To make our readout reaction independent from expensive lab infrastructure, easy to use and storable, we conceived a paper-based solution that complements our fluorescence detector “Lightbringer” in its design. The previously processed patient sample is pumped onto the paper strip, containing the lyophilized Cas13a reaction mix, and the readout reaction takes place. Here we describe the latest prototype of our paper strip.
As regular filter paper shows high auto-fluorescence and therefore conflicts with our detection method, glass fiber filter paper was chosen as substrate for the readout reaction. The glass fiber filter paper is embedded in a sandwich of cellulose filter paper that facilitates transport of the processed sample to the reaction surface via capillary forces. In contrast to glass fiber, cellulose filter paper can be printed with wax5, allowing the creation of fluidic channels in combination with upper and lower layers of tape. The paper strip is composed of layers as depicted below.
Composition of the Paper Sandwich.
Starting with the upper layer the sandwich consists of: One-sided adhesive tape1, cellulose filter paper2, double-sided adhesive tape3, filter paper containing holes for insertion of glass fiber paper4 and printed wax channels, double-sided adhesive tape, cellulose filter paper and again one-sided adhesive tape.
The one-sided adhesive tapes serve to seal the paper sandwich, the top and bottom paper layers are implemented to avoid sticking of the tapes to the fragile loaded glass fiber paper (red). The middle cellulose filter paper is coated with hydrophobic wax (dark grey) applied with a wax printer, forming fluid channels (light grey). For precise cutting of holes into the layers of cellulose paper (white) and adhesive tapes (blue), a laser cutter was used. The assembly is guided by 6 small alignment marks in every layer.
The processed sample and a negative control are applied to the inlets – the large circular holes in the three upper layers. To avoid interferences caused by the light passing through the auto-fluorescent tape, layers were designed with holes above and beneath the glass fiber paper where the detection windows for “Lightbringer” lie.
In a final product, the sealing top- and bottom-layer should be designed to have an easily removable, pre-cut strip instead of the plain holes to avoid contamination by the end-user.