Inorganic phosphate (P_i)–sensing is a key application in many disciplines, and biosensors emerged as powerful analytic tools for use in environmental P_i monitoring, food quality control, basic research, and medical diagnosis. Current sensing techniques exploit either electrochemical or optical detection approaches for P_i quantification. Here, by combining the advantages of a biological P_i–receptor based on the bacterial phosphate binding protein with the principle of thermophoresis, i.e. the diffusional motion of particles in response to a temperature gradient, we developed a continuous, sensitive, and versatile method for detecting and quantifying free P_i in the subnanomolar to micromolar range in sample volumes ≤10 μL. By recording entropy–driven changes in the directed net diffusional flux of the P_i–sensor in a temperature gradient at defined time intervals, we validate the method for analyzing steady–state enzymatic reactions associated with P_i liberation in real–time for adenosine triphosphate (ATP) turnover by myosin, the actomyosin system and for insoluble, high molecular weight enzyme–protein assemblies in biopsy derived myofibrils. Particular features of the method are: (1) high P_i–sensitivity and selectivity, (2) uncoupling of the read–out signal from potential chemical and spectroscopic interferences, (3) minimal sample volumes and nanogram protein amounts, (4) possibility to run several experiments in parallel, and (5) straightforward data analysis. The present work establishes thermophoresis as powerful sensing method in microscale format for a wide range of applications, augmenting the current set of detection principles in biosensor technology.

无机磷酸盐（P _i）感测是许多学科中的关键应用，生物传感器已成为功能强大的分析工具，可用于环境P _i监测，食品质量控制，基础研究和医学诊断。电流感测技术利用电化学或光学检测方法进行P _i量化。在这里，通过组合生物P的优点_我受体基于与热泳原则的细菌磷酸盐结合蛋白上，即响应于温度梯度粒子的扩散运动，我们开发了一种连续的，敏感的，和通用的方法，用于检测和定量游离P _i在亚纳摩尔至微摩尔范围内，样品量≤10μL。通过在定义的时间间隔内记录温度梯度下P _i-传感器的定向驱动净扩散通量的熵驱动变化，我们验证了实时分析与P _i释放相关的稳态酶反应的三磷酸腺苷的方法（ATP）通过肌球蛋白，放线菌素系统以及活检来源的肌原纤维的不溶性，高分子量酶-蛋白质组装进行转换。该方法的特点是：（1）高P _i–灵敏度和选择性，（2）将读出的信号与潜在的化学和光谱干扰分离，（3）最小的样品量和纳克蛋白质量，（4）可以并行进行多个实验，以及（5）简单的数据分析。本工作建立了热泳作为微尺度格式的强大传感方法，可用于广泛的应用范围，从而扩大了生物传感器技术中当前的检测原理集。