Dip Pen Lithography of oligonucleotides on flexible substrates for point-of-care malaria disease testing

Abstract

The first step for prevention and treatment of diseases is the accurate diagnosis. However, proper diagnostic technologies are not available in developing countries due to the lack of reliable electrical power, refrigeration and trained personnel. For this reason, there is an urgent need of low cost, rapid assays not requiring any external support. By coupling such technologies to communication infrastructures, healthcare in areas without access to medical personnel would be possible. “Paper” like substrates are ideal for fabricating such devices since they are cheap, easy to degradate after use and compatible with most of existing printing technologies [1]. We had previously shown the possibility to efficiently deposit oligonucleotides by Dip Pen Lithography(DPL) onto glass surfaces [2]. In this work, we deposited oligonucleotides on nylon substrate for the fabrication of biochips usable for detecting the activity of human topoisomerase I. Subsequently, the chip will be modified to detect the Malariacausing Plasmodium parasites through the detection of Plasmodium topoisomerase I activity [3]. We optimized oligonucleotides printing on nylon substrate, obtaining efficient deposition at 10 - 1 uM oligonucleotide concentrations, 70% relative humidity and 30% glycerol v/v. We obtained circular spots with diameter in the range of 30 - 50 microns, with the dimension being a function of dwell time (1s – 20 s). DPL operation needs ultra tiny amounts of DNA (as low as 0.5 uL, 10 - 1 uM concentration) for printing thousands of spots in a single run so reducing material consumption in comparison with standard bioprinting techniques [4]. In a first set of experiments, the printed oligonucleotides was hybridized with a fluorescence-labelled complementary probe to detect and quantify DNA after DPL deposition. In subsequent experiment, the spotted oligonucleotides generate a topoisomerase substrate, which upon reaction with the enzyme will be coupled to a fluorescently labelled oligonucleotide to allow detection of a signal. In conclusion, the combination of DPL and topoisomerase detection onto nylon substrates would be a suitable solution for point-of-care diagnostic chips fabrication.