the connection of Ru(NH3)63+. A large amount of Ru(NH3)63+ could be adsorbed on the electrode with AuNPs-DNA S3. Furthermore, the produced Ru(NH3)62+ was cycled at the electrode surface in the presence of reductant (Fe(CN)63−), leading to the recycling of Ru(III) redox and a further increase of the cathodic current. So, after the incu- bation with TET1, the current of biosensor was very large. The current difference of biosensor with and without the incubation with TET1 was therefore dependent on the concentration of TET1. TET1 and DNA methylation could then be sensitively determined, which could be attributed to the powerful ampliﬁcation effect of the AuNPs-DNA S3 and Ru(NH3)63+ recycling. Moreover, screening of TET1 inhibitors is very signiﬁcant to diagnosis and treatment of disease because abnormal DNA methylation is closely related to many diseases. In this work, we also used the approach to screen the inhibitors of TET1, which may be helpful for the discovery of relevant anticancer drugs.

3.2. Characterization

3.2.1. Characterization of AuNPs and AuNPs DNA S3
TEM and UV–vis absorption spectra were used to characterize AuNPs and AuNPs-DNA S3. TEM analysis of AuNPs shows that the diameter of AuNPs is about 18 nm (Fig. 1a). That of AuNPs-DNA S3 was almost the same as AuNPs (Fig. 1b) and maybe because the DNA S3 is too short to characterize (only 18 bases). Consequently, UV–vis absorption spectra (Fig. 1c) was carried out to further characterize AuNPs and AuNPs-DNA S3. AuNPs have an absorption maximum located at h = 520 nm (curve i). After the modiﬁcation with DNA S3, a shift of the UV–vis absorption peak was observed, where hmax shifted from 520 to 524 nm (curve ii). Because DNA ligands close to the surface of AuNPs may increase the refractive index of AuNPs’ immediate environment and subsequently cause a red shift. The above results are consistent with previous reports [35,36], indicat- ing DNA S3 and AuNPs were conjugated successfully.

Fig. 1. The TEM images of (a) AuNPs and (b) AuNPs-DNA S3. (c) UV–vis absorption of (i) AuNPs (ii) AuNPs-DNA S3.

the AuNPs/GCE electrode through Au S bond and followed by MCH blocking and hybridization of DNA S2, Ru(NH3)63+ was accumulated as the electrochemical indicator and only a little current responses were observed (Fig. 3a, curve i). When the DNA S2/MCH/DNA S1/AuNPs/GCE electrode hybridized with DNA S3, peak current showed a little higher than that obtained at curve i (Fig. 3a, curve ii). But the current increased signiﬁcantly (Fig. 3a, curve iii) after hybridization of AuNPs-DNA S3 on the DNA S2/MCH/DNA S1/AuNPs/GCE electrode, which due to more Ru(NH3)63+ electrostatically bound to AuNPs-DNA S3. However, a dramatic decrease of current signal was appeared after that the AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrode were only treated with T4 β-GT (50 U/mL) and MspI (200 U/mL) (Fig. 3a, curve v). The decrease of the peak current was mainly derived from the speciﬁc recognition and cleavage of 5r-CmCGG-3r by MspI. Simultaneously, large number of AuNPs-DNA S3 were released

Fig. 2. Cyclic voltammograms of different modiﬁed electrodes in 5 mM
K4 Fe(CN)6 / K3 Fe(CN)6 . (i) GCE; (ii) AuNPs/GCE; (iii) DNA S1/AuNPs/GCE; (iv) MCH/DNA S1/AuNPs/GCE; (v) DNA S2/MCH/DNA S1/AuNPs/GCE; (vi) AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE; (vii) MspI/AuNPs-DNA S3/DNA S2/MCH/DNA
S1/AuNPs/GCE.

3.2.2. Electrochemical characterization of the biosensor
Cyclic voltammetry (CV) was performed to verify the step- wise modiﬁcation of electrodes and the results are recorded in Fig. 2. A pair of well-deﬁned redox peak of Fe(CN)6 3/4− was observed at bare GCE (curve i). Its current increased after AuNPs was deposited on the electrode (curve ii), which attributed to the fact that AuNPs could effectively accelerate the electron transfer. After DNA S1 was dropped onto the AuNPs/GCE electrode, peak cur- rent decreased slightly (curve iii) because the negatively charged DNA S1 would repulse Fe(CN)63/4-. Due to the blocking effect of MCH, decreased peak currents could be observed at the MCH/DNA S1/AuNPs/GCE (curve iv). When the MCH/DNA S1/AuNPs/GCE elec- trode was further modiﬁed with DNA S2, the peak current further decreased (curve v), demonstrating that the double helix DNA was formed (The obtained electrode is denoted as DNA S2/MCH/DNA S1/AuNPs/GCE). After hybridization with AuNPs- DNA S3 (curve vi), the lowest redox peak was obtained due to blocking effect of AuNPs-DNA S3 on electron transfer and exchange between Fe(CN)6 3/4− and the electrode surface, since a single AuNP could be loaded with a huge amount of DNA S3. Finally, peak current was increased sharply after the AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrode was incubated with MspI (curve vii), suggesting success- ful digestion by MspI. All results conﬁrmed that the biosensor was fabricated successfully.

