Instituto de Microelectrónica de Barcelona (IMB-CNM), CSIC, Campus UAB, 08193, Bellaterra, Spain.
Institut Català de La Vinya i el Vi (IRTA-INCAVI), Plaça Àgora 2, 08720, Vilafranca del Penedès, Spain.
Sci Rep. 2020 Nov 10;10(1):19404. doi: 10.1038/s41598-020-76502-7.
During the malolactic fermentation of red wines, L-malic acid is mainly converted to L-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10 to 1 × 10 M (LOD of 3.2 ± 0.3 × 10 M) and from 1 × 10 to 1 × 10 M (LOD of 6.7 ± 0.2 × 10 M) for the L-lactate and the L-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.
在红葡萄酒的苹果酸-乳酸发酵过程中，L-苹果酸主要转化为 L-乳酸。在整个过程中都应精确测量这两种酸，以保证最终葡萄酒的质量，因此实时监测方法在酿酒行业中非常重要。目前应用的是基于实验室程序的传统分析方法，无法在现场部署。在这项工作中，我们报告了一种双参数紧凑型分析流系统的设计和开发，该系统集成了两个电化学生物传感器，可潜在应用于这种情况。该开发的流系统将首次允许在实时和远程方式下，在实际场景中同时测量这两种酸。通过标准光刻技术在硅衬底上制造微型薄膜铂四电极芯片，并进一步将其集成到聚合物流体结构中。这包括一个 15 μL 的流动池以及用于样品和试剂流体管理的所需流体通道。四电极芯片包括对电极和伪参比电极，以及两个工作电极。这些电极依次用聚吡咯电聚合膜修饰，该膜包埋特定的受体，用于选择性地检测两种目标分析物。通过计时安培法研究了两种生物传感器的分析性能，结果表明 L-乳酸的线性范围为 5×10 到 1×10 M（LOD 为 3.2±0.3×10 M），L-苹果酸的线性范围为 1×10 到 1×10 M（LOD 为 6.7±0.2×10 M）。两种生物传感器均具有长期稳定性，在 30 天以上的时间内保留超过初始灵敏度的 90%，这是监测红葡萄酒苹果酸-乳酸发酵全过程（时间在 20 到 40 天之间）的前提。通过对在三种红葡萄酒的苹果酸-乳酸发酵过程中收集的多个葡萄酒样本进行评估，该流系统的性能与标准方法获得的结果非常吻合。
