br Author contributions br Acknowledgements The work was
Acknowledgements The work was supported in part by Natural Science Foundation of China (31601995, 31501028) and an internal research grant of Jianghan University (14042). The authors thank HY of Jingchu University of technology for revising the manuscript.
Introduction As is known to all, post-translation modification (PTM) can conspicuously expand the diversification of protein feature . Histone PTM is capable to adjust the cellular processes more quickly relative to protein translation and gene wnt inhibitor [2,3], which includes acetylation , methylation , phosphorylation  and ADP-ribosylation . It is beneficial to accommodate the subcellular localization, interaction, activity and stability of proteins, leading to the accurate dynamic response to internal and external multiple stimulation. Thereinto, protein acetylation is indispensable to the development of sickness prevention, cell biology and clinic treatment, so it becomes a fashionable histone PTM, presenting great exploring space and significance [, , ]. Histone acetyltransferases (HATs) are able to prompt the histone acetylation within core histone proteins, in which process an acetyl group is transferred from acetyl-CoA (Ac-CoA) to the specific lysine residues . Presently, lots of studies manifest and hint that the abnormity of HAT activity may bring about some hair-raising and enigmatic clinical illnesses, involving cancers, inflammatory, neurological disorders and HIV infection, and so forth [, , , , ]. Medically, HAT themselves are not merely earthshaking clinical markers, but also related inhibitors facilitate the pharmaceutical sciences development and the anti-cancer drugs discovery process. Therefore, the convictive analytical method for probing HAT activity and screening their inhibitors will be conductive to clinical diagnosis and pharmaceutical research. Traditional experiments of HATs depend on radio isotopes and autoradiography, however, there are some disadvantages including complex labeled substrates, radioactive rubbish and multistep steps . Later, various analytical techniques are emerged for HAT activity analysis for instance electrochemistry, chemiluminescence and fluorescence [, , , ]. These methods are effective and yet the study of HAT activity-based biosensor is still at the initial stage, especially, the improvement on sensitivity has much room for further development. Recently, lots of signal amplification technologies have been employed for signal amplification to detect trace targets, referring to enzymes [, , ], nano-materials [, , ] and biomolecules [, , ]. The most common-used traditional method is the enzyme labeled antibodies, presenting a low sensitivity of immunoassay caused by the unseemly ratio for enzyme or nanomaterial and detection antibody. Next, enhances immobilized amount of enzymes and antibodies, improves the sensitivity of immunoassay, but suffers from background signal and high cost due to nonspecific adsorption. Another way to improve sensitivity is the increment of the labeling signal molecules and the extension of outer Helmholtz plane (OHP) [, , , ]. Based on these above, several ultrasensitive immunoassays have been reported and focused on the detection of marine bacterial pathogen [35,36] and microRNA [37,38], while it presents still huge blank for HAT-based biosensor and reveals a broad development prospect. Herein, we designed a recognition and signal probe, graphene oxide (GO) and Au nanocrystals (AuNCs), to label the acetyl-antibody (AbAc, antibody for an acetyl group) and methylene blue (MB, an organic dye with a positively charged), and developed an ultrasensitive mushroom-like electrochemical immunoassay method for probing the HAT activity. HAT p300 was used as the model and the proposed biosensor mainly relied on the following: the synthesis of the GO nanolayer ([email protected]), the immobilization of substrate peptide via AuS bond, the acetylation reaction by transferring an acetyl group from Ac-CoA to lysine and the interaction between acetyl and AbAc. With the binding of antigen-antibody, the GO nanolayers were linked to the electrode surface one by one, which were similar to many mushrooms growing directionally in the ground. We found a significant improvement of sensitivity and suspected it may be the reason of an increase in signal molecule and a high-efficiency electron transfer rate. While these can be attributed to the constructed Faraday cage and the extended outer Helmholtz plane (OHP) on the electrode surface. When the acetylation reaction occurred, the strong electrochemical signal appeared, which was directly proportional to the concentration of the target (p300). Moreover, some inhibitors could hinder the acetylation process, it was of significance on the development of cancer-relevant drug screening.