PMC:4996378 / 14783-25845 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996378","sourcedb":"PMC","sourceid":"4996378","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996378","text":"3. Microintaglio Printing-Based Biomolecular Microarrays\n\n3.1. Printing and Arraying of Ready-To-Print Biomolecular Ink\nRNA 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.\nThe 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.\nFigure 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.\nFigure 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.\n\n3.2. Printing and Arraying of In Situ-Synthesized Biomolecular Ink\nThe limitations of both the leading technologies historically employed to manufacture microarrays, i.e., the synthesis-on-chip approach using photolithography and the delivery-to-chip approach using microcontact printing, have made it impractical to broaden the applicability of microarrays in the postgenomic era. Photolithography is still a very expensive and time-consuming process, and microcontact printing is restricted to a limited number of arrays and also to the deposition of presynthesized biomolecular ink. Although DNA microarrays have achieved commercial success using these approaches, it is important to note that protein microarrays have not been successful, since they are crucially dependent on maintaining the complex 3D quaternary structure of proteins on the microarray surface, which is technically difficult using these conventional approaches (Figure 1). Therefore, the concept of the on-demand in situ creation of microarrays is giving a new meaning to the term ‘custom’ microarrays, which greatly reduce the risk of loss of function when arraying biomolecular ink and also remove the need for handling steps, such as purification and modification, prior to printing. For this purpose, previously, the concept of a DNA-linked protein array was presented, where we adopted a strategy of one-to-one indexing between spatially unknown individual DNA arrays and their encoded protein array products in situ [31,32]. Next, this process was miniaturized by demonstrating the advanced application of µIP for the in situ co-synthesis and printing of protein microarrays on demand. First, magnetic beads carrying a dsDNA sequence encoding double-histidine-tagged GFP were arrayed onto a microchamber array chip. In the next step, a droplet of a cell-free-coupled transcription/translation system was sandwiched between a Ni-NTA-modified glass surface and a bead-incorporated microchamber array chip. Then, the coupled transcription/translation reaction was initiated by increasing the temperature from 4 °C to room temperature and incubating the assembly at 30 °C for 60 min. Figure 6 shows that histidine-tagged GFP was successfully synthesized in situ from the bead-bound template DNA inside the microchamber of the microchamber-array chip and transferred to the Ni-NTA-modified glass substrate to produce a clear 60-µm-scale array pattern that was successfully detected using a confocal laser microscope [22]. These results confirm the successful one-step in situ synthesis and printing of individual proteins from localized DNAs and that this approach can be used to rapidly create large-scale integrated protein microarrays directly from the encoded DNA microarrays.\nAlthough a single protein was arrayed and printed in the above demonstration, in principle, this concept is extendable to the simultaneous printing of multiple proteins per array using BEAMing and self-assembled magnetic bead approaches [23]. This has been demonstrated by fabricating kilo-giga-density DNA microarrays [24] and the on-chip synthesis and arraying of a randomized library of mutant GFP using an ultrahigh-density (144 million) microbead array format [26]. Therefore, the inclusion of an in situ synthesis process in µIP enables the development of a beyond-mega-spot protein microarray format that can be used not only to express whole proteomes on a chip, but also to create novel artificial proteins by massively increasing the repertoire of analyzable proteins up to the level used in molecular-directed evolution.\nFigure 6 Printing and arraying of in situ-synthesized protein molecules. Bright-field icroscopy image of DNA-bead carrier-incorporated microreactor array chip (left image) and fluorescence microscopy image of in situ co-synthesized and patterned GFP spots (right image). The scale bar indicates 100 µm.\n\n3.3. Instrument-Free Arraying of “Kilo-Giga”-Dense Microarrays with High Resolution\nThe printing of higher density protein microarrays is highly desirable for global proteome analysis. Similarly, ultrahigh density DNA or RNA microarrays are a prerequisite for on-chip novel DNA or RNA probe (i.e., aptamer) screening purposes, which deal with huge aptamer libraries with up to 1015 variants. Conventional microarrays fabricated by a robotic spotter or a photolithographic approach have a number of immobilized spots on a single array substrate that is limited to the 105 order. However, µIP can be considered as an ultrahigh-throughput printing tool for the instrument-free and inexpensive printing of arrays of biomolecules. Owing to the fundamental nature of µIP, this technology would allow the printing of spotter-free ultra-small microarrays while maintaining a high resolution and, thus, can overcome the low-medium-density limitations of current methods. Using µIP, we demonstrated the printing of in situ-transcribed messenger RNAs onto part of a 30 mm × 30 mm glass substrate with a density of 40,000 spots per mm2, which to our knowledge, is the highest ever density reported for microarrays. To demonstrate the high-resolution characteristic of µIP, the printing of an arbitrary shape, a pattern comprising the letters “RNA” with a line width of 7.5 µm, was also achieved [31]. Therefore, µIP technology is considered to provide an instrument (spotter)-free platform for generating in situ-synthesized biomolecular (messenger RNA/protein) microarrays with an ultrahigh density (kilo-giga scale) and a high resolution (sub-micrometer).\n","divisions":[{"label":"Title","span":{"begin":0,"end":56}},{"label":"Section","span":{"begin":58,"end":5524}},{"label":"Title","span":{"begin":58,"end":119}},{"label":"Figure caption","span":{"begin":2628,"end":2978}},{"label":"Figure caption","span":{"begin":4609,"end":5523}},{"label":"Section","span":{"begin":5523,"end":9417}},{"label":"Title","span":{"begin":5523,"end":5589}},{"label":"Figure caption","span":{"begin":9111,"end":9416}},{"label":"Section","span":{"begin":9416,"end":11061}},{"label":"Title","span":{"begin":9416,"end":9499}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600226-11283591-69477180","span":{"begin":739,"end":741},"obj":"11283591"},{"id":"27600226-17963362-69477181","span":{"begin":788,"end":790},"obj":"17963362"},{"id":"27600226-22458258-69477182","span":{"begin":941,"end":943},"obj":"22458258"},{"id":"27600226-25106674-69477183","span":{"begin":3189,"end":3191},"obj":"25106674"},{"id":"27600226-25106674-69477184","span":{"begin":4157,"end":4159},"obj":"25106674"}],"attributes":[{"subj":"27600226-11283591-69477180","pred":"source","obj":"2_test"},{"subj":"27600226-17963362-69477181","pred":"source","obj":"2_test"},{"subj":"27600226-22458258-69477182","pred":"source","obj":"2_test"},{"subj":"27600226-25106674-69477183","pred":"source","obj":"2_test"},{"subj":"27600226-25106674-69477184","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#afec93","default":true}]}]}}