| Trinucleotide repeat related-disease refers to a genetic disease caused by expansion of trinucleotide repeat sequences in the pathogenic gene.Trinucleotide repeat expansion is a type of genetic mutation,known as a dynamic mutation,in which the number of triplets in a repeat changes and the length become unstable.To date,nearly 30 disorders caused by trinucleotide repeat expansions have been identified.Such as Fragile X syndrome,myotoruc Dystrophy,Huntington's disease,schizophrenia,autism and so on.The mutations of d?CAG?n occurred in the coding region of the pathogenic gene result in extended mutation of polyglutamine caused bythe encoded protein of the gene.The disease is known as poly glutamine?PolyQ?disease.At present,there are at least nine kinds of PolyQ diseases,such as Huntington's disease?HD?,dentatorubral-pallidoluysian atrophy?DRPLA?and spinocerebellar ataxias?SCA?,etc.Therefore,the detection of d?CAG?n is particularly important.Electrochemical DNA biosensors are valuable analytical tools,which combine the merits of electrochemical and biological technolog.Electrochemical sensors monitor target analytes through the changes of electrochemical signals before and after the binding.They have some advantages including rapid response,high sensitivity,good selectivity,simple operation and low cost,which make them easily realize miniaturization,integration and in situ,real-time,online detection.In this work,some new electrochemical method was studied for the detection of sequence and repeat length of d?CAG?n.The detail contents are mainly as follows:?1?A simple single-signal sensor was constructed to detect the trinucleotide repeat sequence d?CAG?n.The complex of target-report DNA was enriched on the electrode surface by the hybridization between the complex and capture DNA modified on surface of the electrode.Amperometric responses of SA-HRP were recorded after the modified electrode was incubation with SA-HRP through the strong biotin-avidin interaction.The hybridization sequence,temperation and time were optimized in the process.The result indicated that single-signal sensor exhibited a good selectivity for d?CAG?n,and a good linear relationship was obtained between the reduced current and centration of target from 1 pM to 100 nM with a limit of detection as low as 0.21 pM.In addition,there was a good linear relationship between steady state current and n at a DNA concentration of 100 pM.?2?In order to improve reliability and stability of electrochemical sensing,especially for detection of the number of repeat,we present a new double-signal technique and set up a double-signal sensor.The double-signal technique used an electrochemical molecular beacon?ferrocene?,which was directly modified on the surface of a gold electrode,while a reporter probe?horseradish peroxidase?HRP??was hybridized to the target DNA.When the complex of target-reporter DNA was captured on the electrode surface,the electrochemical molecular beacon hairpin DNA opened,resulting in decreased ferrocene current?F?.SA-HRP was bound to the working electrode to get HRP current signal?H?.The results show that the sensor has excellent selectivity and sensitivity.A good linear relationship was obtained in the range of 1pM-100 nM has with a limit of detection as low as 0.15 pM.Importantly,the double-signal sensor was more accurate for the determination of repeat length,which was measured from the ratio of signals for HRP and ferrocene?H/F?.A linear relationship was found between H/F and the number of repeats?n?,H/F=0.1398n+9.89788?n=10-35?,with a correlation coefficient of 0.974.Only 10 nM of target DNA was required for measurements based on the value of H/F in the double-signal technique.These results indicated that this new double-signal electrochemical sensor provides a reliable method for the analysis of CAG trinucleotide repeats.?3?A simple and portable screen-printed biosensor is developed for quantitative detection of trinucleotide repeat sequence d?CAG?n based on magnetic nanoparticles because of the unique advantages of magnetic nanoparticles in the field of biological separation.Capture DNA was modified on the surface of the carboxyl group modified magnetic nanoparticles by amide.The modified magnetic nanoparticles captured target DNA,followed by magnetic separation.The magnetic nanoparticles with targets were directly dropped onto the screen printing electrode surface for detection.Under the optimal conditions,the value of electrochemical signal was linearly correlation versus the concentration of trinucleotide repeat sequence d?CAG?n in the range from 100 pM to 1?M with a limit of detection as low as 42pM.The number of d?CAG?n repeatscan be detected through a good linear relationship between the current and n.In this work,simple and convenient electrochemical sensing system was constructed to detect the trinucleotide repeat sequence d?CAG?n by using nucleic acid hybridization,double signal technique and magnetic nanoparticles.The results show that the sensors have an excellent selectivity,good linear range and the low detection limit.The use of double-signal technique make the detection of n more reliability and stability because the introduction of two variable.These studies provide a a feasible strategy for the the analysis of d?CAG?n repeat.The methods are also expected to be used for the detection of other repeat sequences in neuropathic diseases. |
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