Bacterial communities exhibit complex self-organization that contributes to their survival. To better understand the molecules that contribute to transforming a small number of cells into a heterogeneous surface biofilm community, we studied acellular aggregates, structures seen by light microscopy in Pseudomonas aeruginosa colony biofilms using light microscopy and chemical imaging. These structures differ from cellular aggregates, cohesive clusters of cells important for biofilm formation, in that they are visually distinct from cells using light microscopy and are reliant on metabolites for assembly. To investigate how these structures benefit a biofilm community we characterized three recurrent types of acellular aggregates with distinct geometries that were each abundant in specific areas of these biofilms. Alkyl quinolones (AQs) were essential for the formation of all aggregate types with AQ signatures outside the aggregates below the limit of detection. These acellular aggregates spatially sequester AQs and differentiate the biofilm space. However, the three types of aggregates showed differing properties in their size, associated cell death, and lipid content. The largest aggregate type co-localized with spatially confined cell death that was not mediated by Pf4 bacteriophage. Biofilms lacking AQs were absent of localized cell death but exhibited increased, homogeneously distributed cell death. Thus, these AQ-rich aggregates regulate metabolite accessibility, differentiate regions of the biofilm, and promote survival in biofilms.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen with the ability to cause infection in the immune-compromised. It is well established that P. aeruginosa biofilms exhibit resilience that includes decreased susceptibility to antimicrobial treatment. This work examines the self-assembled heterogeneity in biofilm communities studying acellular aggregates, regions of condensed matter requiring alkyl quinolones (AQs). AQs are important to both virulence and biofilm formation. Aggregate structures described here spatially regulate the accessibility of these AQs, differentiate regions of the biofilm community, and despite their association with autolysis, correlate with improved P. aeruginosa colony biofilm survival.
Pseudomonas Infections , Quinolones , Humans , Quinolones/metabolism , Biofilms , Pseudomonas Infections/microbiology , Virulence , Pseudomonas aeruginosa/metabolism
The advent of 3D printing has facilitated the rapid fabrication of microfluidic devices that are accessible and cost-effective. However, it remains a challenge to fabricate sophisticated microfluidic devices with integrated structural and functional components due to limited material options of existing printing methods and their stringent requirement on feedstock material properties. Here, a multi-materials multi-scale hybrid printing method that enables seamless integration of a broad range of structural and functional materials into complex devices is reported. A fully printed and assembly-free microfluidic biosensor with embedded fluidic channels and functionalized electrodes at sub-100 µm spatial resolution for the amperometric sensing of lactate in sweat is demonstrated. The sensors present a sensitive response with a limit of detection of 442 nm and a linear dynamic range of 1-10 mm, which are performance characteristics relevant to physiological levels of lactate in sweat. The versatile hybrid printing method offers a new pathway toward facile fabrication of next-generation integrated devices for broad applications in point-of-care health monitoring and sensing.
Biosensing Techniques , Lab-On-A-Chip Devices , Microfluidics , Biosensing Techniques/methods , Printing, Three-Dimensional , Lactates
Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a wide range of diseases in immunocompromised patients. In order to better understand P. aeruginosa behavior and virulence and to advance drug therapies to combat infection, it would be beneficial to understand how P. aeruginosa cells survive stressful conditions, especially environmental stressors. Here, we report on a strategy that measures potential-dependent fluorescence of individual P. aeruginosa cells, as a sentinel, for cellular response to starvation, hunger, and oxidative stress. This is accomplished using a micropore electrode array capable of trapping large numbers of isolated, vertically oriented cells at well-defined spatial positions in order to study large arrays of single cells in parallel. We find that conditions promoting either starvation or oxidative stress produce discernible changes in the fluorescence response, demonstrated by an increase in the prevalence of fluorescence transients, one of three canonical spectroelectrochemical behaviors exhibited by single P. aeruginosa cells. In contrast, more modest nutrient limitations have little to no effect on the spectroelectrochemical response when compared to healthy cells in the stationary phase. These findings demonstrate the capabilities of micropore electrode arrays for studying the behavior of single microbial cells under conditions where the intercellular spacing, orientation, and chemical environment of the cells are controlled. Realizing single-cell studies under such well-defined conditions makes it possible to study fundamental stress responses with unprecedented control.
