The Detection Of Ophthalmic Biomarkers MiR-145 And HAase Based On Hydrogel Biosensor

Purpose:Biomarkers have unique advantages in personalized medicine.Finding and discovering valuable biomarkers is one of the current research hotspots in the medical field.At the same time,more and more attention is paid to the detection methods for these specific biomarkers.In this article,for the two common ophthalmic biomarkers microRNA-145(miR-145)and hyaluronidase(Hyaluronidase,HAase),two new hydrogel biosensors were constructed for rapid and convenient detection.Methods:This study is mainly divided into the following two parts:the preparation of DNA hydrogel electrochemiluminescence(ECL)biosensor and its use in the detection of miR-145 in pterygium samples;hyaluronic acid,HA)Preparation of hydrogel membrane biosensor and its application in the detection of HAase in vitreous samples.1)Two oligonucleotide chains modified with amino groups are covalently crosslinked with HA to form HA hydrogel,and the synthesized signal molecule Ru@SiO2 nanoparticles are wrapped in DNA hydrogel to construct DNA hydrogel ECL biology sensor.When the target product(miR-145)and Duplex-specific nuclease(DSN)are added,miR-145 will be fully complementary to one of the oligonucleotide strands.DSN selectively degrades the DNA in the DNA-RNA duplex,so that the DNA hydrogel gradually disintegrates,the Ru@SiO2 nanoparticles are released,and the ECL signal is generated.The concentration of MiR-145 has a linear relationship with the ECL signal within a certain range,which can realize the quantitative detection of the target.The total miRNA of 3 pterygium samples with different severity levels were extracted and added to the prepared DNA hydrogel ECL biosensor for miR-145 detection.2)Spread the activated HA solution on the surface of the mixed cellulose membrane(MCEM)placed in the needle filter,and evenly drop the PEI solution to covalently form the HA hydrogel membrane to prepare Hydrogel membrane biosensor.After adding different concentrations of HAase,HAase can specifically degrade the HA hydrogel membrane and change the permeability of the hydrogel.With the aid of drainage equipment and an electronic balance,the mass of water passing through the HA hydrogel-MCEM composite membrane within 5 minutes was accurately weighed.Within a certain concentration range,the drainage volume is positively correlated with the concentration of hyaluronidase,and the establishment of a linear equation can realize the quantitative detection of HAase.The human hyaluronidase detection kit(HAase ELISA Kit)and the hydrogel membrane biosensor designed in this article were used to detect HAase in human vitreous samples.Result:1)In this experiment,an ECL biosensor based on DNA hydrogel was successfully constructed,which realized the sensitive and convenient detection of miR-145.When the concentration of the target substance is between 1.0 fM and 1.0 nM,there is a good linear relationship between the ECL signal intensity of the DNA hydrogel biosensor and the concentration of miR-145,the linear correlation coefficient is 0.9963,and the detection limit(Limit of Detection,LOD)is 0.42 fM(S/N=3),and the reproducibility relative standard deviation(RSD)of the same batch and different batches are 3.02%and 4.14%,respectively.The results of the detection of miR-145 in pterygium samples showed that the concentration of miR-145 in the sample was negatively correlated with the severity of the patient’s pterygium.This result is consistent with previous reports in the literature.2)In this experiment,a hydrogel membrane biosensor for HAase detection was successfully constructed,which realized the simple and quantitative detection of HAase.The detection range of the biosensor is 1.0 U/mL～36 U/mL.In this concentration interval,the drainage volume within 5 min has a linear relationship with the concentration of HAase.The linear correlation coefficient is 0.9973,the LOD is 0.35 U/mL,the RSD is 3.7%,and the anti-interference and stability are good.Both the method in this experiment and the HAase ELISA Kit detected a small amount of HAase in the liquefied vitreous sample,and the detection results were relatively close.The recovery rate of the hydrogel membrane biosensor in actual sample detection ranges from 95.26%to 105.40%,and the RSD is less than 6.8%,which is good.Conclusion:1)This study successfully constructed a DNA hydrogel ECL biosensor for the sensitive detection of miR-145,which realized the detection of pterygium samples,with good stability,reproducibility and anti-interference.In addition,due to the programmability of the DNA hydrogel biosensor,the detection of other different types of miRNA molecules can be achieved by changing the base sequence of DNA.Therefore,the biosensor designed in this experiment has great application potential and can be applied to the actual detection of multiple targets and multiple samples in the field of clinical medicine.2)This study successfully constructed a new type of biosensor for HAase detection.The sensor combines the permeability changes of the target-responsive hydrogel membrane and the advantages of accurate readings of the electronic balance,and realizes a sensitive and simple detection of vitreous humor.Good stability and antiinterference make it have a wide range of clinical application prospects.