Institute of Optical Materials and Technology, Bulgarian Academy of Sciences, 109 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria.
Institute of Electronics, Bulgarian Academy of Sciences, 72 Tzarigradsko chaussee Blvd., 1113 Sofia, Bulgaria.
Sensors (Basel). 2020 Sep 29;20(19):5572. doi: 10.3390/s20195572.
Immobilization of proteins on a surface plasmon resonance (SPR) transducer is a delicate procedure since loss of protein bioactivity can occur upon contact with the untreated metal surface. Solution to the problem is the use of an immobilization matrix having a complex structure. However, this is at the expense of biosensor selectivity and sensitivity. It has been shown that the matrix-assisted pulsed laser evaporation (MAPLE) method has been successfully applied for direct immobilization (without a built-in matrix) of proteins, preserving their bioactivity. So far, MAPLE deposition has not been performed on a gold surface as required for SPR biosensors. In this paper we study the impact of direct immobilization of heme proteins (hemoglobin (Hb) and myoglobin (Mb)) on their bioactivity. For the purpose, Hb and Mb were directly immobilized by MAPLE technique on a SPR transducer. The bioactivity of the ligands immobilized in the above-mentioned way was assessed by SPR registration of the molecular reactions of various Hb/Mb functional groups. By SPR we studied the reaction between the beta chain of the Hb molecule and glucose, which shows the structural integrity of the immobilized Hb. A supplementary study of films deposited by FTIR and AFM was provided. The experimental facts showed that direct immobilization of an intact molecule was achieved.
蛋白质在表面等离子体共振（SPR）传感器上的固定是一个精细的过程，因为蛋白质的生物活性在与未经处理的金属表面接触时可能会丧失。解决这个问题的方法是使用具有复杂结构的固定基质。然而，这是以牺牲生物传感器的选择性和灵敏度为代价的。已经表明，基质辅助脉冲激光蒸发（MAPLE）方法已成功应用于蛋白质的直接固定（无需内置基质），同时保持其生物活性。到目前为止，MAPLE 沉积尚未在 SPR 生物传感器所需的金表面上进行。在本文中，我们研究了直接固定血红素蛋白（血红蛋白（Hb）和肌红蛋白（Mb））对其生物活性的影响。为此，通过 MAPLE 技术将 Hb 和 Mb 直接固定在 SPR 传感器上。通过 SPR 注册各种 Hb/Mb 功能基团的分子反应，评估以这种方式固定的配体的生物活性。通过 SPR，我们研究了 Hb 分子的β链与葡萄糖之间的反应，这表明了固定化 Hb 的结构完整性。提供了对通过 FTIR 和 AFM 沉积的薄膜的补充研究。实验事实表明，完整分子的直接固定已经实现。
