Polymer Pen Lithography-Fabricated DNA Arrays for Highly Sensitive and Selective Detection of Unamplified Ganoderma Boninense DNA

Abstract

There is an increasing demand for lithography methods to enable the fabrication of diagnostic devices for the biomedical and agri-food sectors. In this regard, scanning probe lithography methods have emerged as a possible approach for this purpose, as they are not only convenient, robust and accessible, but also enable the deposition of “soft” materials such as complex organic molecules and biomolecules. In this report, the use of polymer pen lithography for the fabrication of DNA oligonucleotide arrays is described, together with the application of the arrays for the sensitive and selective detection of Ganoderma boninense, a fungal pathogen of the oil palm. When used in a sandwich assay format with DNA-conjugated gold nanoparticles, this system is able to generate a visually observable result in the presence of the target DNA. This assay is able to detect as little as 30 ng of Ganoderma-derived DNA without any pre-amplification and without the need for specialist laboratory equipment or training.

Sensitive and selective detection of DNA fragments associated with Ganoderma boninense by DNA-nanoparticle conjugate hybridisation

“…The implementation of strand displacement strategies will also be able to achieve clearer differentiation between sequences bearing mismatches [42][43][44][45]. The overall sensitivity of the assay could also be improved, especially with respect to dsDNA, through further modification of the basic approach, such as incorporating the capture of the nanoparticle aggregates on surfaces, by increasing the probe/target ratio or by increasing the size of the AuNP used [21,32]. Engineering and Physical Sciences Research Council for equipment support under Grants EP/K024485/1, and the HEFCE N8 Agri-food Network for travel funds.…”

“…The DNA-AuNP conjugates were prepared using a general procedure previously described [32] with few modifications. Aqueous solutions of disulfide-functionalised oligonucleotides (100 lM, 40 lL) and tris(2-carboxyethyl)phosphine (20 mM, 40 lL) were combined and incubated for 2 h at room temperature (RT) to prepare 50 lM solutions of the oligonucleotides with reduced thiols.…”

Artificial Biosystems by Printing Biology

“…[134] The first approach demonstrating this significant evolution is constituted by PPL. [78,135] It can be defined as a patterning methodology in which pens made of a soft elastomeric polymer (polydimethylsiloxane) deliver inks [136,137] onto solid supports by controlling the movement of the pen array with a scanning probe microscope on large areas (on the order of several cm 2 ) in a single print step. [138] Elastomeric pens are also characterized by an additional factor for controlling feature size since the pens themselves may deform, resulting in a force-dependent pen-surface contact area.…”

“…Tween-20 10 1 µm Protein immobilization by DDI [78] Oligonucleotides Trehalose Detection of fungal pathogen [137] PEG derivatives -10 0 µm ECM protein immobilization by chemisorption [261] Phospholipids --10 1 µm Phospholipid pattern functionalization with DNA Origami [262] Phospholipids -10 0 -10 1 µm Gradient patterns of phospholipids [135] Phospholipids -10 1 µm Cells recruiting by immobilized protein [136] Thiols -10 -1 -10 0 µm Diffraction Gratings Fabrication [263] Thiols -10 0 µm Patterning on gold surfaces [264] Proteins 10-50% w/v glycerol 10 1 µm Glucose oxidase monolayer at silicon dioxide surface [69] Proteins DMSO/glycerol (9:1) 10 2 µm DMSO-rich liquid compartments [187] Proteins 30% w/v glycerol 10 2 µm Glycerol-rich liquid compartments [188] Lipids 2-bis(10,12tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) Fluorophore (0.1 mM), and 5% (v/v) glycerine.…”

Sensitive and Selective Detection of DNA Fragments Associated with Ganoderma Boninense by DNA-Nanoparticle Conjugate Hybridisation

“…The DNA-AuNP conjugates were prepared using a general procedure previously described [32] This process of centrifugation, decanting, and resuspension was carried out three times in total, with the final pellet of AuNP being re-suspended in 100 μL of 10 mM PBS buffer. The OD was then measured by UV-vis spectroscopy at 525 nm.…”