Research On DNA Hydrogel-based Detection Of Creatine Kinase Isoenzyme

The base sequences of DNA have rich structural and functional information,which can be used to precisely construct two-dimensional and three-dimensional macro products through rational design.DNA hydrogels formed by DNA cross-linking have the advantages of biocompatibility,stability,and tunable bio-functionality,and have broad application prospects in the fields of bioanalysis and biomedical engineering.Among them,stimuli-responsive DNA hydrogels or smart DNA hydrogels can produce a reversible and switchable transition from hydrogel to sol under different triggering factors,and have attracted great interest as smart materials for sensing.In this study,a typical fatigue biomarker creatine kinase isoenzyme（CK-MB）was used as a target for detection.By optimizing the detection conditions and combining various signal amplification methods and output signals,two types of DNA hydrogel biosensing systems were constructed,thus realizing the visualization and sensitive detection of creatine kinase isoenzymes.The key research and findings of this paper are summarized below:Chapter 1:Research on the application of DNA hydrogel-based microfluidic smart readout in POCT assay.A target-responsive aptamer cross-linked hydrogel was designed and synthesized for portable visualization and quantification of the fatigue biomarker CK-MB.First,gold nanoparticles（Au NPs）with a particle size of13 nm were synthesized by the reduction of chloroauric acid,and bovine serum albumin（BSA）was modified on Au NPs by a classical modification method to avoid its aggregation.The optimal modification concentration was derived by optimizing the assay conditions.Afterwards,DNA hydrogels coated with Au NPs were synthesized.The hydrogels use aptamers as cross-linking agents,and when the aptamers recognize the targets,the conformational changes cause the DNA hydrogel cross-linked chains to dissociate,the hydrogels disintegrate,releasing the Au NPs to be detected by the naked eye.To make the method applicable to in situ detection,a microfluidic chip is further introduced.The volume change before and after the recognition of the target by the DNA hydrogel is used as the control valve of the microfluidic liquid channel.The addition of the target causes the DNA hydrogel to disintegrate,the valve opens,and the liquid to be measured then flows into the detection area.After taking a picture using a cell phone or a video camera,the image’s average grayscale value could then be performed and analyzed with Image J software.With the increase of destination concentration,the concentration of Au NPs released from the hydrogel increases,making the detection area darker and the average gray value decreases thus achieving portable quantitative detection of the target.The detection range was obtained between0.2 and 625 n M using a microfluidic chip,with a limit of detection of 0.027 n M（S/N=3）.The introduction of microfluidic chip provides an alternative method for the field detection of CK-MB,which is expected to safeguard the fatigue injury of officers and soldiers during the training of troops in peace and war.However,from the experimental results,we could find that the microfluidic chip could meet the requirements of portable detection,but the required target concentration is high due to the volume phase change required for detection.Therefore,the next step in this study is to further improve the detection sensitivity of DNA hydrogels.Chapter 2:A study on the isothermal amplification-based DNA hydrogel detection technique for Cas14a protein cleavage.A novel DNA hydrogel aptamer sensing technique based on exponential amplification reaction（EXPAR）and CRISPR-associated nuclease Cas14a was constructed for highly sensitive detection of fatigue marker CK-MB.The method combines the dual advantages of target pre-amplification and Cas14a enzyme activation.After conversion of the target detection signal to nucleic acid signal by the aptamer,EXPAR amplification is used for first-pass signal amplification,followed by further amplification of the detection signal with the aid of efficient side-branch cleavage activity of Cas14a effector protein.Specifically,CK-MB and the complementary strand（c DNA）are selected to compete for binding aptamers,and the free complementary strand obtained is amplified by EXPAR through magnetic separation to form an exponentially amplified target DNA that is recognized by the Cas14a protein,which activates the protein.On the other hand,DNA hydrogels coated with hybridized nanosheets Pt NPs/Cu-TCPP（Fe）were designed and synthesized.Linear DNA-polyacrylamide polymers PS1 and PS2 were formed by grafting two non-complementary oligonucleotide chains（S1/S2）onto polyacrylamide chains,respectively.We then used a linker chain（linker）that forms a bridge between PS1 and PS2 to crosslink the precursor chains,which contain single-stranded,T-rich Cas14a side branch cleavage sites.Finally,the activated Cas14a can perform efficient multi-turnaround non-specific side branch cleavage of the linker strand of the DNA hydrogel,which disintegrates the hydrogel to release the encapsulant.The encapsulant Pt NPs/Cu-TCPP（Fe）has excellent peroxidase-like activity and can act on3,3’,5,5’-tetramethylbenzidine（TMB）substrate for colorimetric reaction and thus for quantification of the reactants.The method has a low detection limit of 0.157 p M(13.52pg m L^-1)and successfully achieved a highly sensitive detection of CK-MB.The method has good detection performance,requires relaxed experimental conditions,and is simple to operate,which can lay the foundation for the future development of biosensors for the detection of trace non-nucleic acid targets.In this thesis,two simple,stable and specific stimulus-responsive DNA hydrogels were prepared,and their three-dimensional porous structures were used to encapsulate a large number of signal indicators.By combining microfluidic microarrays,streptavidin-biotin,isothermal amplification and CRISPR,two stimulus-responsive DNA hydrogel biosensing methods that can be used for portable detection and highly sensitive detection were constructed,with a view to providing an alternative technical means for the detection of fatigue biomarkers.