Understanding the binding affinities and kinetics of protein-ligand interactions using a label-free method is crucial for identifying therapeutic candidates in clinical diagnostics and drug development. In this work, the IGZO-TFT (thin-film transistor) biosensor integrated with a tailored microfluidic chip was developed to explore binding kinetics of protein-ligand biochemical interactions in the real-time manner. The IGZO-TFT sensor extracts the binding characteristics through sensing biomolecules by their electrical charges. Using lysozyme and tri-N-acetyl-D-glucosamine (NAG3) as an example, we established a procedure to obtain the parameters, such as the dissociation constant, Kd, and association rate constant, ka, that are critical to biochemical reactions. The correlation between the lysozyme concentration and TFT drain current signal was first constructed. Next, solutions of lysozyme and NAG3 of different mixing ratios were prepared. They were pre-mixed for various periods of reaction time before applying to the TFT sensor to extract signals of lysozyme molecules and the concentration remaining. With the knowledge of drain current changes at different reaction times, ka and Kd can be obtained. The values from our experiment are comparable to other methods, which suggests the proposed approach can be employed to explore protein-ligand interaction kinetics in the massively parallel manner if the TFT array is considered.

中文翻译：

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晶体管-微流体集成传感器对蛋白质-配体反应动力学的表征

使用无标记方法了解蛋白质-配体相互作用的结合亲和力和动力学对于确定临床诊断和药物开发中的治疗候选药物至关重要。在这项工作中，开发了与定制微流控芯片集成的 IGZO-TFT（薄膜晶体管）生物传感器，以实时探索蛋白质-配体生化相互作用的结合动力学。IGZO-TFT 传感器通过感应生物分子的电荷来提取结合特征。以溶菌酶和三-N-乙酰-D-葡糖胺 (NAG3) 为例，我们建立了一个程序来获取对生化反应至关重要的参数，例如解离常数 Kd 和结合速率常数 ka。首先构建溶菌酶浓度与TFT漏极电流信号之间的相关性。接下来，制备不同混合比例的溶菌酶和NAG3的溶液。在应用于 TFT 传感器以提取溶菌酶分子和剩余浓度的信号之前，将它们预混合了不同的反应时间。有了对不同反应时间漏电流变化的了解，就可以得到ka和Kd。我们实验的值与其他方法相当，这表明如果考虑 TFT 阵列，则可以采用所提出的方法以大规模并行方式探索蛋白质-配体相互作用动力学。在应用于 TFT 传感器以提取溶菌酶分子和剩余浓度的信号之前，将它们预混合了不同的反应时间。有了对不同反应时间漏电流变化的了解，就可以得到ka和Kd。我们实验的值与其他方法相当，这表明如果考虑 TFT 阵列，则可以采用所提出的方法以大规模并行方式探索蛋白质-配体相互作用动力学。在应用于 TFT 传感器以提取溶菌酶分子和剩余浓度的信号之前，将它们预混合了不同的反应时间。有了对不同反应时间漏电流变化的了解，就可以得到ka和Kd。我们实验的值与其他方法相当，这表明如果考虑 TFT 阵列，则可以采用所提出的方法以大规模并行方式探索蛋白质-配体相互作用动力学。