Study On Nanopore Single-Channel Technology To Detect Melamine In Milk Products

Melamine is an important industrial chemical with high nitrogen level(66%nitrogen by mass).Due to its low price,it has been added illegally into infant formula to artificially increase the crude protein contents.Generally,over 90% of taken melamine could be excreted by the kidneys at a low concentration.However,melamine will combine with cyanuric acid to form the insoluble melamine-cyanurate crystal in the kidneys once it was taken with excessive amount for long period,which will destroy the kidney and urinary system.Therefore,to establish a sensitive and accurate detection method of melamine is one of the important issues in food industry.Up to now,various methods including HPLC,LC-MS,GC-MS,CE and aptamer-based sensors,colorimetric sensor,electrochemical biosensor have been developed for detecting melamine.The present study aimed to propose a novel nanopore-based single channel sensing method for melamine detection by designing the single stranded DNA probes selectively bound to melamine and investigate the signal sources by analyzing the interaction model of DNA-melamine complex,and apply this system to determine melamine in foods such as milk,which would provide a reference for determining melamine in food with significant sensitivity and selectivity.The main obtained results are listed as follows:(1)A specific nanopore single-channel recording system was developed for detecting melamine.By using alpha hemolysin protein nanopore platform,the most effective strand DNA1(C)30(T)20 was selected from 6 designed DNA probes for the following experiments to detect melamine.The temperature and time were further optimized for incubation of melamine and DNA probe as 27 ? and 1 h with incubation buffer(0.1 M PB,0.3 M NaCl).(2)The binding mode of melamine to DNA probe and its possible detection principle were well investigated.The results showed that the melamine concentrations had an effect on amplitudes of characteristic signals,indicating that melamine and DNA probe formed the DNA-melamine complexes with different configurations viavarious binding modes.Melamine at the concentrations of 0.1~50 ?M and 50~200?M bounded to two and three single stranded DNAs,respectively,to form DNA-Melamine complexes.The DNA-Melamine complexes with different configurations could not pass through the protein nanopore which resulting in the irreversible decline of electric current.Melamine was finally detected based on the changes of these signals.(3)The qualitative and quantitative detections of melamine in buffer and fresh milk samples were studied based on the nanopore single-channel recording system.In the buffer,the time of characteristic signal had a good linear relationship with melamine concentrations ranging from 1 ?M to 100 ?M with R2 of 0.9901 and detection limit of 100 nM.In fresh milk samples,the linear detection range was from10 ?M to 90 ?M with R2 of 0.9801 and detection limit of 1 ?M.Lactose,metal ions(Mg2+,Ca2+,Fe3+,Cu2+,Zn2+),Vc and structural analogues of melamine(cyanuric acid,Lys,Cys)had no interference with melamine detection,proving that the developed method had a significant sensitivity and selectivity.