The Research Of Nucleic Acid-Functionalized Hydrogel Materials In Liquid Biopsy

Liquid biopsy is considered as the most attractive alternative to traditional tissue biopsy.The main advantages of this method lie in its noninvasive detection process,rapidity of sample collection,and its potential for early cancer diagnosis and real-time monitoring of disease and treatment response.Liquid biopsy based on functional nucleic acid-modified hydrogel materials has high sensitivity and specificity.In many cases,it has the advantages of simple operation,rapid analysis,low cost,and portability,which make it suitable for the needs of personalized medicine.However,due to the complex composition of blood samples,the high heterogeneity and individual differences of tumors,there is still an urgent need to develop new techniques for liquid biopsy with higher accuracy and higher sensitivity.Therefore,with the goal of developing a new method for tumor liquid biopsy based on functional nucleic acid-modified hydrogel materials,we developed methods for the analysis and detection of extracellular vesicles,circulating tumor cells and tumor-related marker proteins based on nucleic acidfunctionalized DNA hydrogels and hydrogel microspheres.Specifically,this work included the following aspects:1.DNAzyme-functionalized DNA hydrogels for the isolation and enrichment of circulating tumor cellsDue to the low abundance of circulating tumor cells(CTC)in blood,the enrichment of CTC is difficult.Therefore,we established DNAzymefunctionalized DNA hydrogel to capture and release target CTCs.The enrichment of CTCs is achieved by aptamers,and the formation of DNAzyme hydrogels can achieve the sol-gel transition.The release of CTC was achieved by Zn2+(gel-sol transition).The sol-gel-sol transition process allowed selective capture,in situ encapsulation,and gentle release of target cells.The whole enrichment process is protease-free and generates no radical ions.In a proof-of-concept,we successfully achieved selective isolation and release of CEM and MDA-MB-231 cells from cell mixtures using DNA hydrogels composed of sgc8 and PDL1 aptamers.In addition,its potential in clinical applications was evaluated by spike and recovery experiments on CEM and PBMC.Compared with traditional CTC enrichment methods,this DNAzymebased hydrogel method has the following advantages:1.repeated aptamers on the chain could bind to multiple targeted proteins of cells,enhancing the capture efficiency;2.encapsulation of cells within the formed hydrogel prevented them from escaping and physically separated them from the cell mixture;3.the release of captured cells was activated by the addition of nontoxic zinc ions.Minimal damage was done to the enriched cells because of the unique DNA hydrogel properties and mild release conditions,which is attractive in situations where highly viable cells are desired.Therefore,this method is expected to realize the specific enrichment and release of CTC cells in the clinic,providing new ideas for the detection of specific CTCs and the application of CTCs in downstream research.2.Preparation of aptamers functionalized density-encoded hydrogel microcarriers for the detection of multiplex tumor marker proteinsIn order to overcome the shortcomings of the existing encoding technology of microbeads and the insufficient accuracy of single-target protein detection,we successfully prepared density-tunable two-component hydrogel by microfluidic technology and vortexing method.At the same time,the density-encoded hydrogel microcarriers were used for the detection of multiplex tumor-associated proteins.To explore the applicability of DMs,we functionalized DMs with aptamers that selectively recognize specific molecular targets,for the label-free detection and quantification of protein biomarkers.A series of two-component hydrogel microcarriers was prepared at different wt%/v ratios to yield hydrogels with different densities.A label-free molecular beacon is constructed on the microcarriers,which is composed of nucleic acid aptamer and human telomeric repeat expansion sequence.When the target protein exists,the target protein will bind to the nucleic acid aptamer,and the expansion sequence will be destroyed,thereby releasing the human telomere repeat sequence that binds to Thioflavin T fluorescence.The molecular beacons constructed above were linked to density-encoded hydrogel microcarriers to achieve multiplex detection of target proteins.The experimental results demonstrate that the hydrogel microcarriers can achieve the decoding and separation of density-encoded hydrogel microspheres with high fidelity by density gradient centrifugation.Hydrogel microcarriers with a 1%difference in methacrylamide composition can be clearly distinguished using a suitable density gradient centrifuge.An 8×2×4 3D-encoded hydrogel microcarrier library with density,size,and color was successfully constructed,realizing the expansion of the encoding capacity of suspension arrays.When changing the density gradient centrifuge composition and volume,a higher capacity density encoding can be achieved.By labeling functional nucleic acids on density-encoded hydrogel microcarriers,this hydrogel microsphere enables qualitative and quantitative detection of multiple disease-related proteins simultaneously.Furthermore,their encoding and responsiveness can be improved by further reducing the density differences between densityencoded hydrogel microcarriers.We expect that the use of density encoding in combination with other encoding parameters may find broad application in high-throughput biomedical analysis.3.Aptamer-functionalized hydrogel microspheres for visual detection of tumor-derived extracellular vesiclesOn the basis of the aforementioned studies on density-encoded hydrogel microcarriers,we have occasionally found that hydrogel microspheres can be used for the visualization of tumor-derived extracellular vesicles.In this method,the acrydited-modified aptamer is immobilized on PEGDA hydrogel microspheres.When EVs are present in plasma,PEGDA microbeads can capture tumor-derived EVs.Since multiple tumor marker proteins is expressed on one EV’ surface,so multiple aptamer-functionalized hydrogel microspheres may bind to the same EVs,and one aptamer-functionalized hydrogel microsphere will bind to multiple EVs.Therefore,the cross-binding of EVs and aptamer-functionalized hydrogel microspheres will eventually lead to the changes in their density.The hydrogel microspheres will tilt during density gradient centrifugation,and the angle of this tilt is easily observed with the naked eye.As a result,specific detection of tumor-derived EVs was achieved in this way.The instruments required for this work are inexpensive and operate easily,so this method has great potential for clinical application in the early detection of cancer.