A thermophoresis-based biosensor for real-time detection of inorganic phosphate during enzymatic reactions

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.