Abstract
Bacterial infection is a widespread problem in humans that can potentially lead to hospitalization and morbidity. The largest obstacle for physicians/clinicians is the time delay in accurately identifying infectious bacteria, especially their sub-species, in order to adequately treat and diagnose such infected patients. Loop-mediated amplification (LAMP) is a nucleic acid amplification method that has been widely used in diagnostic applications due to its simplicity of constant temperature, use of up to 4 to 6 primers (rendering it highly specific), and capability of amplifying low copies of target sequences. Use of interfacial effect-based monitoring is expected to dramatically shorten the time-to-results of nucleic acid amplification techniques. In this work, we developed a LAMP-based point-of-care platform for detection of bacterial infection, utilizing smartphone measurement of contact angle from oil-immersed droplet LAMP reactions. Whole bacteria (Escherichia coli O157:H7) were assayed in buffer as well as 5% diluted human whole blood. Monitoring of droplet LAMP reactions was demonstrated in a three-compartment, isothermal proportional-integrated-derived (PID)-controlled chip. Smartphone-captured images of droplet LAMP reactions, and their contact angles, were evaluated. Contact angle decreased substantially upon target amplification in both buffer and whole blood samples. In comparison, no-target control (NTC) droplets remained stable throughout the 30 min isothermal reactions. These results were explained by the pre-adsorption of plasma proteins to an oil-water interface (lowering contact angle), followed by time-dependent amplicon formation and their preferential adsorption to the plasma protein-occupied oil-water interface. Time-to-results was as fast as 5 min, allowing physicians to quickly make their decision for infected patients. The developed assay demonstrated quantification of bacteria concentration, with a limit-of-detection at 102 CFU/μL for buffer samples, and binary target or no-target identification with a limit-of-detection at 10 CFU/μL for 5% diluted whole blood samples.
Highlights
Www.nature.com/scientificreports culturing systems, where growth curves in enriched media are monitored via carbon dioxide levels, fluorescence, or colorimetric photometry
Recent attempts to decrease time-to-results for bacterial identification include the integration of molecular diagnostic techniques such as hybridization [i.e. fluorescent in situ hybridization (FISH)], nucleic acid amplification [i.e. broad range and multiplexed polymerase chain reaction (PCR)], mass spectroscopy [i.e. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)], and protein analysis[9]
Escherichia coli O157:H7 and methicillin-sensitive Staphylococcus aureus (MSSA; part #0801675; ZeptoMetrix Corp.) samples were cultured in lysogeny broth (LB) Miller’s formula (Molecular Biological International Inc., Irvine, CA, USA) at 37 °C for 8 hours. 1 mL of final concentrated bacterial stocks (105 CFU/μL) were centrifuged at 5.6 g for 10 min and resuspended in 1 mL of nuclease-free water
Summary
Www.nature.com/scientificreports culturing systems, where growth curves in enriched media are monitored via carbon dioxide levels, fluorescence, or colorimetric photometry. Recent attempts to decrease time-to-results for bacterial identification include the integration of molecular diagnostic techniques such as hybridization [i.e. fluorescent in situ hybridization (FISH)], nucleic acid amplification [i.e. broad range and multiplexed polymerase chain reaction (PCR)], mass spectroscopy [i.e. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)], and protein analysis[9]. By using such molecular techniques, the time-to-results is expedited since blood samples can be directly analyzed, bypassing the culture-enrichment process. It is possible to relate this interfacial tension to molecular self-assembly at the interface (e.g. water-oil) and protein adsorption/kinetics towards the interface[31,32,33,34,35], and eventually to the quantification of amplified products
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