In this study, we use nanopore arrays as a platform for detecting and characterizing individual nanoparticles (NPs) in real time. Dark-field imaging of nanopores with dimensions smaller than the wavelength of light occurs under conditions where trans-illumination is blocked, while the scattered light propagates to the far-field, making it possible to identify nanopores. The intensity of scattering increases dramatically during insertion of AgNPs into empty nanopores, owing to their plasmonic properties. Thus, momentary occupation of a nanopore by a AgNP produces intensity transients that can be analyzed to reveal the following characteristics: (1) NP scattering intensity, which scales with the sixth power of the AgNP radius, shows a normal distribution arising from the heterogeneity in NP size, (2) the nanopore residence time of NPs, which was observed to be stochastic with no permselective effects, and (3) the frequency of AgNP capture events on a 21 × 21 nanopore array, which varies linearly with the concentration of the NPs, agreeing with the frequency calculated from theory. The lower limit of detection (LOD) for NPs was 130 fM, indicating that the measurement can be used in applications in which ultrasensitive detection is required. The results presented here provide valuable insights into the dynamics of NP transport into and out of nanopores and highlight the potential of nanopore arrays as powerful, massively parallel tools for nanoparticle characterization and detection.
Hydrophobic gating in biological transport proteins is regulated by stimulus-specific switching between filled and empty nanocavities, endowing them with selective mass transport capabilities. Inspired by these, solid-state nanochannels have been integrated into functional materials for a broad range of applications, such as energy conversion, filtration, and nanoelectronics, and here we extend these to electrochemical biosensors coupled to mass transport control elements. Specifically, we report hierarchically organized structures with block copolymers on tyrosinase-modified two-electrode nanopore electrode arrays (BCP@NEAs) as stimulus-controlled electrochemical biosensors for alkylphenols. A polystyrene-b-poly(4-vinyl)pyridine (PS-b-P4VP) membrane placed atop the NEA endows the system with potential-responsive gating properties, where water transport is spatially and temporarily gated through hydrophobic P4VP nanochannels by the application of appropriate potentials. The reversibility of hydrophobic voltage-gating makes it possible to capture and confine analyte species in the attoliter-volume vestibule of cylindrical nanopore electrodes, enabling redox cycling and yielding enhanced currents with amplification factors >100× when operated in a generator-collector mode. The enzyme-coupled sensing capabilities are demonstrated using nonelectroactive 4-ethyl phenol, exploiting the tyrosinase-catalyzed turnover into reversibly redox-active quinones, then using the quinone-catechol redox reaction to achieve ultrasensitive cycling currents in confined BCP@NEA sensors giving a limit-of-detection of â¼120 nM. The mass transport controlled sensing platform described here is relevant to the development of enzyme-coupled multiplex biosensors for sensitive and selective detection of biomarkers and metabolites in next-generation point-of-care devices.
