Application Of Magnetic Nanoparticles In The Dection Of CGG Trinucleotide Repeat

The sequence detection of trinucleotide repeats is of great significance in gene diagnosis because the expansion of trinucleotide repeats was correlated with a lot of neurodegenerative diseases.For example,the dynamic expansion of CGG trinucleotide repeat in the5’-untranslated region of the FMR1 gene engendered Fragile X syndrome,the most common cause of inherited mental impairment.Nanomaterials have been widely applied in DNA detection.Among these nanomaterials,magnetic nanoparticles（MNPs）have received tremendous attention in diverse fields extensively,such as biomedical sciences,biochemical sensing,magnetic separation,imaging,targeted drug delivery and chemical engineering.The advantages were attributed to the unique nanoscale physicochemical properties of MNPs,especially their larger specific surface area,magnetic properties,low toxicity and biocompatibility.Considering that nucleic acids recognition molecule-naphthyridine derivative can be used for the preparation of bifunctional nanoprobe,electrochemical sensor based on the bifunctional nanoprobe and MNPs as nanocarriers was further studied for the detection of CGG trinucleotide repeat.On the other hand,MNPs can be used to separate and purify the target DNA immobilized on their surface,which can effectively improve the detection sensitivity.Thus the fluorescent method was designed for the detection of CGG trinucleotide repeat.The main contents of this paper include two aspects as follows:（1）A novel bifunctional nanoprobe was successfully prepared.Then,a simple,rapid electrochemical sensor was developed based on the bifunctional nanoprobe for the selectively detection of CGG trinucleotide repeat.The bifunctional nanoprobe was synthesized by using carboxyl functionalized Fe₃O₄ MNPs as carriers of naphthyridine derivatives（NC-linker）and ferrocene derivatives（AFFA）.Both the small molecules with terminal amino group were immobilized on the surface of MNPs via amide bond.The bifunctional nanoprobe has both the function of recognition and contribution of electrochemical signal as the redox probe because naphthyridine derivatives can selectively recognize CGG trinucleotide repeat and ferrocene derivatives has electrochemical activity.An electrochemical sensor based on the new bifunctional nanoprobe was established for CGG trinucleotide repeat detection.A remarkable electrochemical signal can be detected by DPV,attributing to ferrocene derivatives as redox probe.These results demonstrated that the sensing strategy was simple,rapid,easy to operate,label-free,sensitive and environmentally friendly for the selectively recognition of CGG trinucleotide repeat.Furthermore,this proposed method would provide great promise for early diagnosis and treatment of neurodegenerative diseases associated with trinucleotide repeats.（2）A simple and sensitive fluorescent method was successfully designed based on nucleic acid modified MNPs for CGG trinucleotide repeat detection.The streptavidin was firstly modified onto the surface of carboxyl functionalized Fe₃O₄ MNPs via amide bond.Then,the biotinylated capture DNA was immobilized on the surface of streptavidin modified MNPs via biotin-avidin conjugation,which served as the magnetic capture probe.In the presence of target DNA molecules,the carboxyfluorescein-labeled DNA as fluorescent signal probe and magnetic capture probe were respectively complementary to the part of target DNA to form a sandwich structure,followed by magnetic separation to remove excess DNAs.The fluorescent signal probe was then released from the surface of MNPs via dehybridization under high temperature and separated from the MNPs by magnetic separation.The fluorescence detection of the supernatant was performed.The fluorescence intensity increased with the increase of the concentration of target DNA.The fluorescence intensity was linearly correlation versus the concentration of target DNA in the range from 100 pM to 150 nM with a detection limit as low as 86.5 pM.These results demonstrated that the fluorescence intensity increased with the increase of the copy number（n value）in CGG trinucleotide repeat.This proposed method was simple,sensitive and has good selectivity.It could provide a very promising option for early diagnosis and treatment of disease-related genes.