Temperature controlled valves for paper-based microfluidic systems

A method for testing a sample for the presence or absence of a nucleic acid of said sample comprising the steps of:

• providing an assay device comprising:
• a first thermally reversible barrier, wherein the first thermally reversible barrier defines an assay area, and a plurality of heating and temperature control components comprising a conductive ink printed resistor operatively positioned proximate said first thermally reversible barrier, wherein the first thermally reversible barrier includes an associated inlet and an associated outlet across a membrane and selectively operates according to four states:
• 1) an un-actuated state, where the first thermally reversible barrier is placed adjacent the membrane whereby fluid is free to travel across the membrane;
• 2) an actuated state when temperature of the first thermally reversible barrier is raised to a first predetermined threshold, where the first thermally reversible barrier melts and permeates into the membrane whereby fluid is stopped from travelling across the membrane, after which the temperature is allowed to be at or below the first predetermined threshold;
• 3) a closed state when temperature of the first thermally reversible barrier is below a second predetermined threshold, where the first thermally reversible barrier remains permeated in the membrane thereby fluid is stopped from travelling across the membrane;
• and 4) an open state when temperature of the first thermally reversible barrier is at or above the second predetermined threshold, where the first thermally reversible barrier re-melts whereby fluid in presence of a fluid pressure differential across the membrane is allowed to travel across the membrane;
• and
• a second thermally reversible barrier disposed proximate a second conductive ink printed resistor, wherein the first thermally reversible barrier is positioned between a sample application area and an amplification area and the second thermally reversible barrier is positioned between the amplification area and a detection area, wherein the second thermally reversible barrier includes an associated inlet and an associated outlet and operates according to the four states of the first thermally reversible barrier, wherein the amplification area allows for amplification of a biological molecule of interest present in a sample;
• placing the first thermally reversible barrier in the closed state next to the sample application area;
• placing the second thermally reversible barrier in the closed state between the amplification area and the detection area;
• placing a sample in the sample application area;
• opening the first thermally reversible barrier whereby temperature of the first thermally reversible barrier is raised to at or above the second predetermined threshold, whereby the sample is allowed to flow through the first thermally reversible barrier into the amplification area;
• closing the first thermally reversible barrier by reducing the temperature of the first thermally reversible barrier to below the second predetermined threshold;
• amplifying the biological molecule of interest;
• opening the second thermally reversible barrier whereby temperature of the second thermally reversible barrier is raised to at or above the second predetermined threshold;
• and
• determining the presence or absence of the biological molecule of interest in the detection area.