Biosensing Techniques , Nanopores , Monophenol Monooxygenase , Electrodes , Oxidation-Reduction , Phenols
Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen responsible for a number of healthcare-associated infection. It is currently difficult to assess single cell behaviors of P. aeruginosa that might contribute to acquisition of antibiotic resistance, intercellular communication, biofilm development, or virulence, because mechanistic behavior is inferred from ensemble collections of cells, thus averaging effects over a population. Here, we develop and characterize a device that can capture and trap arrays of single P. aeruginosa cells in individual micropores in order to study their behaviors using spectroelectrochemistry. Focused ion beam milling is used to fabricate an array of micropores in a Au/dielectric/Au/SiO2-containing multilayer substrate, in which individual micropores are formed with dimensions that facilitate the capture of single P. aeruginosa cells in a predominantly vertical orientation. The bottom Au ring is then used as a working electrode to explore the spectroelectrochemical behavior of parallel arrays of individual P. aeruginosa cells. Application of step-potential or swept-potential waveforms produces changes in the fluorescence emission that can be imaged and correlated with applied potential. Arrays of P. aeruginosa cells typically exhibit three characteristic fluorescence behaviors that are sensitive to nutritional stress and applied potential. The device developed here enables the study of parallel collections of single bacterial cells with well-defined orientational order and should facilitate efforts to elucidate methods of bacterial communication and multidrug resistance at the single cell level.
Pseudomonas aeruginosa (P. aeruginosa) is commonly implicated in hospital-acquired infections where its capacity to form biofilms on a variety of surfaces and the resulting enhanced antibiotic resistance seriously limit treatment choices. Because surface attachment sensitizes P. aeruginosa to quorum sensing (QS) and induces virulence through both chemical and mechanical cues, we investigate the effect of surface properties through spatially patterned mucin, combined with sub-inhibitory concentrations of tobramycin on QS and virulence factors in both mucoid and non-mucoid P. aeruginosa strains using multi-modal chemical imaging combining confocal Raman microscopy and matrix-assisted laser desorption/ionization-mass spectrometry. Samples comprise surface-adherent static biofilms at a solid-water interface, supernatant liquid, and pellicle biofilms at an air-water interface at various time points. Although the presence of a sub-inhibitory concentration of tobramycin in the supernatant retards growth and development of static biofilms independent of strain and surface mucin patterning, we observe clear differences in the behavior of mucoid and non-mucoid strains. Quinolone signals in a non-mucoid strain are induced earlier and are influenced by mucin surface patterning to a degree not exhibited in the mucoid strain. Additionally, phenazine virulence factors, such as pyocyanin, are observed in the pellicle biofilms of both mucoid and non-mucoid strains but are not detected in the static biofilms from either strain, highlighting the differences in stress response between pellicle and static biofilms. Differences between mucoid and non-mucoid strains are consistent with their strain-specific phenology, in which the mucoid strain develops highly protected biofilms.
Anti-Bacterial Agents , Quinolones , Anti-Bacterial Agents/pharmacology , Pseudomonas aeruginosa , Quinolones/pharmacology , Mucins , Biofilms , Tobramycin/pharmacology , Virulence Factors
We report a closed bipolar electrode (CBE)-based sensing platform for the detection of diagnostic metabolites in undiluted whole human blood. The sensor is enabled by electrode chemistry based on: (1) a mixed layer of blood-compatible adsorption-resistant phosphorylcholine (PPC) and phenylbutyric acid (PBA), (2) ferrocene (Fc) redox mediators, and (3) immobilized redox-active enzymes. This scheme is designed to overcome nonspecific protein adsorption and amplify sensing currents in whole human fluids. The scheme also incorporates a diffusing mediator to increase electronic communication between the immobilized redox enzyme and the working electrode. The use of both bound and freely diffusing mediators is synergistic in producing the electrochemical response. The sensor is realized by linking the analyte cell, containing the specific electrode surface architecture, through a CBE to a reporter cell containing the electrochromic reporter, methyl viologen (MV). The colorless-to-purple color change accompanying the 1e- reduction of MV2+ is captured using a smartphone camera. Subsequent red-green-blue analysis is performed on the acquired images to determine cholesterol, glucose, and lactate concentrations in whole blood. The CBE blood metabolite sensor produces a linear color change at clinically relevant concentration ranges for all metabolites with good reproducibility (â¼5% or better) and with limits of detection of 79 µM for cholesterol, 59 µM for glucose, and 86 µM for lactate. Finally, metabolite concentration measurements from the CBE blood metabolite sensor are compared with results from commercially available FDA-approved blood cholesterol, glucose, and lactate meters, with an average difference of â¼3.5% across all three metabolites in the ranges studied.