3.3. Feasibility and selectivity

The feasibility of the electrochemical biosensor was conﬁrmed by DPV under different conditions. Nanostructured ﬁlm at elec- trode surface and nanoparticles modiﬁed oligonucleotides were employed as signal ampliﬁcation elements for Ru(NH3)63+ recy- cling. As shown in Fig.3, when DNA S1 was self-assembled on

from the electrode surface, less Ru(NH3)63+ was adsorbed on hybridization composites. Nevertheless, if the AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrode was ﬁrstly oxidized by TET1, then glycosylated by T4 β-GT and lastly cleaved by MspI (200 U/mL), only a small decrease of Ru(NH3)63+ current happened (Fig. 3a, curve iv). Because most of the digestion effect by MspI was blocked after the methylated cytosine was reversed by TET1. It indicated that the site of 5r-CmCGG-3r in hybridization compos- ite was successfully demethylated by TET1. These results show the viability of the biosensor for evaluation of TET1 activity.
Furthermore, DNA S4 without the methyl group was selected to investigate the methylation speciﬁcity. DNA S4 was hybridized with DNA S2 and AuNPs-DNA S3, and then treated with TET1 and T4 β-GT. The peak current was very small when DNA S4/DNA S2/AuNPs-DNA S3 (unmethylated) was cleaved by MspI (Fig. S1, curve i), and the signal was almost unchangeable compared to the DNA S1/DNA S2/AuNPs-DNA S3 which were only incubated with T4 β-GT (50 U/mL) and MspI (Fig. 3a, curve v), suggesting that DNA S4 without the methyl group could not be oxidized by TET1 and then be selectively cleaved by MspI. These results demonstrated that the fabricated biosensor had a very high selectivity to methy- lated DNA and therefore could be applied to determination of TET1 demethylation activity.

3.4. Optimization of experimental conditions

To improve detection sensitivity, the inﬂuences of hybridiza- tion time of AuNPs-DNA S3, TET1 incubation time, glycosylation reaction time and MspI concentrations were all systematically investigated. The DPV signal of Ru(NH3)63+ increases with the increase of hybridization time from 10 to 150 min, and then plateaus on prolonging the hybridization time (Fig. 4a). Accord- ingly, 120 min was selected as ﬁnal hybridization time. TET1 incubation time is a key parameter for performance of the biosen- sor. Fig. 4b shows that the peak current increases with reaction time up to 60 min and then remains constant thereafter. There- fore, a 60 min reaction period was deemed sufﬁcient for subsequent

Fig. 3. DPV signals of the AuNPs modiﬁed electrode at different stages. (i) DNA S2/MCH/DNA S1/AuNPs/GCE electrode; (ii) capture of DNAS3; (iii) AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrode; (iv) incubation with TET1 (21 ng/µL) and T4 β-GT (50 U/mL), followed by incubation with MspI (200 U/mL); (v) incubation with T4 β-GT (50 U/mL) and MspI (200 U/mL).

Fig. 4. Optimization of experimental conditions. (a) Hybridization time of AuNPs-DNA S3; (b) Incubation time of TET1; (c) Glycosylation reaction time of T4 β–GT; (d) MspI concentration.

experiments. Similarly, the optimal glycosylation reaction time was 30 min (Fig. 4c). As shown in Fig. 4d, with enhancing MspI concen- trations from 0 to 250 U/mL, the current of biosensor decreased before 200 U/mL and then tended to be constant. Thus 200 U/mL MspI was employed throughout subsequent work.

3.5. Performance of the biosensor to TET1

The optimized experimental condition ensures a sensitive TET1 assay through the MspI-assisted restriction digestion. As illustrated in Fig. 5a, when TET1 concentration changes from 3.5–21 ng/µL, the current of biosensor increases progressively. These results revealed a good linear relationship between the peak currents and TET1 con- centrations in Fig. 5b, with a correlation coefﬁcient (R) of 0.995. The linear ﬁtting equation is Y = 0.144X + 4.202 (where Y represents the peak current and X represents the concentration of TET1) and limit of detection is 0.33 ng/µL. This biosensor was compared with other methods for TET1 determination in terms of linear range and detec-

tion limit, which are listed in Table S2. The results show that the performance is satisfactory because of wide linear range and the lowest detection limit.

