Food and Biobased Research, Biomolecular Sensing and Diagnostics, Wageningen University and Research Centre, Bornse Weilanden 9, 6708 AA Wageningen, The Netherlands; Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands.
Food and Biobased Research, Biomolecular Sensing and Diagnostics, Wageningen University and Research Centre, Bornse Weilanden 9, 6708 AA Wageningen, The Netherlands.
Talanta. 2015 Jan;131:541-7. doi: 10.1016/j.talanta.2014.08.001. Epub 2014 Aug 12.
The distribution of inkjet-printed biomolecules in porous nitrocellulose substrates often results in a non-homogeneous spot morphology commonly referred to as 'doughnut-shaped' spots. We have studied the influence of Pluronic F127 (an amphiphilic surfactant) on the functionality of inkjet-printed primary antibody molecules and on the final assay result by performing a one-step antibody binding assay in the nitrocellulose substrate. The primary antibody was printed with and without Pluronic, followed by the addition of double-labelled amplicons as antigen molecules and a fluorophore-labelled streptavidin as detection conjugate. The distribution of the fluorescence intensity down into the nitrocellulose substrate was investigated by confocal laser scanning microscopy in 'Z' stacking mode. Each horizontal slice was further analysed by applying a concentric ring format and the fluorescence intensity in each slice was represented in a colour-coded way. The mean and total fluorescence intensity of the antibody binding assay (fluorescent streptavidin) showed a peak at 0.2% (w/v) Pluronic F127. In addition, an improved spot morphology was observed also peaking at the same Pluronic concentration. Subsequently, we investigated the direct influence of Pluronic F127 on the location of the primary antibody molecules by labelling these molecules with the fluorophore Alexa-488. Our results show that upon increasing the concentration of Pluronic F127 in the printing buffer, the spot diameter increased and the number of primary antibody molecules bound in the spot area gradually decreased. This was confirmed by analysing the distribution of fluorescently labelled primary antibody molecules down into the membrane layers. We conclude that a particular ratio between primary antibody and Pluronic F127 molecules in combination with available substrate binding capacity results in an optimal orientation, that is Fab-UP, of the primary antibody molecules. Consequently, an increased number of antigen molecules (in our case the labelled amplicons) and of the fluorescent detection conjugate (streptavidin) will give an optimal signal. Moreover, distribution of the primary antibody molecules was more homogeneous at the optimal Pluronic F127 concentration, contributing to the better spot morphology observed.
喷墨打印的生物分子在多孔硝化纤维素基质中的分布通常会导致斑点形态不均匀，通常称为“甜甜圈形”斑点。我们研究了 Pluronic F127（一种两亲性表面活性剂）对喷墨打印的一抗分子的功能以及对最终测定结果的影响，方法是在硝化纤维素基质中进行一步法抗体结合测定。在没有和有 Pluronic 的情况下打印一抗，然后加入双标记的扩增子作为抗原分子和荧光标记的链霉亲和素作为检测结合物。通过共焦激光扫描显微镜在“Z”堆叠模式下研究荧光强度在硝化纤维素基质中的分布。每个水平切片进一步通过应用同心环格式进行分析，并且以颜色编码的方式表示每个切片中的荧光强度。抗体结合测定（荧光链霉亲和素）的平均和总荧光强度在 0.2%（w/v）Pluronic F127 时出现峰值。此外，在相同的 Pluronic 浓度下观察到斑点形态得到改善。随后，我们通过用荧光染料 Alexa-488 标记这些分子来研究 Pluronic F127 对一抗分子位置的直接影响。我们的结果表明，随着打印缓冲液中 Pluronic F127 浓度的增加，斑点直径增加，并且结合在斑点区域中的一抗分子数量逐渐减少。通过分析荧光标记的一抗分子在膜层中的分布证实了这一点。我们得出结论，一抗和 Pluronic F127 分子之间的特定比例与可用的基质结合能力相结合导致一抗分子的最佳取向，即 Fab-UP。因此，增加抗原分子（在我们的情况下是标记的扩增子）和荧光检测结合物（链霉亲和素）的数量将产生最佳信号。此外，在最佳 Pluronic F127 浓度下，一抗分子的分布更加均匀，有助于观察到更好的斑点形态。
