Fabrication Of The Electrochemical Biosensor Based On Nucleic Acid Amplification Technology And Nanomaterials

With the vigorous development of life sciences,people pay more attention to health,food and other life-relevant issues,and explore these issues are often carried out by means of detection,thus requiring detection technology with higher sensitivity and accuracy.Due to its low concentration in the living body,the nucleic acid cannot be detected quantitatively directly.Recently,electrochemical biosensor has attracted much attention in quantitative detection of nucleic acid due to its sensitivity and specificity.With the rapidly development of nanotechnology,nanomaterials with controlled size and shape as well as unique physical/chemical properties can be prepared.Functionalized nanomaterials not only have a large specific surface area to hold more signal molecules and probes,but also can be used as catalysts to catalyze signal molecules and amplify signals,and are therefore widely used in electrochemical biosensors.In this paper,the electrochemical biosensor with amplificated signal has been built,through selecting the appropriate nucleic acid amplification technology to expand the target nucleic acid and combining with functionalized nanomaterials,which has high specificity and ultra sensitivity.The details are as follows:1.Preparation of electrochemical biosensor for miR-21 detection with CoFe2O4 magnetic nanoparticles and padlock exponential rolling circle amplification.In this report,an ultrasensitive electrochemical biosensor for miR-21 detection was designed on the basis of a padlock exponential rolling circle amplification(P-ERCA)assay and CoFe2O4 magnetic nanoparticles(CoFe2O4 MNPs)by nanoelectrocatalysis for signal amplification.Nanocatalyst(CoFe2O4 MNPs)and redox molecule(Tb)were co-immobilized onto the graphene(Gra)surface,and Au@CoFe2O4/Tb-Gra probe was formed.The obtained probe exhibits high-performance Tb catalysis attributed to the reduced interaction distance between CoFe2O4 MNPs and Tb.Simultaneously,amplification of miR-21 was performed by using P-ERCA,large number of P-ERCA amplification products were obtained by repeating polymerization and digestion,the detector content are therefore increased,which in turn further improve the biosensor's sensitivity to achieve the ultra-sensitive detection of miR-21.The as-prepared biosensor showed a wide dynamic range of 1 fM to 2 nM with a low detection limit of 0.3 fM for miR-21 detection and exhibited high specificity and sensitivity.The proposed assay was universal for the detection of other miRNAs or DNA,only in accordance with the corresponding probe sequence of target.2.Preparation of electrochemical biosensor for detection of gene CYP707A1 in Triticum aestivum based on RT-PCR and the nanocomposites of gold nanostar/graphene/deposition gold nanoparticles.Electrochemical biosensors based on nucleic acid amplification technology are popular in the present study.As a nucleic acid amplification technology with high efficiency and specificity—polymerase chain reaction(PCR)has rarely been used in electrochemical biosensors.Therefore,we proposed a PCR-based electrochemical biosensor for the detection of gene 8'-hydroxylase(CYP707A1)in Triticum aestivum.The amplification of the target gene was carried out by PCR,which two single stranded DNA modified biotin and thiol groups at the 5' end as forward primer and reverse primer were used,respectively,and thionine was added to the amplified products.Streptavidin-modified magnetic beads were used to separate-enrich the amplified functionalized DNA fragments by the strong force of streptavidin and biotin,and a bioconjugate(Thi-DNA@SA-MB)was formed.The bioconjugate was incubated to the surface of glassy carbon electrode modified gold nanostar/grapheme/deposited gold nanoparticles(AuNs/Gra/DpAu/GCE)and tested.The result shows that the streptavidin-modified magnetic beads exhibit strong electrocatalysis to the redox molecule(Thi).The proposed demonstrated good properties,with a broad detection range of 5 orders magnitude,and the detection limit was lowered to 13 pM for the gene CYP707A1 detection.The biosensor had good properties with a broad detection range of 5 orders magnitude and the detection limit was lowered to 13 pM for the gene CYP707A1 detection.The result also showed that the efficiency and specificity of biosensor is consistent with that of qRT-PCR in the detection of gene expression of T.aestivum.