Biosensing Techniques , Blood Chemical Analysis , Humans , Biosensing Techniques/methods , Electrodes , Enzymes, Immobilized , Glucose , Lactic Acid , Paraquat , Reproducibility of Results , Blood Chemical Analysis/instrumentation
Indium-tin oxide (ITO) is used in a variety of applications due to its electrical conductivity and optical transparency. Moreover, ITO coated glass is a common working electrode for spectroelectrochemistry. Thus, the ITO substrates should exhibit well-understood spectroscopic characteristics. Here, we report anomalous potential-dependent luminescence emission from three structurally-dissimilar electrofluorogenic probe on ITO coated glass. The three probes, flavin mononucleotide, resorufin, and Nile blue, show the expected fluorescence modulation between their oxidized, emissive forms and their reduced, non-fluorescent forms at low laser irradiance and/or high concentrations. However, at high irradiance and/or low concentration, the emission intensity increases at reducing potentials, contrary to expectations. In addition, a strong interplay between probe molecule concentration and laser irradiance is observed. We attribute the anomalous behavior to a combination of (1) irradiance-dependent ITO carrier dynamics, and (2) interaction of the fluorescent probe with ITO at reducing potentials resulting in a charge transfer state with altered emission behavior. Thus, the potential- and irradiance-dependent behavior of ITO and the resulting charge transfer state may not only interfere with the observation of potential-dependent fluorescence from redox probes but can completely reverse the polarity of the potential-dependent luminescence, especially at high irradiance and low concentration.
Understanding functional states of individual redox enzymes is important because electron-transfer reactions are fundamental to life, and single-enzyme molecules exhibit molecule-to-molecule heterogeneity in their properties, such as catalytic activity. Zero-mode waveguides (ZMW) constitute a powerful tool for single-molecule studies, enabling investigations of binding reactions up to the micromolar range due to the ability to trap electromagnetic radiation in zeptoliter-scale observation volumes. Here, we report the potential-dependent fluorescence dynamics of single glutathione reductase (GR) molecules using a bimodal electrochemical ZMW (E-ZMW), where a single-ring electrode embedded in each of the nanopores of an E-ZMW array simultaneously serves to control electrochemical potential and to confine optical radiation within the nanopores. Here, the redox state of GR is manipulated using an external potential control of the Au electrode in the presence of a redox mediator, methyl viologen (MV). Redox-state transitions in GR are monitored by correlating electrochemical and spectroscopic signals from freely diffusing MV/GR in 60 zL effective observation volumes at single GR molecule average pore occupancy, ⟨n⟩ â¼ 0.8. Fluorescence intensities decrease (increase) at reducing (oxidizing) potentials for MV due to the MV-mediated control of the GR redox state. The spectroelectrochemical response of GR to the enzyme substrate, i.e., glutathione disulfide (GSSG), shows that GSSG promotes GR oxidation via enzymatic reduction. The capabilities of E-ZMWs to probe spectroelectrochemical phenomena in zL-scale-confined environments show great promise for the study of single-enzyme reactions and can be extended to important technological applications, such as those in molecular diagnostics.
