The lipidic liquid-crystalline cubic phase (LCP) is a membrane-mimetic material useful for the stabilization and structural analysis of membrane proteins. Here, we focused on the incorporation of the membrane ATP-hydrolysing sodium/potassium transporter Na⁺/K⁺-ATPase (NKA) into a monoolein-derived LCP. Small-angle X-ray scattering was employed for the determination of the LCP structure, which was of Pn3m symmetry for all the formulations studied. The fully characterized NKA-LCP material was immobilized onto a glassy carbon electrode, forming a highly stable enzyme electrode and a novel sensing platform. A typical NKA voltammetric signature was monitored via the anodic reaction of tyrosine and tryptophan residues. The in situ enzyme activity evaluation was based on the ability of NKA to transform ATP to ADP and free phosphate, the latter reacting with ammonium molybdate to form the ammonium phosphomolybdate complex under acidic conditions. The square-wave voltammetric detection of phosphomolybdate was performed and complemented with spectrophotometric measurement at 710 nm. The anodic voltammetric response, corresponding to the catalytic ATP-hydrolysing function of NKA incorporated into the LCP, was monitored at around + 0.2 V vs. Ag/AgCl in the presence or absence of ouabain, a specific NKA inhibitor. NKA incorporated into the LCP retained its ATP-hydrolysing activity for 7 days, while the solubilized protein became practically inactive. The novelty of this work is the first incorporation of NKA into a lipidic cubic phase with consequent enzyme functionality and stability evaluation using voltammetric detection. The application of LCPs could also be important in the further development of new membrane protein electrochemical sensors and enzyme electrodes.

脂质液晶立方相（LCP）是一种膜模拟材料，可用于稳定和分析膜蛋白。在这里，我们集中于将膜ATP水解钠/钾转运蛋白Na ⁺ / K ⁺ -ATPase（NKA）掺入单油精衍生的LCP中。小角X射线散射用于确定LCP结构，该结构对所有研究的制剂均具有Pn3m对称性。完全表征的NKA-LCP材料固定在玻璃碳电极上，形成高度稳定的酶电极和新型传感平台。通过酪氨酸和色氨酸残基的阳极反应监测了典型的NKA伏安签名。在原位酶活性评估基于NKA将ATP转化为ADP和游离磷酸盐的能力，后者在酸性条件下与钼酸铵反应生成磷酸钼酸铵络合物。进行了钼酸磷的方波伏安法检测，并辅以710 nm的分光光度法测量。阳极伏安法响应（对应于结合到LCP中的NKA的催化ATP水解功能）在+ 0.2 V相对于0.2 V的条件下进行监测。在有或没有哇巴因（一种特殊的NKA抑制剂）存在下的Ag / AgCl。掺入LCP的NKA保留了其ATP水解活性7天，而溶解的蛋白质实际上变得无活性。这项工作的新颖性是首次将NKA掺入脂质立方相中，从而使用伏安法检测酶的功能和稳定性。LCP的应用在新型膜蛋白电化学传感器和酶电极的进一步开发中也可能很重要。