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Supporting Information forElectrochemiluminescence biosensor for DNA hydroxymethylation detection based on enzyme-catalytic covalent bonding reaction of –CH2OH and thiol functionalized Fe3O4 magnetic beadsChengji Sui, Huanshun Yin *, Lingsong Wang, Yunlei Zhou, Shiyun Ai * College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, People’s Republic of China* Corresponding author.E-mail: yinhs@sdau. (Huanshun Yin), ashy@sdau. (Shiyun Ai).Reagents and apparatusFeCl3?6H2O, ethylene glycol, sodium acetate trihydrate (NaAc), polyethylene glycol, dopamine hydrochloride, N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) were provided by Aladdin (Shanghai, China). 4-Mercaptophenylboronic acid was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Bis(hexafluorophosphate)(Ru(bpy)2(phen-5-NH2)(PF6)2) (Ru) was bought from Sigma-Aldrich (USA). 2′-Deoxycytidine-5′-triphosphate(dCTP), 2′-deoxythymidine-5′-triphosphate(dTTP), 2′-deoxyguanosine-5′-triphosphate(dGTP) and 2′-deoxyadenosine-5′-triphosphate (dATP) were obtained from Sangon Biotech (Shanghai). N6-methyl-2′-adenosine-5′-triphosphate (m6-ATP), 5-methyl-2′-deoxycytidine-5′-triphosphate (5m-dCTP) and 5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate (5hm-dCTP) were ordered from Trilink BioTechnologies (San Diego, USA). M. HhaI methyltransferase (M. HhaI) was bought from NEB (USA). Buffer solution used in this work were as follow. M. HhaI reaction buffer, 50 mM Tris-HCl (pH 7.4), 10 mM EDTA. Wash buffer, 10 mM Tris-HCl (pH 7.4). Detection buffer, 100 mM K2S2O8, 10 mM PBS (pH 7.4).Transmission electron microscopy (TEM) was detected on Tecnai G2 20 (FEI, USA). X-Ray diffractometer equipment (XRD) was performed on Smartlab SE (Rigaku, Japan). Scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) were carried out on a QUANTA250 (FEI, USA). Electrochemical impedance spectroscopy (EIS) was obtained from CHI660C electrochemical workstation (CH instruments, Austin, USA). The ECL intensities were measured by MPI-E multifunctional electrochemical and chemiluminescence analytical system (Xi’an Remax Analytical Instrument Ltd. Co. China), with an ECL analyzer between -1.5 to 0 V at 100 mV s-1 with a photomultiplier tube voltage of 600 V in 8 mL of detection buffer.Real sample preparation5hmC level in genomic DNA of chicken embryo fibroblast cell infected with avian leukosis virus subgroup J (ALV-J) was detected. Initially, DF-1 cells were seeded in a 6-well-plate at 200 k cells per well, after cultured for 12 h, cells were treated by ALV-J at the concentration of 106 PFU/mL per well. Every treatment was repeated for three times. And then all DF-1 cells proceed to culture at cell incubator. Ultimately, cells were respectively harvested at 24 h, 48 h, and 72 h. Afterword, the genomic DNA was extracted by Animal Genomic DNA Extraction Kit (TianGen, Beijing, China) according to instruction of the manufacturer. The concentration of the genomic DNA was detected by Quawell Q3000 micro-volume UV spectrophotometer (USA). Finally, the genomic DNA was dilute to uniform concentration and degraded to individual nucleotide components using DNA Degradase? (Zymo Research, USA). In addition, the 5hmC content in genomic DNA of rice seedling treated with antibiotics and heavy metal composite pollutants was detected by this biosensor. Firstly, the rice seeds were sterilized with 2% sodium hypochlorite solution followed washing and soaking with sterilized water overnight. Then, the rice seeds were transferred to the gauze on the breeding tray in the thermostatic light box with the temperature of 25 °C and successive light on and off cycles every 12 hours. After 7 days, the rice seedling was treated with the solution containing 100 