Glutathione Reductase , Glutathione , Nanotechnology , Single Molecule Imaging , Diffusion , Fluorescence , Glutathione Disulfide , Glutathione Reductase/chemistry , Oxidation-Reduction , Single Molecule Imaging/methods
Wetting and dewetting behavior in channel-confined hydrophobic volumes is used in biological membranes to effect selective ion/molecular transport. Artificial biomimetic hydrophobic nanopores have been devised utilizing wetting and dewetting, however, tunable mass transport control utilizing multiple transport modes is required for applications such as controllable release/transport, water separation/purification and energy conversion. Here, we investigate the potential-induced wetting and dewetting behavior in a pH-responsive membrane composed of a polystyrene-b-poly(4-vinylpyridine) (PS-b-P4VP) block copolymer (BCP) when fabricated as a hierarchically-organized sandwich structure on a nanopore electrode array (NEA), i.e. BCP@NEA. At pH < pKa(P4VP) (pKa â¼ 4.8), the BCP acts as an anion-exchange membrane due to the hydrophilic, protonated P4VP cylindrical nanodomains, but at pH > pKa(P4VP), the P4VP domains exhibit charge-neutral, hydrophobic and collapsed structures, blocking mass transport via the hydrophobic membrane. However, when originally prepared in a dewetted condition, mass transport in the BCP membrane may be switched on if sufficiently negative potentials are applied to the BCP@NEA architecture. When the hydrophobic BCP membrane is introduced on top of 2-electrode-embedded nanopore arrays, electrolyte solution in the nanopores is introduced, then isolated, by exploiting the potential-induced wetting and dewetting transitions in the BCP membrane. The potential-induced wetting/dewetting transition and the effect on cyclic voltammetry in the BCP@NEA structures is characterized as a function of the potential, pH and ionic strength. In addition, chronoamperometry and redox cycling experiments are used to further characterize the potential response. The multi-modal mass transport system proposed in this work will be useful for ultrasensitive sensing and single-molecule studies, which require long-time monitoring to explore reaction dynamics as well as molecular heterogeneity in nanoconfined volumes.
Nanopores , Electrodes , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Nanotechnology
Myxococcus xanthus is a common soil bacterium with a complex life cycle, which is known for production of secondary metabolites. However, little is known about the effects of nutrient availability on M. xanthus metabolite production. In this study, we utilize confocal Raman microscopy (CRM) to examine the spatiotemporal distribution of chemical signatures secreted by M. xanthus and their response to varied nutrient availability. Ten distinct spectral features are observed by CRM from M. xanthus grown on nutrient-rich medium. However, when M. xanthus is constrained to grow under nutrient-limited conditions, by starving it of casitone, it develops fruiting bodies, and the accompanying Raman microspectra are dramatically altered. The reduced metabolic state engendered by the absence of casitone in the medium is associated with reduced, or completely eliminated, features at 1140 cm-1, 1560 cm-1, and 1648 cm-1. In their place, a feature at 1537 cm-1 is observed, this feature being tentatively assigned to a transitional phase important for cellular adaptation to varying environmental conditions. In addition, correlating principal component analysis heat maps with optical images illustrates how fruiting bodies in the center co-exist with motile cells at the colony edge. While the metabolites responsible for these Raman features are not completely identified, three M. xanthus peaks at 1004, 1151, and 1510 cm-1 are consistent with the production of lycopene. Thus, a combination of CRM imaging and PCA enables the spatial mapping of spectral signatures of secreted factors from M. xanthus and their correlation with metabolic conditions.
Myxococcus xanthus/metabolism , Cell Culture Techniques , Culture Media/chemistry , Culture Media/metabolism , Metabolome , Myxococcus xanthus/chemistry , Myxococcus xanthus/growth & development , Spectrum Analysis, Raman
Microbes, such as bacteria, can be described, at one level, as small, self-sustaining chemical factories. Based on the species, strain, and even the environment, bacteria can be useful, neutral or pathogenic to human life, so it is increasingly important that we be able to characterize them at the molecular level with chemical specificity and spatial and temporal resolution in order to understand their behavior. Bacterial metabolism involves a large number of internal and external electron transfer processes, so it is logical that electrochemical techniques have been employed to investigate these bacterial metabolites. In this mini-review, we focus on electrochemical and spectroelectrochemical methods that have been developed and used specifically to chemically characterize bacteria and their behavior. First, we discuss the latest mechanistic insights and current understanding of microbial electron transfer, including both direct and mediated electron transfer. Second, we summarize progress on approaches to spatiotemporal characterization of secreted factors, including both metabolites and signaling molecules, which can be used to discern how natural or external factors can alter metabolic states of bacterial cells and change either their individual or collective behavior. Finally, we address in situ methods of single-cell characterization, which can uncover how heterogeneity in cell behavior is reflected in the behavior and properties of collections of bacteria, e.g. bacterial communities. Recent advances in (spectro)electrochemical characterization of bacteria have yielded important new insights both at the ensemble and the single-entity levels, which are furthering our understanding of bacterial behavior. These insights, in turn, promise to benefit applications ranging from biosensors to the use of bacteria in bacteria-based bioenergy generation and storage.