3.6. The inhibition on TET 1 activity

DNA methylation is known to be responsible for various path- ways involved in the tumorigenic process. The TET family enzymes are vital to catalyze the reverse process that is DNA demethyla- tion by recognizing 5-methylcytosine and oxidizing the methyl group. So, the inhibitors of TET1 have promising applications for anticancer therapeutics. According to a new research from ACS Medicinal Chemistry Letters [37], a promising cytosine-based lead compound, Bobcat339, that has mid-µM inhibitor activity against TET1 and TET2. We demonstrated the validity of our method in evaluating the inﬂuence of Bobcat339 on TET1 activity. The results are shown in Fig. 6a. The activity of TET1 continuously decreased with an increase in Bobcat339 concentration. According to the data

Fig. 5. (a) Differential pulse voltammograms of the AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrodes treated by (i) 0 (ii) 3.5 (iii) 7 (iv) 10.5 (v) 14 (vi) 17.5 (vii) 21 ng/µL TET1. (b) Linear relationship between the DPV peak current changes and TET1 concentrations.

Fig. 6. (a) Differential pulse voltammograms of the AuNPs-DNA S3/DNA S2/MCH/DNA S1/AuNPs/GCE electrodes to Bobcat339 with concentrations of 0, 10, 20, 30, 40, 50, 80 µM in the presence of 21 ng/µL TET1. (b) The relative activity of TET1 after inhibited by different concentrations of Bobcat339.

Fig. 7. DPV current changes of biosensor with respectively addition of ﬁve com- pounds in the presence of 21 ng/µL TET1 including taxol, 5-ﬂuorouracil, resveratrol, gallic acid and luteolin (100 µg/mL).

in Fig. 6b, IC50 was calculated to be 38 µM for Bobcat339 and it was almost consistent with the reported value of 32 µM. These results further proved that the proposed strategy has huge potential in application to inhibitors screening of TET1 and anticancer drugs.
Moreover, this biosensor for inhibitors screening was further evaluated by other ﬁve compounds including taxol, 5-ﬂuorouracil, resveratrol, gallic acid and luteolin. Taxol and 5-ﬂuorouracil are representative of anticancer drugs used in clinical trials. Resvera- trol, gallic acid and luteolin are active constituents of traditional Chinese medicine. As displayed in Fig. 7, DPV currents have a slight

decrease compared to the control group but not signiﬁcant. The results showed that these compounds had no obvious effect on TET1 activity in vitro. Maybe these compounds could not act on the TET1 active site due to their structure.

3.7. Precision, reproducibility and stability

Three independent systems with 3.5 ng/µL TET1 were prepared in order to test precision and relative standard deviation (RSD) was calculated to be 5.43 % in Fig. S2a, which shows a high pre- cision. Moreover, the reproducibility is very important parameter to evaluate the performance of the biosensor. Five sensors fabri- cated with same process were used to detect 3.5 ng/µL TET1 and the RSD was about 6.35 % in Fig. S2b, indicating that the developed biosensor has a desirable reproducibility. Furthermore, the stabil- ity of the biosensor was also investigated by reserving under 4 ◦C for 10 days. The fabricated biosensor still remained 93.6 % of the initial current response in Fig. S2c, suggesting satisfactory stabil- ity of the biosensor for practical application. The scan stability was further investigated through three consecutive scans, the peak cur- rent decreased about 7% and became stable after the second scan as shown in Fig. S3. These results indicated that the biosensor has excellent stability and availability.

4. Conclusions

In summary, a triple-ampliﬁed electrochemical biosensor was constructed for highly sensitive TET1 detection taking advantages of the restriction digestion of endonuclease enzyme MspI, the ampliﬁcation capacity of AuNPs-DNA S3 and Ru(III) redox recy- cling. Compared to existing methods, a relatively lower detection limit of 0.33 ng/µL and a wide linear range from 3.5–21 ng/µL were achieved by this assay. Importantly, the feasibility of this biosensor for inhibitors screening was evaluated by Bobcat339 as a known

inhibitor of TET1 with an IC50 of 38 µM and further conﬁrmed by other compounds including taxol, 5-ﬂuorouracil, resveratrol, gal- lic acid and luteolin. Notably, simple experimental operation and label-free electrochemical biosensor not only allows TET1 quantiﬁ- cation to be achieved, but also shows great potential for anticancer drug screening and clinical diagnostics.

Author’s contributions

Ying Cheng: Investigation, Methodology, Writing – original draft, Visualization, Software. Jing Tang: Investigation, Method- ology. Xue Chen: Investigation, Data curation. Fang Wang: Supervision, Funding acquisition, Project administration, Concep- tualization, Writing – review & editing. Zilin Chen: Supervision, Writing – review & editing.

Funding

This work was supported by the National Natural Science Foun- dation of China (21778041 and 91753201).

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

Authors thank for the help of Yafen Wang and Professor Weng, College of Chemistry and Molecular Sciences, Key Laboratory of Biochemical Polymers of Ministry of Education. The Institute for Advanced Studies, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2021. 114228.

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