Fabrication of three-dimensional microfluidic channels in a single layer of cellulose paper

Three-dimensional microfluidic paper-based analytical devices (3D-mu PADs) represent a promising platform technology that permits complex fluid manipulation, parallel sample distribution, high throughput, and multiplexed analytical tests. Conventional fabrication techniques of 3D-mu PADs always involve stacking and assembling layers of patterned paper using adhesives, which are tedious and time-consuming. This paper reports a novel technique for fabricating 3D microfluidic channels in a single layer of cellulose paper, which greatly simplifies the fabrication process of 3D-mu PADs. This technique, evolved from the popular wax-printing technique for paper channel patterning, is capable of controlling the penetration depth of melted wax, printed on both sides of a paper substrate, and thus forming multilayers of patterned channels in the substrate. We control two fabrication parameters, the density of printed wax (i.e., grayscale level of printing) and the heating time, to adjust the penetration depth of wax upon heating. Through double-sided printing of patterns at different grayscale levels and proper selection of the heating time, we construct up to four layers of channels in a 315.4-mu m-thick sheet of paper. As a proof-of-concept demonstration, we fabricate a 3D-mu PAD with three layers of channels from a paper substrate and demonstrate multiplexed enzymatic detection of three biomarkers (glucose, lactate, and uric acid). This technique is also compatible with the conventional fabrication techniques of 3D-mu PADs, and can decrease the number of paper layers required for forming a 3D-mu PAD and therefore make the device quality control easier. This technique holds a great potential to further popularize the use of 3D-mu PADs and enhance the mass-production quality of these devices.