Study Of Homogeneous Biosensing Method Based On Zeta Potential Technology And Its Applications In Biochemical Analysis

Biomarkers are characteristic indicators that can be objectively measured and evaluated in normal physiological and pathological processes.They can be used for diagnosis and classification of diseases,monitoring of disease development,evaluation of clinical response to treatment intervention,drug development and design of personalized disease management plan.The analysis of biomarkers plays a significant role in many fields from basic biological researches to clinical medicine.Nowadays,in addition to the widely used enzyme linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR),a variety of new biochemical analysis methods based on different biosensing technologies have emerged.Although some of them have achieved good detection results,however,there is still an urgent need to develop more analytical methods with better performance to meet the requirements of different biological applications and clinical testing needs.Zeta potential is a parameter commonly used to characterize the stability and surface properties of dispersed nanoparticles,which can sensitively reflect the change of surface charge of nanoparticles.Therefore,it has the potential to be utilized as a tool for quantitative analysis of biomolecules.Besides,zeta potential measurement is facile,fast and labelfree.Although this technology has many advantages,its application in biochemical analysis has rarely been reported.This is mainly because nanoparticles are easy to interact with impurities such as proteins in complex samples,the adsorption of which will interfere with the recognition of targets and mask the signals.Meanwhile,the nanoparticles dispersed in the solution are often charged,which will lead to high background.Moreover,changing the surface charge of nanoparticles may seriously affect the stability of the detection system,resulting in incredible measurement results.In this work,a zwitterionic lipid bilayer coated magnetic nanoparticle is designed to deal with the above-mentioned challenges,and homogeneous and highly sensitive sensing platforms for nucleic acid and protein biomarkers based on zeta potential technology have been successfully constructed.The major contents of the thesis are as follows:1.The construction of antifouling nanoparticle and its application in highly sensitive detection of nucleic acidsIn this section of the thesis,we first built an antifouling nanoparticle,which consists of three components,namely,a magnetic nanoparticle core,DOPC lipid double layer coated outside the core,and peptide nucleic acids(PNAs)inserted into the lipid membrane through cholesterol groups.The nanoparticle has the function of target recognition and separating the binding complex from the media.It also has excellent performance of resisting the nonspecific adsorption of protein impurities in complex biological samples.The electroneutral DOPC shell and PNAs effectively shield the negative charges of the magnetic nanoparticles,leading to low background.In a typical assay,the testing sample was incubated with fabricated nanoparticles.When the target nucleic acids with highly negative charges exist in the solution,they can be recognized by PNAs and bind to the nanoparticles,giving rise to significant change of the surface charge of the nanoparticles.Therefore,the nucleic acid quantitation can be realized through zeta potential measurement.Moreover,by integrating catalytic hairpin assembly,the sensitivity of the assay method can be further improved,and the limit of detection is as low as 12.5 f M.The proposed detection method satisfactorily achieved highly sensitive,one-step detection of nucleic acids in complex media,exhibiting promising application prospects of zeta potential technology in biochemical analysis.2.Target-triggered hydrophobic assembly of two functional nanoparticles for homogeneous detection of proteinsIn this section of the thesis,we constructed two kinds of functionalized nanoparticles,including magnetic nanoparticles(ANPs)coated with DOPC lipid shell,which have good antifouling properties,and gold nanoparticles(GNPs)modified with DNA hairpins containing aptamer sequence of the target protein(take interferon-γ as an example).There is a cholesterol group labeled at the end of the DNA hairpin,which is buried inside and faces the surface of the gold nanoparticles due to the rigid conformation of the hairpin,and the steric hindrance effect makes it unable to interact with the ANPs.When there is interferon-γ in the solution,it can bind to the aptamer and open the hairpin,exposing the enclosed cholesterol to the surrounding solution.Afterwards,the GNPs can bind to the surface of the ANPs by cholesterol-mediated hydrophobic interaction.GNPs carrying many nucleic acid molecules and small organic acid molecules have a large number of negative charges,which can cause obvious change in ANPs’ surface charge,so they can produce potential responses even to very low concentrations of target protein.Meanwhile,the ANPs solution with adsorbed GNPs will appear red,so when the concentration of target protein is relatively high,naked eye detection can be realized.This work took advantage of efficient hydrophobic interaction-mediated nanoparticle assembly and achieved rapid,facile,homogeneous detection of proteins in complex samples with dual-readout signals and wide detection range.