Huanshun Yin,Zhiqing Yang,Haiyan Wang,Yunlei Zhou,Shiyun Ai
A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and Hpa II restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe 3 O 4 nanoparticle, the glycosylation reaction of hydroxymethylated cytosine can be performed with the catalytic effect of T4 β-glucosyltransferase using uridine diphosphoglucose as glucose donor. Then, 4-mercaptophenylboronic acid (4-MPBA) can be further reacted with glucose and captured on Fe 3 O 4 nanoparticle surface. After the dsDNA was digested by Hpa II, the fragment with 4-MPBA can be released from Fe 3 O 4 nanoparticle surface. After magnetic separation, this fragment can be further modified on gold electrode surface through the interaction between Au and −SH, which can result in a decreased electrochemical signal of [Fe(CN) 6 ] 3− . According to this detection strategy, the electrochemical biosensor showed high sensitivity with the low detection limits of 0.14 nM and 0.27 unit/mL for hydroxymethylated DNA and T4 β-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 β-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 β-glucosyltransferase activity assay and its inhibition screening.
A photoelectrochemical method was proposed for DNA hydroxymethylation determination using black TiO 2 (B-TiO 2 ) nanosphere as photoactive material and ZnO as photoactivity inhibitor. After hydroxymethylated DNA (5hmC-DNA) was captured on the probe modified B-TiO 2 /ITO electrode surface through hybridization, a glycosyl can be then transferred from uridine diphosphoglucose to 5hmC-DNA and formed a covalent structure with –CH 2 OH in the presence of T4 β-glucosyltransferase (β-GT). Afterwards, based on a series of covalent reaction, amino functionalized ZnO nanoparticles are further immobilized to the surface of the electrode. Due to the capacity to expend the irradiation light and the photogenerated electron of electron donor, the modified ZnO nanoparticles can result in a decreased photocurrent. The developed method shows wide linear ranges from 0.05-200 nM for hydroxymethylated DNA and 1-220 unit·mL −1 for T4-β-glucosyltransferase. The corresponding determination limits were 0.013 nM and 0.24 unit·mL −1 , respectively. The enzyme activity inhibited by 4-phenylimidazole was evaluated. This photoelectrochemical method shows high specificity for 5hmC-DNA (compared to 5fC, 5mC, m6A, control) and β-GT (compared to β-AGT, UGT2B7), and shows excellent stability for testing 5hmC (RSD = 2.75%).
Schematic representation of photoelectrochemical method for DNA hydroxymethylation and β-glucosyltransferase detection based on the glycosylation reaction of –CH 2 OH in 5-hydroxymethylcytosine and the inhibition activity of ZnO to the photoactivity of black TiO 2 nanospheres.
5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS 2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS 2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface . Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation , B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent . The PEC biosensor afforded high selectivity , excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS 2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.
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