Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing

Abstract

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

An Electrochemical Biosensor Platform for Rapid Immunoanalysis of Physiological Fluids

“…In the future, additional antibody pairs could be incorporated for immunoprofiling and the biosensor system could be automated by microfluidics for minimizing the manual handling of samples and be applicable to other physiological fluids, such as whole blood. The immunoanalysis system can also be combined with other microfluidic analysis systems for a comprehensive analysis of infections and inflammation [27], [28].…”

The future of microbiome analysis: Biosensor methods for big data collection and clinical diagnostics

“…These particles are useful in biosensors; for example, Kurt, Yüce, Hussain, and Budak (2016) developed a dual‐excitation sensing method using aptamer‐functionalized QDs and UCNPs to obtain fluorescent emission spectra of two distinct pathogens. Other examples of nanomaterials used for pathogen detection include carbon‐based nanomaterials such as carbon nanotubes (CNTs; Gao et al., 2019), graphene oxide nanosheets (Chen & Nugen, 2019) and carbon nanodots (Yang et al., 2018). Furthermore, metal nanoparticles (mNPs), such as gold, platinum and silver nanoparticles (NPs), possess intrinsic properties including surface plasmon resonance (SPR) and Raman scattering properties, resulting in colour change upon particle aggregation (Sashidhar et al., 2019).…”

“…Both light and bacterial cells are confined within the fibre, and flowing cells can generate unique spectral events, allowing infection quantification and qualification (Hunter et al, 2019). A variety of other substrates have also been utilized in microfluidic platforms, including glass (Zuo, Li, Dominguez, & Ye, 2013), polypropylene (Gao et al, 2019), polydimethylsiloxane (PDMS; Sadat Mousavi et al, 2020), metal-coated surfaces (Tokel et al, 2015) and paper substrates (Fava et al, 2019). Microarrays have been used to identify antibiotics in milk (Kloth et al, 2009), cancer biomarkers in serum (Premaratne et al, 2019;Zhou et al, 2015), enteric pathogens in faecal samples (Yang et al, 2019), etc.…”

Applications of Microwave Energy in Medicine

“…This delivery and id method can be used directly on samples, and the whole process from the initi microscopic identification can take as little as 30 min. NAME can identify path as those mentioned previously at a single cell level enabling accurate quant cell counts of fluorescent cells under the microscope, however further instru opment is required before this method can be clinically evaluated [71,72]. When comparing the state of the art microwave based DNA extraction m current commercially available DNA extraction kits, the overall opinion is that methods are more efficient, cost-effective and simpler, so could be implemen the need for specialised training [63,73].…”