Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Dielectrophoretic Monitoring and Interstrain Separation of Intact <i>Clostridium difficile</i> Based on Their S(Surface)-Layers

<i>Clostridium difficile</i> (<i>C. difficile</i>) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic <i>C. difficile</i> strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic <i>C. difficile</i> strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular <i>C. difficile</i> strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed <i>C. difficile</i> samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic <i>C. difficile</i> strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes

Electrokinetic Preconcentration and Detection of Neuropeptides at Patterned Graphene-Modified Electrodes in a Nanochannel

Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5–15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery

Correlating Antibiotic-Induced Dysbiosis to <i>Clostridioides difficile</i> Spore Germination and Host Susceptibility to Infection Using an <i>Ex Vivo</i> Assay

Antibiotic-induced microbiota disruption and its persistence create conditions for dysbiosis and colonization by opportunistic pathogens, such as those causing Clostridioides difficile (C. difficile) infection (CDI), which is the most severe hospital-acquired intestinal infection. Given the wide differences in microbiota across hosts and in their recovery after antibiotic treatments, there is a need for assays to assess the influence of dysbiosis and its recovery dynamics on the susceptibility of the host to CDI. Germination of C. difficile spores is a key virulence trait for the onset of CDI, which is influenced by the level of primary vs secondary bile acids in the intestinal milieu that is regulated by the microbiota composition. Herein, the germination of C. difficile spores in fecal supernatant from mice that are subject to varying degrees of antibiotic treatment is utilized as an ex vivo assay to predict intestinal dysbiosis in the host based on their susceptibility to CDI, as determined by in vivo CDI metrics in the same mouse model. Quantification of spore germination down to lower detection limits than the colony-forming assay is achieved by using impedance cytometry to count single vegetative bacteria that are identified based on their characteristic electrical physiology for distinction vs aggregated spores and cell debris in the media. As a result, germination can be quantified at earlier time points and with fewer spores for correlation to CDI outcomes. This sets the groundwork for a point-of-care tool to gauge the susceptibility of human microbiota to CDI after antibiotic treatments

Real-Time Electrochemical Monitoring of Adenosine Triphosphate in the Picomolar to Micromolar Range Using Graphene-Modified Electrodes

We report on a competitive electrochemical detection system that is free of wash steps and enables the real-time monitoring of adenosine triphosphate (ATP) in a quantitative manner over a five-log concentration range. The system utilizes a recognition surface based on ATP aptamer (ATPA) capture probes prebound to electroactive flavin adenine dinucleotide (FAD) molecules, and a signaling surface utilizing graphene (Gr) and gold nanoparticle (AuNP) modified carbon paste electrode (Gr–AuNP–CPE) that is optimized to enhance electron-transfer kinetics and signal sensitivity. Binding of ATP to ATPA at the recognition surface causes the release of an equivalent concentration of FAD that can be quantitatively monitored in real time at the signaling surface, thereby enabling a wide linear working range (1.14 × 10^–10 to 3.0 × 10^–5 M), a low detection limit (2.01 × 10^–11 M using graphene and AuNP modified glassy carbon), and fast target binding kinetics (steady-state signal within 12 min at detection limit). Unlike assays based on capture probe-immobilized electrodes, this double-surface competitive assay offers the ability to speed up target binding kinetics by increasing the capture probe concentration, with no limitations due to intermolecular Coulombic interactions and nonspecific binding. We utilize the real-time monitoring capability to compute kinetic parameters for target binding and to make quantitative distinctions on degree of base-pair mismatch through monitoring target binding kinetics over a wide concentration range. On the basis of the simplicity of the assay chemistry and the quantitative detection of ATP within fruit and serum media, as demonstrated by comparison of ATP levels against those determined using a standard high-performance liquid chromatography (HPLC)-UV absorbance method, we envision a versatile detection platform for applications requiring real-time monitoring over a wide target concentration range