Institute of Photonics and Electronics, Czech Academy of Sciences , Chaberská 57, Prague 182 51, Czech Republic.
Institute of Macromolecular Chemistry, Czech Academy of Sciences , Heyrovského nám. 2, Prague 162 00, Czech Republic.
Anal Chem. 2017 Mar 21;89(6):3524-3531. doi: 10.1021/acs.analchem.6b04731. Epub 2017 Mar 10.
Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.
从血浆等复杂生物流体到生物识别元件（BRE）功能化涂层的污染阻碍了亲和生物传感器技术在医学诊断中的应用。在这里，我们报告了参与低污染羧基功能化涂层功能化的分子机制对 BRE 容量和抗血浆污染的影响。使用偏振调制红外反射/吸收光谱、X 射线光电子能谱和表面等离子体共振方法研究了最近开发的超低污染羧基甜菜碱聚合物和共聚物刷（pCB）以及传统羧基末端聚乙二醇基烷硫醇自组装单层（OEG-SAMs）上 EDC/NHS 激活羧基、BRE 附着和残留活化基团失活的具体机制。结果表明，BRE 功能化 pCB 涂层的抗污染性受影响刷的超低污染分子结构和表面电荷的失活方法强烈影响。结果表明，与游离羧基末端 OEG-SAMs 相反，只有在 pCB 刷中通过自发水解才能部分失活 EDC/NHS 活化的两性离子羧基。已显示出，仅通过将氨基酸失活剂共价连接到 pCB 的残留活化羧基，才能恢复活化/BRE 功能化 pCB 的抗污染性。所开发的失活程序进一步与随机共聚物羧基甜菜碱甲基丙烯酰胺（CBMAA）和 N-（2-羟丙基）甲基丙烯酰胺（HPMAA）的超低污染刷结合使用，优化的 CBMAA 含量（15%）提供了一种具有优于各种羧基功能化低污染涂层的抗污染性的 BRE 功能化涂层，包括均聚物 pCB 刷和 OEG-SAMs。通过在血浆中灵敏地检测 microRNA 癌症生物标志物（miR-16），证明了 pHPMAA-CBMAA（15%）的生物识别能力。
