Printed microwells with highly stable thin-film enzyme coatings for point-of-care multiplex bioassay of blood samples

A paper-based colorimetric biosensor suitable for point-of-care bioassay of blood samples is developed using highly stable enzyme thin-film coatings confined within inkjet printed polymeric microwells. The microwells are developed through a simple one-step inkjet printing of hydrophobic polystyrene on paper, with walls formed by the polymer that fills the gaps inside the paper body. The microwells can also be patterned to be interlinked with printed microchannels for multiplex bioassays. Thin film enzyme coatings confined within the microwells are then constructed, thereby constituting biosensors that work like traditional microwell plates, yet allow easy colorimetric readouts with naked eyes or portable devices, such as smart phones. The efficiency of the paper-based sensor was demonstrated for colorimetric assays of glucose and lactate, both as individual analytes or mixed, as well as samples with red blood cells. Such sensors showed good sensitivities within the concentration ranges of the analytes in human blood (0.5-10 mM), with a visible sensitivity of <0.5 mM detectable by naked eyes for a sample size as small as 1 μL. More accurate digital readouts were shown to be feasible with computerized scanners or smartphones. The thin-film coating format affords the paper biosensors an extended lifetime, and they could retain 100% performance over 6 months of storage at room temperature, or up to one month heated at 50°C, which promises refrigeration-free storage of the sensor. The simple preparation, high enzyme stability and ease-of-use of the paper-based sensor promise low-cost and reliable point-of-care multiplex bioassay for biomedical diagnostics.