| Alzheimer's disease?AD?is a progressive neurodegenerative disease,the most common type of dementia,accounting for 60-80%in the number of dementia.Due to extension of human longevity,the number of patients present a rapid increase trend,leading to a great burden for community family.AD has become one of the major diseases jeopardizing the health of elderly.Its main clinical symptoms are cognitive dysfunction,behavioral abnormalities and memory loss.There is no effective treatment and drug to reverse or cure AD.Up to now,the pathogenesis of AD is not clear,and the deposition and clearance of A???-Amyloid?is the major factor.The abnormally deposits in the brain mainly comprise A??1-40?and A?1-42,where the evolution of A?oligomerization initiates years or decades before the onset of AD.Thus,A??1-40?or A?1-42has been reviewed as a biomarker of AD,and accurate detection of A? is helpful for early diagnosis or treatment of AD.In addition,self-association of A?1-42can form a series of species from low molecular weight A?1-42oligomer(LMW A?1-42,2-4 mer)to high molecular weight A?1-42oligomer(HMW A?1-42,14-50 mer),all of which can bind to cellular receptor and cause synaptic and neurotoxic effects.However,different A? species seem to trigger differential synapse impairment and neurotoxicity in AD,and the specific mechanism remains to be elucidated.Dual Polarization Interferometry?DPI?is a high-resolution interface analysis technology,mainly using for real-time characterization and quantification of biomolecule interactions.In this paper,based on aforementioned research background,we constructed two colorimetric immunosensor methods for A? detection and used DPI to study the kinetic information of different A? species with cell membrane receptor.1.Based on the specificity of antibody,the optical properties of silver nanoparticles?AgNPs?and the binding of A? with Cu2+,we designed a colorimetric immunoseneor for detecting A? We firstly conjugated AgNPs surface with the antibody of A?,named Antibody-AgNPs?Ab-AgNPs?,keeping a yellow color.Upon the addition of the mixtures of A??1-40/1-42?and Cu2+,Ab-AgNPs bound to A??1-40/1-42?by its surface antibody,and simultaneously Ab-AgNPs aggregated via the=[' binding of Cu2+ to the imidazoles of His6,His13,and His14 residues of A?.With the increase of A??1-40/1-42?,Ab-AgNPs was gradually aggregated,accompanying with obvious color change from yellow to red.The UV absorption change in this process was proportional to the concentration of A??1-40/1-42?,ranging from 0.25 to 150 nM.The limit of detection?LOD,3?/s?was as low as 86 pM.This method had several merits of high selectivity and sensitivity,visualization,cost-effectiveness and simple operations.2.Combining the optical properties of AuNPs and N-or C-terminal antibody of A?1-42,we established a dual antibody-modified AuNPs colorimetric immunosensor for the detection of A?1-42.We individually conjugated AuNPs surface with C/N-terminal antibody of A?1-42via the combination of ionic and hydrophobic interactions?C-Antibody?1-42?,C-Ab?1-42?;N-Antibody?1-42?,N-Ab?1-42??.Subsequently,the AuNPs@C/N-Ab?1-42?was acquired by mixing the solution of C-Ab?1-42?-AuNPs and N-Ab?1-42?-AuNPs with equal amounts?1:1?.A certain concentration of A?1-42was added into the prepared AuNPs@C/N-Ab?1-42?,resulting in aggregating owing to the high specificity of C-terminal and N-terminal antibody simultaneously bound to A?1-42,forming a sandwich structure.With the increase of A?1-42,AuNPs@C/N-Ab?1-42?was gradually aggregated,accompanying with obvious color change from red to blue.A good linear relationship between ?A value and concentrations of A?1-42in the range of 7.5-300 nM was obtained.The limit of detection was calculated to be around 2.3 nM?3?/s?.Our proposed strategy for the detection of A?1-42retained the powerful features inherent in ELISA?e.g.,quantitative,sensitive and selective?,and meanwhile showed other additional advantages of visualization,simplicity and simple operation.3.We used dual polarization interferometry?DPI?tool to evaluate the binding events of the three AP?1-42?species such as monomeric A?1-42,low molecular weight A?1-42oligomer(LMW A?1-42),and high molecular weight A?1-42oligomer(HMW A?1-42)with extracellular D1D2 domain of leukocyte immunoglobulin like receptor B2 receptor?ED132L?.The EDID2L receptor was precisely coated onto amino-modified by BS3.Then,different morphologies of A?1-42were individually injected into the probe surface.Several detailed measurements of layer mass,thickness and density could be simultaneously collected.Based on the in-depth evaluation of binding curves in mass,the kinetics parameters of ka and kd values were precisely measured for the first time.By comparing k,values,the association rate of HMW A?1-42(1.34 × 105 M-1·s-1)was nearly 5-fold faster than that of monomeric A?1-42(2.85 × 104 M-1·s-1),which was somewhat lower than that of LMW A?1-42(4.52 ×104M-1·s-1).The dissociation rate of HMW A?1-42(5.34 × 10-4 s-1)for ED1D2L receptor was nearly 30-fold lower than that of monomeric A?1-42?1.79 × 10-2 S-1?and LMW A?1-42(2.09 × 10 2 s-1).Thus,HMW A?1-42showed the strongest affinity with ED1D2L receptor,and a longer time for tight combination with ED1D2L receptor was detrimental,leading to abnormal downstream signal transduction and toxic effect.Our work hopefully contributed to provide explicit direction for drug or antibody design for AD treatment.Based on these kinetic parameters,they greatly helped us understand how HMW A?1-42causes toxic effects in AD,and they also contribute to drug discovery and screen in the future. |
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