Double Imprinting Based Electrochemical Technology In Exosomes Analyzing

Cells release exosomes（with a diameter of 30-150 nm）to surrounding environment,being stable in body fluids,which represents an important mode of intercellular communication by serving as vehicles for transfering proteins,lipids,DNA and RNA between cells.Exosomes are rich in lots of glycoproteins and glycolipids.Changes of these surface glycoconjugates are related to many diseases such as cancer,thus these surface glycoconjugates appear to be promising cancer biomarkers.Exosomes originated from cancer cells are usually more concentrated and larger than that from normal cells.Therefore,it is of great importance to analyze the particle size distribution（PSD）and concentration of exosomes.Current exosomes analyzing technologies mostly need expensive instruments and antibodies.Molecular imprinting polymers（MIPs）are artificial receptors with special sites that lock to the targets based on the shape and size matching.With the good chemical stability,heat resistance,solvent resistance and low costs,MIPs are widely used in the drug,DNA,RNA,proteins,cells and virus analysis.However,it is hard to distinguish exosomes from the interferences such as liposomes with similar size and shape relying on the current molecular imprinting technology.Since the structure of surface biomacromolecules composite of the surface ultrastructure which varies with the kinds of targets,a double imprinting technology based on the size and morphology（including the shape and surface ultrastructure）double recognition was proposed to analyze exosomes.Combing the double selectivity of double imprinting and the advantages of sensitive and quick response of electrochemical sensor,we constructed the double imprinting（DIP）based sensor for the PSD and concentration analyzing.（1）First,double imprinting-based electrochemical detection strategy has been developed for analyzing the particle size distribution of mimetic exosomes—SiO₂@HRP,relying on the both size and morphology of the nanoparticles.50 nm,100 nm and 150 nm SiO₂@HRP were prepared and mixed at a ratio similar to the exosomes,which were employed as mimetic exosomes.The double imprinting polymers（DIP-50,DIP-150 and DIP-150）were formed on the gold nanoparticles and reduced graphene oxide（Au NPs-GO）modified glassy carbon electrode,using SiO₂@HRP at different sizes as templates.After rebinding the target—SiO₂@HRP,the signal amplification tag—SiO₂@Ag/MPBA was bound to SiO₂@HRP by the formation of boric acid ester between 4-mercaptophenylboronic acid（MPBA）and horseradish peroxidase（HRP）.The relationship between current intensity and concentration of mimetic exosomes was deduced asΔI₅₀=-1.52+0.50×lgc₅₀（2.89×10⁴-2.89×10⁹ particles/mL）,ΔI₁₀₀=-1.61+0.48×lgc₁₀₀（2.89×10⁴-2.89×10⁹particles/mL）andΔI₁₅₀=-1.54+0.49×lgc₁₅₀（5.75×10⁴-5.75×10⁹ particles/mL）with the limit of detection of 1.44×10³ particles/mL,5.68×10² particles/mL and7.70×10² particles/mL,respectively.It also performed good in the PSD detection of mixed SiO₂@HRP,obtaining a ratio of 50 nm:100 nm:150nm=5.00%:42.50%:52.50%with relatively small error compared to the real PSD.This double imprinting-based method showed high potential in the separation and detection of real exosomes.（2）Then the DIP based sensor was used to quantify the mimetic exosomes mixed at the ratio of 50 nm:100 nm:150 nm=5.00%:42.50%:52.50%and the ratio of 50nm:100 nm:150 nm=1:57:168.With the excellent performance in quantifying particles with different sizes,the DIP film was further used in the concentration detection of exosomes.With exosomes as templates and the biocompatible materials poly methacrylic acid and vinylphenylboronic acid（poly MAA-VPBA）,the prosed DIP film exhibited a wide linear range from 1.20×10³ particles/mL to 1.20×10⁸particles/mL with a low detection limit of 140 particles/mL.The proposed method performed excellent in the quantifying of exosomes with high sensitivity and good selectivity.