Phosphorodiamidate Morpholino Oligomers Functionalized Nanochannel Biosensor For Highly Sensitive Detection Of Coronavirus Nucleic Acid

Coronavirus is a kind of infectious disease pathogen widely existing in nature.It can spread across species among vertebrates and pose a serious threat to human and animal health.Due to the relatively wide range of hosts,the particularity of nucleic acid structure,the survival competition between different viruses and the rapid replication of the virus in the host cell,the virus continues to mutate.Coronavirus has caused three major outbreaks since it was isolated from human body in 1965,including COVID-19outbreak in late December 2019,which has a great impact on the economy and human health all over the world.Therefore,rapid and accurate identification of pathogenic coronavirus plays a vital role in selecting appropriate treatment methods,saving lives and preventing the re outbreak of the epidemic.With the progress of molecular biology technology,nucleic acid detection method has become the leading technology of virus detection.The method based on reverse transcription quantitative PCR（q RT-PCR）has the characteristics of fast detection speed,high sensitivity and specificity.It is considered as the"gold standard"of nucleic acid detection.Although q RT-PCR is a mature technology,the pollution of amplification environment is easy to cause a large number of false positive results in clinical application.In order to prevent the large-scale spread of the virus,there is an urgent need for a simple,highly sensitive,specific and economical detection method suitable for large-scale screening of coronavirus at the grass-roots level.Nano-biosensors have been widely used in the detection of disease-related molecular markers because of their high sensitivity,good specificity and fast analysis speed.Among them,the nanochannel biosensor has developed into a representative nano sensing platform:（1）High sensitivity.Because the nanochannel has high specific surface area and three-dimensional spatial structure,which may increases the probability of interaction between the probe and target,thus this sensor has high sensitivity;（2）Variety of materials and shapes.Nanochannels can be designed into different configurations and pore sizes according to experimental needs;（3）The surface chemistry is controllable and can be chemically modified by a variety of functional groups.In view of the above advantages,nanochannel is a excellent detection platform for the construction of biosensors.We constructed a Phosphorodiamidate Morpholino Oligomers（PMO）functionalized nanochannel biosensor to achieve highly sensitive detection of coronavirus RdRp gene.Due to the 3D finite space structure,high specific surface area and low background signal of electrically neutral PMO,the hybridization efficiency between probe and target RNA is greatly improved.The highly sensitive detection of coronavirus RdRp gene was realized by monitoring the charge changes on the surface of nanochannels before and after hybridization.The sensor is label free,does not need signal amplification,has high sensitivity and good specificity,and provides a new idea for large-scale screening.The work is divided into two parts:PartⅠ:Construction of PMO functionalized nanochannels biosensorIn this work,we used polyethylene terephthalate（PET）as the base material to construct the nanochannel sensor.Firstly,conical nanochannels were prepared by asymmetric chemical etching.The field emission scanning electron microscopy（FE-SEM）images indicated that the prepared nanochannels were conical,and the pore diameters of the large and small ends were about 700 nm and 44 nm,respectively.The surface of the etched nanochannels exposed a large number of carboxyl groups（-COOH）,PMO molecules were covalently immobilized to the surface of the nanochannels through EDC/NHS.At the same time,various characterizations are used to further illustrate the successful construction of PMO functional nanochannel biosensor,which can be used for the detection of RdRp gene in the next step.PartⅡ:PMO functionalized nanochannels biosensor for highly sensitive detection of coronavirus RdRp geneThe PMO functionalized nanochannels biosensor was used to detect coronavirus RdRp gene.Firstly,we investigated the specificity of the sensor.The results indicated that the sensor can distinguish target RNA from other non-complementary sequences and has good specificity.The concentration of RdRp gene has a good linear correlation with the current change ratio（I-I₀/I₀）,and the detection limit is as low as 60.7 a M.This sensor can be used for the detection of target RNA in throat swab samples.In order to investigate the accuracy and repeatability of the sensor,the recovery experiment was carried out.The data showed that the recovery efficiency of the sensor was 81%～123%.Finally,the sensor was applied to analysis of clinical quality control samples.The results show that the sensor can effectively distinguish positive and negative quality control samples.In conclusion,PMO functionalized nanochannels biosensor has potential clinical application value in coronavirus detection.