Study Of Paper Based Biosensor Based On A Toner-binding DNA Aptamer And A Type ? DNAzyme

Functional nucleic acids include ribozymes and DNAzymes with catalytic properties,as well as RNA and DNA aptamers with specifically binding abilities,which can be obtained through in vitro selection,and their most extensive application is to construct biosensors.Paper sensors based on functional nucleic acids are mainly composed of fixed elements on paper,signal recognition elements,and signal conversion and amplification elements.They are particularly concerned because of their safety,low cost,portability,and environmental protection.Immobilization of FNAs on paper is the basis of paper biosensors.In this study,in view of the lack of stability of existing methods or the need for chemical modification,we selected and obtained DNA aptamers that can specifically bind to laser printing toner as the nucleic acid immobilization element,and established a new method of functional nucleic acid immobilization on paper.After 13 rounds of selecion,more than 50000 sequences were obtained from a DNA library containing10¹⁴different DNAs by combining traditional cloning and high-throughput sequencing.Six representative DNA sequences were obtained by sequence alignment analysis,of which YG2 had the best binding capacity(up to 40%).YG2 can bind multi-color(black,red,yellow,blue)toners,and has strong binding abilities with bothblack and red toners.In addition,YG2 has a high specificity,had much higher binding affinity on toner compared with cellulose(the main component of paper).Finally,we successfully bound the fluorescein-labeled YG2 on the paper printed with toner,thereby realizing the immobilization of nucleic acid on the paper.This immobilization method is simple and fast,and the nucleic acid and paper do not need complicated chemical modifications,which avoids the shortcomings of the current immobilization methods.Copper ion(Cu²⁺)plays an important role in the physiological process of organisms.Detection of copper ions with low cost and high sensitivity is of great significance to human health and the environment.Type II DNA fragmentation deoxyribozyme is a kind of catalytic DNA that relies on copper ions,which can be used as a recognition element for copper ions to construct sensors.However,this DNAzyme and its substrate need complex modifications offluorescent molecule and quencher,and the detection sensitivity(21.1 nM)is still low.To solve these problems,we used a Cu²⁺-dependent type II DNA cleavage DNAzyme as the copper ion recognition element,rolling circle amplification as the signal amplification method,and the fluorescent reaction of G-quadruplex with thioflavin(Th T)as the signal output to construct a non-labeled,highly sensitive copper ion sensor.This sensor has a great detection specificity.A strong fluorescent signal was only observed for Cu²⁺,among 9divalent metal ions(Cu²⁺,Mg²⁺,Ca²⁺,Mn²⁺,CO²⁺,Ni²⁺,Zn²⁺,Ba²⁺,Fe²⁺).In the range of 1-1000 n M Cu²⁺,there was a linear relationship between copper ion concentration and relative fluorescence intensity(F-F0)/F0,with a detection limit as low as 0.82 nM.In addition,this sensor has good anti-interference.100–fold Ca²⁺inhibited the detection of copper ions,while up to 1000–fold Mg²⁺only slightly interfered with copper ions detection.A new nucleic acid immobilization element-YG2 aptamer and the construction of a non-labeled,highly sensitive copper ion sensor will lay a foundation for the development of low-cost and portable paper sensor platform.