Bacteria , Mass Gatherings , Bacteria/genetics , Humans
Pseudomonas aeruginosa produces a number of phenazine metabolites, including pyocyanin (PYO), phenazine-1-carboxamide (PCN), and phenazine-1-carboxylic acid (PCA). Among these, PYO has been most widely studied as a biomarker of P. aeruginosa infection. However, despite its broad-spectrum antibiotic properties and its role as a precursor in the biosynthetic route leading to other secondary phenazines, PCA has attracted less attention, partially due to its relatively low concentration and interference from other highly abundant phenazines. This challenge is addressed here by constructing a hierarchically organized nanostructure consisting of a pH-responsive block copolymer (BCP) membrane with nanopore electrode arrays (NEAs) filled with gold nanoparticles (AuNPs) to separate and detect PCA in bacterial environments. The BCP@NEA strategy is designed such that adjusting the pH of the bacterial medium to 4.5, which is above the pKa of PCA but below the pKa of PYO and PCN, ensures that PCA is negatively charged and can be selectively transported across the BCP membrane. At pH 4.5, only PCA is transported into the AuNP-filled NEAs, while PYO and PCN are blocked. Structural characterization illustrates the rigorous spatial segregation of the AuNPs in the NEA nanopore volume, allowing PCA secreted from P. aeruginosa to be quantitatively determined as a function of incubation time using square-wave voltammetry and surface-enhanced Raman spectroscopy. The strategy proposed in this study can be extended by changing the nature of the hydrophilic block and subsequently applied to detect other redox-active metabolites at a low concentration in complex biological samples and, thus, help understand metabolism in microbial communities.
Metal Nanoparticles , Nanopores , Electrodes , Gold , Phenazines , Pseudomonas aeruginosa , Pyocyanine
Pseudomonas aeruginosa is an opportunistic human pathogen implicated in both acute and chronic diseases, which resists antibiotic treatment, in part by forming physical and chemical barriers such as biofilms. Here, we explore the use of confocal Raman imaging to characterize the three-dimensional (3D) spatial distribution of alkyl quinolones (AQs) in P. aeruginosa biofilms by reconstructing depth profiles from hyperspectral Raman data. AQs are important to quorum sensing (QS), virulence, and other actions of P. aeruginosa. Three-dimensional distributions of three different AQs (PQS, HQNO, and HHQ) were observed to have a significant depth, suggesting 3D anisotropic shapes-sheet-like rectangular solids for HQNO and extended cylinders for PQS. Similar to observations from 2D imaging studies, spectral features characteristic of AQs (HQNO or PQS) and the amide I vibration from peptide-containing species were found to correlate with the PQS cylinders typically located at the tips of the HQNO rectangular solids. In the QS-deficient mutant lasIrhlI, a small globular component was observed, whose highly localized nature and similarity in size to a P. aeruginosa cell suggest that the feature arises from HHQ localized in the vicinity of the cell from which it was secreted. The difference in the shapes and sizes of the aggregates of the three AQs in wild-type and mutant P. aeruginosa is likely related to the difference in the cellular response to growth conditions, environmental stress, metabolic levels, or other structural and biochemical variations inside biofilms. This study provides a new route to characterizing the 3D structure of biofilms and shows the potential of confocal Raman imaging to elucidate the nature of heterogeneous biofilms in all three spatial dimensions. These capabilities should be applicable as a tool in studies of infectious diseases.
