Application Of Functionalized MXene In Biosensors And Protein Function Regulation

With the increasingly deepening application research of nanotechnology in the biomedicine field,the combination of two-dimensional(2D)nanomaterials with biomacromolecules(such as proteins and nucleic acids),which could integrate their respective superior performance and created a large number of nanocomposite materials with biomedical application value,has gradually become a new research hotspot.Two-dimensional transition metal carbides/nitrides(MXenes)are ultra-thin nanomaterials derived from MAX phase ceramics and show great application prospects in the field of biomedicine due to their adjustable chemical composition,unique physical and chemical properties,and excellent biological effects.Herein,we combine MXenes with functional proteins and nucleic acids to construct a variety of new biological nanomaterials and explore their potential applications in biosensors and protein function regulation by utilizing the high-efficiency fluorescence quenching ability and excellent photothermal conversion performance of MXenes.The main research contents are as follows:1.Construction of the dual-response nanofluorescent imaging probe based on nucleic acid-functionalized Ti₃C₂MXenes.In this section,we use carboxyl-rich polyacrylic acid(PAA)to functionalize Ti₃C₂MXene,which not only improves its stability in complex physiological environments but also introduces a large number of covalent coupling sites on its surface.Subsequently,we covalently coupled the dual fluorescently labeled chimeric DNA probe(dcDNA)to the surface of Ti₃C₂MXenes to construct a dcDNA-Ti₃C₂composite nanoprobe.Due to the efficient fluorescence quenching ability of Ti₃C₂MXenes,the dual fluorescence signal is quenched,whereas their combination with the target MUC1 and miR-21 could restore the corresponding fluorescence signal.Under the optimized conditions,the LOD(limits of detection)of dcDNA-Ti₃C₂composite nanoprobe for MUC1 and miR-21 were 6 nM and 0.8 nM,respectively.In addition,we use the dcDNA-Ti₃C₂composite nanoprobe for multi-target imaging in live cells,which can not only realize the surface-to-inside,real-time,and in situ imaging of the cell membrane surface glycoproteinMUC1 and the cytoplasmic miR-21 at the single-cell level,but also monitor changes in the expression levels of multiple disease markers in cells mediated by different drugs,confirming the application potential of MXenes in biosensing.2.Near-infrared light-controllable MXene-hydrogel for tunable on-demand release of the therapeutic protein.The MXene@hydrogel/protein system was prepared by encapsulating a photothermal active agent,MXene,and certain therapeutic proteins into the agarose hydrogel matrix.MXene could convert the energy of NIR light into heat,which induces reversible phase transition of the matrix to release the pre-loaded protein drugs and activate specific receptor-mediated cell signaling pathways.The pattern and release profile of protein drugs can be facilely and precisely controlled by the internal(Ti₃C₂MXene and agarose concentration)and external parameters(NIR light intensity and irradiation time).We fabricated a hepatic growth factor(HGF)preloaded composite hydrogels system(MXene@agarose/HGF)to remotely activate the c-Met-mediated signaling for regulating cell diffusion,migration,and proliferation with high spatiotemporal control in vitro and promoting wound healing in the skin-injured animal model.By replacing the encapsulated protein with tumor necrosis factor-?(TNF-?),NIR light can trigger the activation of the apoptosis signaling pathway in tumor cells,enables the exogenous eradication of the tumors in a xenograft model.This NIR light-responsive MXene/hydrogel system provides a new strategy for controlled protein drug release and has great application prospects in tissue regeneration and tumor therapy.3.MXene/Cas12a core processor for logical regulation of CRISPR gene editing.This multi-functional MXene/Cas12a core processor was consisted by the delicate assembling of Ti₃C₂MXene,DNA logic computing unit,and CRISPR Cas12a system.With further surface modification,efficient intracellular delivery of the CRISPR Cas12a gene editing system can be achieved.The stable hybrid between the capture DNA(c DNA)and the cr RNA keeps the CRISPR-Cas12a ribonucleoprotein(RNP)fixed on the surface of Ti₃C₂MXene,preventing the Cas12a RNP from binding and editing to the target gene,thus the Cas12a system is inactive.Upon receiving different signal inputs,the MXene/Cas12a core processor releases the Cas12a RNP from the Ti₃C₂MXene surface through precise logical operations and then activates gene editing.Due to the programmability of the DNA circuit,we design a series of 1-input(YES),2-input(AND,OR,N-IMPLY),and 3-input(AND/OR,N-IMPLY/AND)logic-controlled MXene/Cas12a Core processor.Through the design and optimization of the DNA logic computing unit,this non-genetic MXene/Cas12a core processor is expected to integrate more signal inputs to meet more complex and accurate biomedical requirements.4.The dual-lock MXene/Cas12a core processor enables tumor-specific killing and treatment.Taking advantage of the high photothermal conversion performance of MXenes and the DNA strand displacement reactions(SDR),we designed a double-lock MXene/Cas12a core processor that can activate the gene-editing activity of CRISPR Cas12a only both in the presence of exogenous stimuli(NIR light)and intracellular signaling(tumor biomarker miRNA).This dual-lock MXene/Cas12a core processor can specifically activate CRISPR Cas12a gene editing activity in tumor cells under NIR light irradiation,kill tumor cells without damaging normal cells,and further achieve a good therapeutic effect in tumor-bearing mice.This well-designed"double-lock"MXene/Cas12a core processor overcomes the limitations of the previous light-activated CRISPR system,which was unable to distinguish between target and non-target cells upon the light irradiation,and provides a safer method for therapeutic gene editing in vivo.