Yap Woon Teck, Song W Kelsey, Chauhan Niharika, Scalise P Nina, Agarwal Radhika, Miller Stephen D, Shea Lonnie D
Department of Biomedical Engineering, Northwestern University, Evanston, IL; Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, IL.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL; Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, IL.
Biotechniques. 2014 Jul 1;57(1):39-44. doi: 10.2144/000114190. eCollection 2014 Jul.
Particle-based technologies are increasingly being used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) (PLG) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), while dimethyl sulfoxide (DMSO) is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on bicinchoninic acid (BSA), Coomassie Plus, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the CBQCA assay is a highly sensitive and accurate substitute for radioactivity-based assays that is suitable for measuring multiple particle-bound or particle-encapsulated peptides or proteins in the presence of EDC or PLG in DMSO, compounds that interfere with the more commonly used BSA and Coomassie Plus assays. Our strategy enables the accurate quantification of peptides or proteins loaded onto or into particles-an essential component of particle-based platform design for diagnostics and therapeutics.
基于颗粒的技术在诊断和治疗领域的应用越来越广泛。这些应用中使用的颗粒通常由聚（丙交酯-共-乙交酯）（PLG）等聚合物组成，并通过肽或蛋白质进行功能化修饰。肽或蛋白质与颗粒的偶联通常使用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）来实现，而二甲基亚砜（DMSO）则用于通过溶解颗粒来回收表面附着或包裹的肽或蛋白质。我们研究了基于二辛可宁酸（BCA）、考马斯亮蓝Plus和3-（4-羧基苯甲酰基）喹啉-2-甲醛（CBQCA）测定法的策略，用于定量表面附着或包裹的肽或蛋白质。我们确定，CBQCA测定法是基于放射性的测定法的一种高度灵敏且准确的替代方法，适用于在DMSO中存在EDC或PLG的情况下测量多种与颗粒结合或包裹在颗粒中的肽或蛋白质，这些化合物会干扰更常用的BCA和考马斯亮蓝Plus测定法。我们的策略能够准确量化负载在颗粒上或颗粒内的肽或蛋白质，这是基于颗粒的诊断和治疗平台设计的一个重要组成部分。