Biofilms/drug effects , Pseudomonas aeruginosa/drug effects , Quinolones/pharmacology , Biofilms/growth & development , Microscopy, Confocal , Quinolones/chemistry , Spectrum Analysis, Raman
Quinolone, pyocyanin, and rhamnolipid production were studied in Pseudomonas aeruginosa by spatially patterning mucin, a glycoprotein important to infection of lung epithelia. Mass spectrometric imaging and confocal Raman microscopy are combined to probe P. aeruginosa biofilms from mucoid and nonmucoid strains grown on lithographically defined patterns. Quinolone signatures from biofilms on patterned vs unpatterned and mucin vs mercaptoundecanoic acid (MUA) surfaces were compared. Microbial attachment is accompanied by secretion of 2-alkyl-4-quinolones as well as rhamnolipids from the mucoid and nonmucoid strains. Pyocyanin was also detected both in the biofilm and in the supernatant in the mucoid strain only. Significant differences in the spatiotemporal distributions of secreted factors are observed between strains and among different surface patterning conditions. The mucoid strain is sensitive to composition and patterning while the nonmucoid strain is not, and in promoting community development in the mucoid strain, nonpatterned surfaces are better than patterned, and mucin is better than MUA. Also, the mucoid strain secretes the virulence factor pyocyanin in a way that correlates with distress. A change in the relative abundance for two rhamnolipids is observed in the mucoid strain during exposure to mucin, whereas minimal variation is observed in the nonmucoid strain. Differences between mucoid and nonmucoid strains are consistent with their strain-specific phenology, in which the mucoid strain develops highly protected and withdrawn biofilms that achieve Pseudomonas quinolone signal production under limited conditions.
Pseudomonas aeruginosa , Pyocyanine , Biofilms , Biopolymers , Lung
The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal-insulator-metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM ãnporeãâ¼ 0.082 per nanopore - a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. The NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.
Nanopores , Pseudomonas aeruginosa , Electrodes , Oxidation-Reduction , Phenazines , Pseudomonas aeruginosa/metabolism , Pyocyanine
In this work, we develop a label-free electrochemical immunosensor for the detection of interleukin-6 (IL-6) in human cerebrospinal fluid (CSF) and serum for diagnostic and therapeutic monitoring. The IL-6 immunosensor is fabricated from gold interdigitated electrode arrays (IDEAs) that are modified with IL-6 antibodies for direct antigen recognition and capture. A rigorous surface analysis of the sensor architecture was conducted to ensure high structural fidelity and performance. Electrochemical characterization was conducted by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and sensing was performed using differential pulse voltammetry (DPV). The DPV peak current was used to quantify IL-6 in buffer, CSF, and serum in the range 1 pg mL-1 < [IL-6] < 1 µg mL-1. The IL-6 IDEA sensor achieved a limit of detection (LOD) of 1.63 pg mL-1 in PBS, 2.34 pg mL-1 in human CSF, and 11.83 pg mL-1 in human serum. The sensor response is linear in the concentration range 10 pg mL-1 < [IL-6] < 10 ng mL-1, and the sensor is selective for IL-6 over other common cytokines, including IL-10 and TNF-α. EIS measurements showed that the resistance to charge transfer, R CT, decreases upon IL-6 binding, an observation attributed to a structural change upon Ab-Ag binding that opens up the architecture so that the redox probe can more easily access the electrode surface. The IL-6 IDEA sensor can be used as a point-of-care diagnostic tool to deliver rapid results (â¼3 min) in clinical settings for traumatic brain injury, and potentially address the unmet need for effective diagnostic and prognostic tools for other cytokine-related illnesses, such as sepsis and COVID-19 induced cytokine storms. Given the interdigitated electrode form factor, it is likely that the performance of the sensor can be further improved through redox cycling.
