In a method for initialization of a microfluidic device including a first substrate including a microfluidic channel disposed therein, a valve seat disposed in a microfluidic channel and protruding from the first substrate, and a second substrate disposed opposite to the first substrate and including a space formed therein and corresponding to the valve seat, and a polymer film disposed between the first and second substrates, the method includes separating the polymer film from a surface of the valve seat by applying a positive pressure into the microfluidic channel of the first substrate and applying a negative pressure into the space of the second substrate.
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