Abstract
We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.
Highlights
Klebsiella pneumoniae (KPN) is a Gram-negative bacterium and opportunistic pathogen belonging to the family Enterobacteriaceae
We found that devices printed from NextDent Ortho Clear (NXOC) that were subjected to either no post curing or post curing at ambient temperature displayed very low
In this work we expanded the state-of-the-art by demonstrating a digital light processing (DLP) 3D printing approach that is suitable for the manufacture of microfluidic devices for real-time recombinase polymerase amplification (RPA)
Summary
Klebsiella pneumoniae (KPN) is a Gram-negative bacterium and opportunistic pathogen belonging to the family Enterobacteriaceae. A recent study [31] of common materials used for FDM found that only a few brands of deep black ABS and PLA showed somewhat acceptable fluorescence behavior in a limited wavelength range from 300–400 nm, which falls outside of the Micromachines 2020, 11, 595 spectra of the most commonly used fluorescent labels used for nucleic acid detection, such as those based on fluorescein. These findings show that FDM, unless new materials are developed, is unsuitable for the construction of monolithic microfluidic systems as the manufacture of optically clear parts with low autofluorescence and fluorescence drift is not possible. We demonstrate highly sensitive detection of both the khe and blaNDM-1 genes by rtRPA
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