mg/L different antibiotics and heavy metal composite pollutants for another 4 days. Subsequently, the seedlings were randomly harvested, and their tissue of leaf, stem and root were ground into power by liquid nitrogen freezing. Afterword, the genomic DNA was extracted using Plant Genomic DNA Extraction Kit (TianGen, Beijing, China) according to instruction of the manufacturer. The concentration of the genomic DNA was detected by Quawell Q3000 micro-volume UV spectrophotometer (USA). Finally, the genomic DNA was dilute to uniform concentration and degraded to individual nucleotide components using DNA Degradase? (Zymo Research, USA).Optimization of detection conditionsFig S1. Effect of M. HhaI reaction time (A), phos-tag-biotin reaction time (B), avidin reaction time (C), Ru immobilization time (D) on ECL intensity.ECL mechanism of Ru/K2S2O8 systemBased on the previous reported worksPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaTwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJlY051
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ADDIN EN.CITE.DATA (Jiang et al. 2018; Li et al. 2017; Zhuo et al. 2014), we proposed the following mechanism of ECL process:S2O82- + e- → SO42- + SO4?- (1)Ru(bpy)2(phen-5-NH2)2+ + e- →Ru(bpy)2(phen-5-NH2)+ (2a)Ru(bpy)2(phen-5-NH2)+ + SO4?- →Ru(bpy)2(phen-5-NH2)2+* + SO42- (2b)Ru(bpy)2(phen-5-NH2)2+ + SO4?- →Ru(bpy)2(phen-5-NH2)3+ + SO42- (3a)Ru(bpy)2(phen-5-NH2)3+ + e- →Ru(bpy)2(phen-5-NH2)2+* (3b)Ru(bpy)2(phen-5-NH2)2+* → Ru(bpy)2(phen-5-NH2)2+ + hν (4).The CV diagram of the ruthenium-based compoundFig. S2. Cyclic voltammetry for bare GCE (a) and Fe3O4-5hmC-Phos-Ru/GCE (b) in 5 mM Fe(CN)63-/4- (1:1) solution containing 0.1 M KCl over the potential range of -0.2 to 0.6 V with a scan rate of 100 mV/s.Cyclic voltammetry (CV) was used study the assembly of the biosensor on the GCE. Fig. S2 shows the electrochemical behaviors of the bare GCE and Fe3O4-5hmC-Phos-Ru/GCE in Fe(CN)63-/Fe(CN)64- solution containing 0.1?M KCl in potential range of -0.2 to 0.6?V and at a scan rate of 100?mV/s. The bare GCE (curve a) possessed a pair of well-defined voltammetric peaks, corresponding to the reduction of Fe(CN)63- and oxidation of Fe(CN)64-. However, the Fe3O4-5hmC-Phos-Ru/GCE exhibited a lower peak current than the bare electrode. This decrease in the peak current is due to the steric hindrance effect of Fe3O4-based composite, which can block the diffusion of Fe(CN)63?/4?.Fig S3. Cyclic voltammograms of GCE (a), Fe3O4-5hmC-Phos/GCE (b), Fe3O4-5hmC-Phos-Ru/GCE (c) in 10 mM PBS containing 100 mM K2S2O8. Scan rate: 100 mV/s.To investigate the electrochemical performance of the ruthenium-based compound, the cyclic voltammograms of different modified electrode was measured in 10 mM PBS containing 100 mM K2S2O8. As shown in the Fig S3, no electrochemical response was obtained on the bare GCE and Fe3O4-5hmC-Phos/GCE (curve a and b). However, the CV of the Fe3O4-5hmC-Phos-Ru/GCE (curve c) showed a pair of quasi-reversible redox peaks with a peak separation of 0.12 V due to the electrochemical transition between ruthenium-based compound. Furthermore, an oxidation peak at 1.08 V and a reduction peak at 0.96 V corresponded with the electrochemistry behavior of ruthenium-based compound in PBS, which indicated that ruthenium-based compound was fixed on the Fe3O4 successfully ADDIN EN.CITE <EndNote><Cite><Author>Gao</Author><Year>2013</Year><RecNum>707</RecNum><DisplayText>(Gao et al. 2013)</DisplayText><record><rec-number>707</rec-number><foreign-keys><key app="EN" db-id="0zwzp0w5kw5zeeeetrmp2a5ktsea2ve25ws2">707</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gao, Wenhua</author><author>Chen, Yunsheng</author><author>Xi, Jing</author><author>Lin, Shaoyu</author><author>Chen, Yaowen</author><author>Lin, Yuejuan</author><author>Chen, Zhanguang</author></authors></contributors><titles><title>A novel electrochemiluminescence ethanol biosensor based on tris(2,2′-bipyridine) ruthenium (II) and alcohol dehydrogenase immobilized in graphene/bovine serum albumin composite film</title><secondary-title>Biosensors and Bioelectronics</secondary-title></titles><periodical><full-title>Biosensors and Bioelectronics</full-title><abbr-1>Biosens Bioelectron</abbr-1><abbr-2>Biosens. Bioelectron.