Biological Nanopores Are Used For Amino Acid Recognition And Differentiation

Nanopore electrochemical detection is a new single molecule detection technology developed in the 1970 s.It can accurately identify single molecule electrochemical characteristics and structure activity by detecting characteristic electrochemical signals in the space of nanopore channel limit.It has attracted wide attention due to its advantages of high sensitivity,low cost and no labeling.In this paper,wild type aerolysin nanopores were used to identify and distinguish 20 proteinogenic amino acids.We designed two kinds of bipolar peptide probe aimed to identify proteinogenic amino acids in this paper,namely positively charged bipolar peptide probe DDDDXRRRRR(D4XR5,X)and negatively charged bipolar peptide probe DDDDDDXRRRRR(D6XR5,X-),with amino acid X in the middle.The N-terminus of the amino acid was chemically linked to a peptide with aspartic acid residues(D)and the C-terminus of the amino acid was chemically connected to a peptide with arginine residues(R)to form bipolar peptide probes.With the help of the bipolar peptide probe D4XR5,the amino acids under test pass through the wild-type aerolysin nanopore to produce specific signals of blocking degree and duration.The results showed that 7 amino acids(D,M,E,R,L,W and Y)could be identified by current blocking Gaussian fitting,and 2 amino acids N and A could be distinguished by blocking time Exponential fitting.The overall charge of peptides probe was reversed by adding two aspartic acid residues to the N-terminus of D4XR5,and the obtained probe D6XR5(X-)was employed to distinguish the five amino acids I,F,G,P and Q,and two amino acids,namely G and F,were identified by the degree of blocking.By changing the electrolyte buffer solution,in which 10 m M Tris-HCl buffer was replaced by 25 m M HEPES,the ionic composition of the solution was changed to distinguish the above 5 amino acids.Two amino acids,Q and P,were distinguished according to the degree of blocking.In addition,we found that the charge and length of the polypeptide probe affect its transport in aerolysin nanopores,steric hindrance effect is not a dominant factor for amino acid discrimination.The degree of blocking generated by negatively charged bipolar peptide probes is greatly improved,thus improving the ability of aerolysin nanopores to differentiate between different amino acids.The degree of blocking generated by negatively charged bipolar peptide probes is greatly improved.Thus,the ability to distinguish different amino acids by aerolysin nanopores was improved.Those findings promoted the application of aerolysin nanopores in proteinogenic amino acids recognition.nanopore technology can make a the sequencing in the determination of protein primary structure.