3.1. Printing and Arraying of Ready-To-Print Biomolecular Ink
RNA microarrays, such as RNA aptamer microarrays for the detection and quantization of protein biomarkers in biological samples, are a powerful tool for analyzing RNA-related phenomena. DNA microarrays are commonly used and fabricated by well-developed methodologies. However, a similar methodology may not be applicable to the fabrication of RNA microarrays, because of the extremely labile nature of RNA molecules. The previously reported methods of fabricating RNA microarrays were performed by the direct attachment of a messenger RNA onto a substrate after chemically-modifying the RNA using thiol-gold chemistry [27] or by mechanical transfer from a DNA master [28]. Recently, an on-chip enzymatic synthesis process has also been reported as a means of fabricating RNA aptamer microarrays in a microfluidic format [29]. These methods are successful when applied to a limited set of arrays, although the need for chemical modification is a concern. µIP enables the fabrication of high-density transcribed RNA microarrays with high throughput. In addition, since µIP enables the parallel in situ co-synthesis and immobilization of biological molecules, the need for chemical modification and the risk of degrading labile RNA can be reduced.
The first demonstration of µIP was the successful patterning of a 5-µm-diameter spot of presynthesized messenger RNAs at a density of 10,000 per mm2 [21]. Figure 4 shows a fluorescence micrograph of a 5-µm-scale array pattern of the full-length transcribed messenger RNA acquired by confocal laser scanning microscopy. As can be seen in Figure 4a, high background noise (i.e., low contrast between the foreground (spots) and background) is observed, which is approximately 250 RFU (relative fluorescence units), i.e., nearly one-third of the actual signal intensity. This suggests that the printing of ready-to-print ink molecules (i.e., presynthesized messenger RNAs in this case) must be improved for high-resolution patterning using the µIP approach. Note that there is a risk of part of the ink molecules (i.e., messenger RNA molecules in this case) remaining in the non-array region (outside the grooves on the printing mold) while achieving a tight seal between the microchamber-array chip and the substrate. Thus, the ink molecules remaining in contact with the non-array region before being forced out by the sealing step may also be undesirably printed in the non-array region on the substrate, leading to high background noise or low-contrast printing.
Figure 4  Printing and arraying of presynthesized transcribed RNA molecules. (a) Microscopy image of the printed pattern of full-length messenger RNA with 5-µm array features using the µIP approach; (b) Fluorescence distribution plot for a single row of patterned messenger RNA (indicated by the red arrow in (a)). RFU, relative fluorescence units.   To accomplish high-contrast µIP, we have recently introduced an advanced form of µIP by controlling the adsorption and/or printing reaction of the presynthesized ink molecules via a thermal trigger (Figure 5) [30]. This is based on a controllable hybridization reaction between a presynthesized messenger RNA and a substrate-immobilized complementary DNA probe by switching the ability of the messenger RNA to fold into a secondary structure via the temperature during µIP processing. At a low temperature, the messenger RNA remains in a folded state, in which it is unable to hybridize, whereas a high temperature can convert the folded state into the unfolded state required for the denaturation of the secondary structure of messenger RNA molecules, thus inducing the hybridization reactions. Therefore, temperature switching before and after the press-sealing step in the µIP process can prevent the undesired printing of presynthesized messenger RNA molecules in the non-array region (Figure 5a). Using this method, we reported the fabrication of transcribed RNA microarrays with a diameter of 1.5 µm and a density of 40,000 spots per mm2 with high contrast (Figure 5b,c) [30]. Most importantly, we also demonstrated that the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude is not affected, and thus, this method can be used for the miniaturization of arrays to obtain high-density nanoarrays. As shown in Figure 5d, a printing mold having microchambers with various diameters of 4–50 µm and similar heights generated pattern intensities with a comparable efficiency.
Figure 5  Advanced temperature-controlled microintaglio printing (TC-µIP). (a) Schematic of TC-µIP. Biomolecular ink is first applied on a printing mold, and the mold is pressed with a capturing probe-modified substrate at a low temperature. The binding interaction between the biomolecular ink and the probe, which is suppressed at a low temperature, can then be activated by temperature control; (b) Microscopy images and printed patterns of full-length messenger RNA molecules with (b) and without (c) using the TC-µIP approach. The signal/noise ratios were calculated from the intensity along the yellow line in each image; (d) Fluorescence intensities per pixel of fluorescence-labeled messenger RNA spots with several diameters using the TC-µIP approach. The printed intensities were comparable from diameters of 4–50 µm. Error bars represent the mean ± SD (n = 9–24 spots). The scale bar indicates 100 µm.

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