Dielectrophoresis assisted rapid, selective and single cell detection of antibiotic resistant bacteria with G-FETs

Abstract

Time-consuming, expensive and low sensitivity diagnostic methods used for monitoring bacterial infections lead to unnecessary or delays in prescription of the right antibiotic treatment.Determining an optimal clinical treatment requires rapid detection and identification of pathogenic bacteria and their sensitivity to specific antimicrobials. However, diagnostic devices that meet all of these criteria have proven elusive thus far. Graphene field effect transistors (G-FET) are a promising solution, since they are highly sensitive to chemical/biological modification, can have fast detection times and can be placed on different substrates. Here, by integrating specific peptide probes over G-FETs, we present a proof-of-concept study for species and strain specific label-free detection of clinical strains of pathogenic bacteria with high specificity and sensitivity. We found that pyrene-conjugated peptides immobilized on G-FETs were capable of detecting pathogenic Staphylococcus aureus at the single-cell level and discriminate against other gram-positive and gram-negative bacterial pathogens. A similar device was able to discriminate between antibiotic resistant and sensitive strains of Acinetobacter baumannii, suggesting that these devices can also be used for detecting antibiotic resistive pathogens. Furthermore, a new means of enhancing attachment, electric-field assisted binding, reduced the detection limit to 10 4 cells/ml and the detection time to below 5 minutes. The combination of single step attachment, inexpensive production, rapid, selective and sensitive detection suggest G-FETs plus pyrene-conjugated peptides are a new platform for solving major challenges faced in point of care diagnostics to fight infectious diseases and antimicrobial resistance.Bacterial infections cause a wide range of diseases and significant mortality 1 . While antibiotics are key in controlling disease severity and reducing mortality, their over prescription and misuse are some of the most important factors in the surge of antibiotic resistant cases around the world. [2][3][4] In order to solve this crisis, diagnostic methods are needed that can rapidly and accurately identify the bacterium causing the infection and determine its associated antibiotic resistance profile. Antibiotic susceptibility testing (AST) is mostly carried out by phenotypic methods that require prior identification of bacterial pathogens from patients (at the species and/or strain level) and incubation under antibiotic conditions, 5, 6 a lengthy process that can take up to 24 hours to a month depending on the species. 7 Moreover, both species/strain identification and AST require trained specialists, specific laboratory environments and often expensive instrumentation. 5, 6, 8 Since these conditions limit widespread application and implementation into actual treatment strategies at most points of care, there is much room for improvement to develop new diagnostic devices that have the potential for adoption across a large variety of use cases. Ideally these dev...

Microscale electrokinetic‐based analysis of intact cells and viruses

“…Shangguan et al [72] combined eDEP with aptamer–fluorescent silica nanoparticle recognition achieving the detection of S. aureus at concentrations as low as 93 and 270 CFU/mL in deionized water and spiked water samples, respectively. A more recent study by Kumar et al [73] presented a DEP‐based label‐free graphene biosensor using as target S. aureus and antibiotic‐resistant Acinetobacter baumannii . In this device, DEP was employed to facilitate the binding of the target cells to the sensor, lowering the limit of detection to only 10 4 cells/mL with detection times in the order of 5 min.…”

Analysis of microorganisms with nonlinear electrokinetic microsystems

“…Examination of pathogenic bacteria is of vital importance since bacterial species continually affect millions of humans around the world due to their persistent, hardy nature and their ability to develop antibiotic resistance [67,68]. A great example of this is E. coli capable of multiplying quickly within the body and has slowly mutated and evolved to circumvent physiological barriers [37,68]. Just as the field of medicine advances, so do the bacterial species, challenging scientists to create more innovative and alternative solutions [43,69].…”

Detection of a multi‐disease biomarker in saliva with graphene field effect transistors

“…Further, to prevent false positives that could occur due to nonspecific adsorption of CA1, it is crucial to obtain uniform functionalization of aptamers on the graphene surface. As such, the attachment process was optimized using atomic force microscopy (AFM) to determine attachment and coverage of the aptamers (Kumar et al, 2020). Patterned graphene chips were incubated for 30 minutes with high concentration (10 mM) PBASE linker dissolved in DMF (Wang et al, 2015;Xu et al, 2017).…”

“…sensitivity, scalability, biocompatibility, ease of functionalization via a non-covalent attachment process and compatibility with various substrates Gao et al, 2016;Kumar et al, 2020;Ohno et al, 2009Ohno et al, , 2010Ping et al, 2016;Wang et al, 2015;Xu et al, 2017). To determine the possibility of POC detection of CA, it is crucial to investigate the mechanisms of interaction between G-FETs and CA1, their detection limit, sensitivity, selectivity and utility in a clinically relevant environment (Kostarelos & Novoselov, 2014).…”

“…To achieve high sensitivity, we produced clean and reproducible G-FETs by fabricating them in our cleanroom in a glovebox using the same process that produced devices for selective detection of antibiotic-resistant bacteria (Kumar et al, 2020). Our facility allows rapid prototyping with minimal reduction in graphene quality from unwanted impurities that subsequently reduce the accuracy and sensitivity (Macedo et al, 2019).…”