</abbr-2></periodical><pages>776-782</pages><volume>41</volume><keywords><keyword>Bovine serum albumin</keyword><keyword>Electrochemiluminescence</keyword><keyword>Ethanol biosensor</keyword><keyword>Graphene</keyword></keywords><dates><year>2013</year><pub-dates><date>2013/03/15/</date></pub-dates></dates><isbn>0956-5663</isbn><urls><related-urls><url>;(Gao et al. 2013). Meanwhile, higher current of oxidation peak in curve c was observed in comparison to curve b, demonstrating that the biosensor had excellent electrochemical activity.The efficiency of binding between Fe3O4 magnetic beads and Thiol, as well as the efficiency of -CH2OH and -SH Fig. S4. FT-IR spectra of Fe3O4 (a) and Fe3O4-SH (b).In order to prove the successful modification of Fe3O4, the FT-IR was measured. As present in the Fig. S4, the peak at 1110 and 1620 cm-1 are the stretching vibration of C-O-C and indole structure of PDA PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5KdTwvQXV0aG9yPjxZZWFyPjIwMTE8L1llYXI+PFJlY051
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ADDIN EN.CITE.DATA (Ju et al. 2011; Yu et al. 2017). This demonstrates that the PDA was successfully coated on the surface of Fe3O4. The new peak of Fe3O4-SH at 1350 cm-1 is attributed to the characteristic band of B-O, the peaks at 727 and 829 cm-1 are C-H flexural vibration, the peak at 2556 cm-1 is ascribed to S–H stretching vibrations PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5aaHU8L0F1dGhvcj48WWVhcj4yMDEyPC9ZZWFyPjxSZWNO
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ADDIN EN.CITE.DATA (Chang et al. 2017; Zhu et al. 2012). All the above results prove the successful modification of sulfhydryl.To investigate the efficiency of binding between -CH2OH and -SH, the content of 5hmC before and after magnetic beads capturing was detected by liquid chromatography ADDIN EN.CITE <EndNote><Cite><Author>Mehta</Author><Year>2016</Year><RecNum>712</RecNum><DisplayText>(Mehta et al. 2016)</DisplayText><record><rec-number>712</rec-number><foreign-keys><key app="EN" db-id="0zwzp0w5kw5zeeeetrmp2a5ktsea2ve25ws2">712</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mehta, Angad P.</author><author>Li, Han</author><author>Reed, Sean A.</author><author>Supekova, Lubica</author><author>Javahishvili, Tsotne</author><author>Schultz, Peter G.</author></authors></contributors><titles><title>Replacement of 2′-deoxycytidine by 2′-deoxycytidine analogues in the E. coli genome</title><secondary-title>Journal of the American Chemical Society</secondary-title></titles><periodical><full-title>Journal Of The American Chemical Society</full-title><abbr-1>J Am Chem Soc</abbr-1><abbr-2>J. Am. Chem. Soc.</abbr-2></periodical><pages>14230-14233</pages><volume>138</volume><number>43</number><dates><year>2016</year><pub-dates><date>2016/11/02</date></pub-dates></dates><publisher>American Chemical Society</publisher><isbn>0002-7863</isbn><urls><related-urls><url>;(Mehta et al. 2016). The remaining 5hmC content after the capturing of magnetic beads is only 10.43% of the initial content. This result proves that the 5hmC can be captured by the magnetic beads by the reaction between -CH2OH and -SH. This also proved that -CH2OH and -SH have good bonding efficiency.The chemical reactions involved in the process