The Construction Of Electrochemical Sensing Platform Based On Composite Hydrogel For Monitoring Nitric Oxide Released By 3D Cultured Breast Cancer Cells

As a signaling molecule involved in various physiological and pathological processes in human body,NO is closely related to the occurrence,metastasis and diffusion of tumors.Therefore,real-time monitoring of NO released by cells in situ is of great significance for revealing the pathogenesis of related disease and developing therapeutic drugs.The NO released by cells has the characteristics of short half-life,low concentration and high reactivity.Selecting the appropriate electrochemical sensing method can realize the highly sensitive,unlabeled and highly selective detection of NO.At present,most monitoring studies on NO released by cells are mainly based on two-dimensional（2D）cell culture,but three-dimensional（3D）cell culture can simulate the microenvironment in vivo to a greater extent,and more truly shows the state and function of cells.When combining 3D cell culture with electrochemical sensor platform,it is necessary to consider the potential damage of sensor interface in the actual operation process while considering the realization of highly sensitive monitoring.It is also necessary to pay attention to the simulation of the extracellular microenvironment with different stiffness for better screening drug and cruing disease.To meet the research needs in practical applications,we designed the electrochemical sensing platforms based on composite hydrogel to realize real-time monitoring of NO released by 3D cultured breast cancer cells.The main research contents of this paper are as follows:（1）Based on the potential physical damage of the sensing interface in practical applications,the electrochemical sensing platform based on self-healing hydrogel was constructed for monitoring NO released by breast cancer cells.Oxidized sodium alginate/hydrazide polyethylene glycol（OSA/PEG-DH）hydrogel with good self-healing ability was prepared by the dynamic covalent cross-linking between the aldehyde group of oxidized sodium alginate（OSA）and the hydrazide of hydrazide polyethylene glycol（PEG-DH）.Mn（III）meso-tetra（N-methyl-4-pyridyl）porphyrin（Mn TMPy P）with good biocompatibility was selected as the sensor element.Through the electrostatic interaction between Mn TMPy P and OSA,the stable recombination of Mn TMPy P in hydrogel was realized.OSA/Mn TMPy P/PEG-DH hydrogel was prepared successfully.The self-healing hydrogel not only simulates the dynamic 3D microenvironment of cells in the body,but also enables the sensing interface to self-repair after physical damage.The electrochemical sensing platform based on self-healing hydrogel has a high sensitivity(1.031μAμM^-1 cm^-2)and a low detection limit（17 n M）for the detection of NO,and the current response to NO can be recovered in a short time after the surface is damaged by cutting.The sensor platform was also successfully applied to monitor NO released by MDA-MB-231 cells.（2）Based on the necessity of monitoring the release of active small molecules from cells cultured in the microenvironment with different stiffness in practical application,the electrochemical sensing platform based on the short peptide composite hydrogel was constructed to realize the monitoring of NO released by 3D cultured breast cancer cells in the microenvironment with different stiffness.By the self-assembly process of Fmoc-FF dipeptide in alginate（Alg）solution and the formation of Alg ion crosslinking network through further adding calcium chloride（Ca Cl₂）,Fmoc-FF/Alg composite hydrogel with adjustable stiffness was obtained.Gold nanoparticles（Au NPs）were selected as sensing elements.The titanium mesh electrode（TME）was used as the working electrode to construct the electrochemical sensor platform,which was used to monitor NO released by MCF-7 cells cultured in 3D for different time under two microenvironments with a stiffness difference of 5 times.Excluding the influence of cell proliferation on the experimental results,the cell individuals cultured in the stiffer microenvironment could release more NO under the same culture time.In this paper,the electrochemical sensing platforms based on different composite hydrogel were build according to the specific requirements of practical applications,and realized the monitoring of NO released by 3D cultured breast cancer cells,which also expands the idea for the subsequent research on the pathogenesis of cancer and drug development.