Karp Natasha A, Edwards Paul R, Leatherbarrow Robin J
Affinity Sensors, Saxon Way, Bar Hill, Cambridge, UK.
Biosens Bioelectron. 2005 Jul 15;21(1):128-34. doi: 10.1016/j.bios.2004.08.027.
Recent improvements in sensitivity have enabled direct binding studies of small molecules with evanescent wave biosensors, which monitor binding by measuring refractive index changes close to the sensing surface. The universal solvent for small molecules, dimethylsulfoxide has a high refractive index; consequently, on ligate addition a large non-specific solvent effect is seen which can mask the specific signal. It has been previously noted that different sensor surfaces can respond differently to the same buffer change. The difference is proposed to arise from differences in buffer space and contraction and swelling of the surface hydrogel. Within this paper, a number of calibration approaches are investigated and tested using warfarin binding to human serum albumin as a model system. A number of recommendations are made for accurate referencing for non-specific effects. Changes to the ionic strength of the running buffer had little effect, whilst changes to the charge density of the carboxylmethyl dextran significantly affected how well the control surface reflects the non-specific signal. An amended 'calibration method' can be used, however, it is an additional complex step that was found to overcorrect in the presence of non-specific binding. Matching immobilisation levels between control and active surface significantly reduces solvent differences allowing accurate correction providing solvent compositional changes are minimised in experimental design. Under these circumstances, the traditional method of simple subtraction of the control from the active response is the most appropriate method of correction.
灵敏度的近期提升使得小分子与倏逝波生物传感器的直接结合研究成为可能，这种传感器通过测量传感表面附近的折射率变化来监测结合情况。小分子的通用溶剂二甲基亚砜具有高折射率；因此，加入配体时会出现很大的非特异性溶剂效应，这可能会掩盖特异性信号。此前已经注意到，不同的传感器表面对相同的缓冲液变化可能有不同的反应。有人提出这种差异源于缓冲液空间以及表面水凝胶的收缩和膨胀的差异。在本文中，以华法林与人血清白蛋白的结合作为模型系统，研究并测试了多种校准方法。针对非特异性效应的准确参照提出了一些建议。运行缓冲液离子强度的变化影响不大，而羧甲基葡聚糖电荷密度的变化则显著影响对照表面反映非特异性信号的程度。然而，可以使用一种修正的“校准方法”，但这是一个额外的复杂步骤，发现在存在非特异性结合的情况下会过度校正。在对照表面和活性表面之间匹配固定化水平可显著降低溶剂差异，从而在实验设计中将溶剂组成变化最小化的情况下实现准确校正。在这种情况下，从活性响应中简单减去对照的传统方法是最合适的校正方法。