of the assembly of the Ruthenium derived compoundFig. S5. (A) The (bio)chemical reactions involved in the process of the assembly of the Ruthenium derived compound, (B) The structural formula of bis(hexafluorophosphate)(Ru(bpy)2(phen-5-NH2)(PF6)2).As shown in Fig. S5A, we evenly coated a layer of polydopamine on the surface of Fe3O4 by the self-polymerization effect of dopamine. Then, the -SH was introduced by the covalent chemical reaction between the boracic acid structure of 4-mercaptophenylboronic acid and vicinal diol of PDA. Afterword, the 5hmC was captured on the Fe3O4-SH by the covalent chemical reaction between -SH and -CH2OH of 5hmC under the catalysis of M. HhaI. Next, the phos-tag-biotin was fixed on the Fe3O4-5hmC by the reaction between phos-tag and phosphate group of 5hmC. After that, the avidin was immobilized on the Fe3O4-5hmC-Phos by the reaction between biotin and avidin. Finally, Bis(hexafluorophosphate)(Ru(bpy)2(phen-5-NH2)(PF6)2) (Ru) was captured by the covalent chemical reaction between -COOH of avidin and -NH2 of Ru.To further illustrate the reactions between Ruthenium derived compound and avidin, the structural formula of bis(hexafluorophosphate)(Ru(bpy)2(phen-5-NH2)(PF6)2) was given. As shown in the Fig. S5B, there are -NH2 in the Ruthenium derived compound. We all known that there are -COOH in the avidin. Moreover, the -NH2 and -COOH can react under the activation of EDC and NHS. So, the Ruthenium derived compound was captured on the magnetic beads by the reaction between -NH2 of Ruthenium derived compound and -COOH of avidin.The reproducibility of the biosensorFig. S6. The reproducibility of the biosensor with 100 nM 5hmC.To investigate the reproducibility of the biosensor, the ECL response of seven Fe3O4-5hmC-Phos-Ru/GCE prepared with the same condition were measured. As shown in the Fig. S6, there was no significant difference in the ECL intensity, the RSD was 2.56%, indicating an excellent stability. The statistical data of sample detectionIn order to make the experimental results more obvious, we carried out statistical analysis of the actual sample test data. As shown in the Fig. S7, the 5hmC relative expression of the sample before and after treatment changed significantly. However, the 5hmC relative expression detected by ECL and ELISA haven no obvious difference through the calculation of P value.Fig. S7. The statistical data of different antibiotics and Cd2+ complex pollutants on 5hmC expression in the genomic DNA of rice seedling roots (A), stems (B) and leaves (C). The statistical data of ALV-J on 5hmC expression in the genomic DNA of chicken embryo fibroblast cell for 24, 48, 72 h (D).Table 2. The statistical evaluation of this method compared to ELISA.P valueThe compare of the relative expression between ECL and ELISA0.4601amoxicillin and Cd2+ complex pollutants treated rice seedling roots0.6570tobramycin and Cd2+ complex pollutants treated rice seedling roots0.2879chloramphenicol and Cd2+ complex pollutants treated rice seedling roots 0.2479amoxicillin and Cd2+ complex pollutants treated rice seedling stems0.0249tobramycin and Cd2+ complex pollutants treated rice seedling stems0.0512chloramphenicol and Cd2+ complex pollutants treated rice seedling stems0.4601amoxicillin and Cd2+ complex pollutants treated rice seedling leaves0.5659tobramycin and Cd2+ complex pollutants treated rice seedling leaves0.1108chloramphenicol and Cd2+ complex pollutants treated rice seedling leaves0.2879ALV-J treated chicken embryo fibroblast cell for 24 h0.1108ALV-J treated chicken embryo fibroblast cell for 48 h0.1081ALV-J treated chicken embryo fibroblast cell for 72 hReferences ADDIN EN.REFLIST Chang, L., He, X., Chen, L., Zhang, Y., 2017. 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