# Parallel DNA Synthesis to Produce Multi-Usage Two-Dimensional Barcodes
## 3 Results
### Barcode Design and Manipulation  
We designed three types of DNA-based QR codes that respond differently based on the chemical input (Figure 3). These are labeled \"persist\", \"appear\" and \"fade\" (Figure 3A). The chemical compositions of the black and white pixels of the barcode are shown below for each of the three QR types. The \"persist\" QR is a permanent barcode whose black and white pixels are composed of non-cleavable DNA, i.e., a sequence synthesized over a standard dT\({}_{5}\) linker. White pixels receive an additional Cy3 tag at the 5' end (Figure 3C). Such a persisting QR code is visible immediately after microarray synthesis and remains visible during and after the chemical treatment. The \"appear\" QR is a fully Cy3-labeled code, but the black pixel DNA is synthesized over a base-cleavable dT monomer. After microarray synthesis, the DNA is deprotected under basic conditions, typically using ethylenediamine (EDA). This EDA treatment reacts with the 3'-O ester functionality of the cleavable unit, detaching the DNA from the surface (Figure 3B). This process leads to fluorescence loss on the \"appear\" black pixels but also serves to retrieve the DNA library from the surface of the array [32]. This QR code is therefore expected to produce a large, fluorescent, featureless square after synthesis and to only reveal itself after EDA treatment. The \"fade\" QR is an evanescent code, visible after synthesis but disappearing after deprotection. To achieve this, white pixels are Cy3-labeled and black pixels are left unlabeled. Additionally, DNA synthesized on the white pixels of the code contains the base-cleavable dT, leading to the detachment of the DNA and therefore a loss of fluorescence after EDA treatment. Figure 2: Photolithographic process of barcoded microarray synthesis. (**A**) Two separate lists of DNA sequences are used to populate the two specified areas of microarray synthesis, one for the DNA pool and one for the barcode strip. The DNA sequences and their Cartesian coordinates serve to create a series of instructions for parallel DNA synthesis using photolithography, yielding a fluorescently labeled DNA microarray. (**B**) Toolbox of phosphoramidites employed in the synthesis of barcoded DNA arrays: standard photoprotected (benzyl-nitrophenylpropyloxycarbonyl, Bz-NPPOC) DNA phosphoramidites (top) and base-cleavable succinyl-dT and Cy3 phosphoramidites (bottom). We also designed a data matrix barcode as an example of a non-QR 2D pattern. Primer information was encoded in a small 142 \(\times\) 142 pixel data matrix (Figure S1), and a \"persist\" formula was chosen for this alternative barcode. Figure 3: Chemical design of the “persist”, “appear” and “fade” QR codes. (**A**) Expected behavior of the three barcode types after synthesis (top) and after DNA deprotection (bottom). (**B**) The outcome of the EDA treatment, besides the removal of all protecting groups, was the hydrolysis of the ester functionality, which released all cleavable DNA from the surface. This allowed for the library to be retrieved as well as for fluorescence to massively decrease on all cleavable spots. (**C**) Schematic representation of the chemical composition of black and white pixels for all three QR types. The hollow black square represents the cleavable dT unit, and the tag is the fluorescent Cy3 marker. We then synthesized the corresponding DNA microarray, containing an amplifiable, cleavable 97 mer, and a barcode strip, including all three QR types and a data matrix. Immediately after synthesis (pre-treatment), the array was scanned for Cy3 fluorescence. In the pre-treatment stage, with every DNA molecule attached to the surface and still carrying protecting groups, the QR codes behave as expected (Figure 4A). The \"persist\" QR is already visible and scannable, and so is the \"fade\" QR. The \"appear\" QR is invisible at this stage and instead shows up as a large fluorescent square with homogenous signal distribution.