PMC:4996378 / 10296-14782 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":"2. Concept of Microintaglio Printing\nThis method is based on the principle of the gravure printing (roll-to-roll printing) process using a dense array of microengraved plates (micromold plates). Figure 3 outlines the major steps of the procedure used for µIP: (i) the fabrication of a microchamber-array chip; (ii) the self-assembly and arraying of biomolecular “ink”; and (iii) the in situ synthesis and patterning of microarrays.\nFigure 3 Schematic illustration of key steps in microintaglio printing (µIP) for the in situ fabrication of microarrays. A microengraved intaglio plate (micromold plate) consisting of a dense array of microchambers (a) is filled with the precursors of biomolecular “ink” (e.g., single-DNA molecule-amplified bead carrier or messenger RNA-immobilized bead carrier) (b); A glass substrate, one surface of which is modified to capture the biomolecular ink, is used to sandwich the cell-free system (e.g., coupled transcription/translation system or translation system) and seal the microchambers (c); The filled microchambers are placed in contact at a temperature for the in situ synthesis of the biomolecular ink within the microchambers (d); and the synthesized biomolecular ink (e.g., proteins) is then diffused and captured on the glass substrate (e). The assembly is peeled off to release the printed protein microarrays from the corresponding DNA microarrays.\n\n2.1. Fabrication of Arrays of Microchambers\nA microchamber-array chip is a basic requirement for soft lithography-based µIP. Micromold plates with desired features can be designed by computer software and prepared by photolithography or ordered from commercial suppliers. These plates are then used to fabricate arrays of microchambers using soft lithography by the replica molding of monolithic slabs of PDMS. To demonstrate the concept of µIP, microchambers of 1.5–100 µm in diameter and depth, giving a density of 40,000–20,000 uniformly-distributed independent microchambers per mm2, were employed.\n\n2.2. Self-Organization and Arraying of Precursors of Biomolecular Ink\nBiomolecular libraries, such as proteomes in a cell or tissue lysate, which are anticipated to be composed of over 100,000 proteins, exist as mixtures in a solution and must first be individualized and then organized into a one-to-one format prior to analysis using a microarray approach. This can be carried out by several techniques, including photolithography and contact printing. However, these are not high-throughput and robust ways of dealing with a full-length library. µIP uses a BEAMing (beads, emulsion, amplification, magnetic) approach [25] to first conduct a single-molecule PCR on a single magnetic micrometer-sized bead carrier using a water-in-oil emulsion. The construct of the DNA is modified to express the proteins with a double-histidine tag. These single-molecule-amplified DNA bead carriers are then self-aligned onto intaglio plates (PDMS micromold plates with dense arrays of microchambers) through an external dynamic magnetic force that is applied briefly by sliding a permanent magnet horizontally along the bottom of the micromold plates. The beads are arranged with only one bead in each microchamber owing to physical constraints in the design of the beads and microchambers. We have recently reported the rapid assembly of ultrahigh-density (144 million) DNA microbead arrays using a magnetic field [23,24,26].\n\n2.3. In Situ Synthesis and Patterning of Microarrays\nThis step enables the co-synthesis and immobilization of biomolecular ink from individual microchambers arrays to corresponding positions on a glass substrate. A droplet of a cell-free-coupled transcription/translation system is sandwiched between a Ni-NTA-modified glass surface and a bead-incorporated microchamber array chip by applying pressure. To ensure perfect contact between the microchamber array chip and the glass substrate, the chip is vertically pressed onto the glass using a bench-mounted hand press on ice. Then, the coupled transcription/translation reaction is initiated by increasing the temperature from 4 °C to room temperature and incubating the assembly at 30 °C for 60 min. Multiple copies of a messenger RNA are transcribed from the bead-bound DNAs in the individual compartments of the microchamber-array chip, which is followed by the immediate translation of the double-histidine-tagged protein, which diffuses to the glass surface and is captured by the Ni-NTA-modified glass surface.\n","divisions":[{"label":"Title","span":{"begin":0,"end":36}},{"label":"Figure caption","span":{"begin":432,"end":1398}},{"label":"Section","span":{"begin":1398,"end":2000}},{"label":"Title","span":{"begin":1398,"end":1441}},{"label":"Section","span":{"begin":2002,"end":3416}},{"label":"Title","span":{"begin":2002,"end":2071}},{"label":"Section","span":{"begin":3418,"end":4485}},{"label":"Title","span":{"begin":3418,"end":3470}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600226-16791214-69477179","span":{"begin":2623,"end":2625},"obj":"16791214"}],"attributes":[{"subj":"27600226-16791214-69477179","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#e9ec93","default":true}]}]}}