A paper based graphene-nanocauliflower hybrid composite for point of care biosensing

Graphene paper has diverse applications in printed circuit board electronics, bioassays, 3D cell culture, and biosensing. Although development of nanometal-graphene hybrid composites is commonplace in the sensing literature, to date there are only a few examples of nanometal-decorated graphene paper for use in biosensing. In this manuscript, we demonstrate the synthesis and application of Pt nano cauliflower-functionalized graphene paper for use in electrochemical biosensing of small molecules (glucose, acetone, methanol) or detection of pathogenic bacteria (Escherichia coli O157:H7). Raman spectroscopy, scanning electron microscopy and energy dispersive spectroscopy were used to show that graphene oxide deposited on nanocellulose crystals was partially reduced by both thermal and chemical treatment. Fractal platinum nanostructures were formed on the reduced graphene oxide paper, producing a conductive paper with an extremely high electroactive surface area, confirmed by cyclic voltammetry and electrochemical impedance spectroscopy. To show the broad applicability of the material, the platinum surface was functionalized with three different biomaterials: 1) glucose oxidase (via chitosan encapsulation); 2) a DNA aptamer (via covalent linking), or 3) a chemosensory protein (via his linking). We demonstrate the application of this device for point of care biosensing. The detection limit for both glucose (0.08 ± 0.02 μM) and E. coli O157:H7 (1.3 ± 0.1 CFU mL-1) were competitive with, or superior to, previously reported devices in the biosensing literature. The response time (6 sec for glucose and 10 min for E. coli) were also similar to silicon biochip and commercial electrode sensors. The results demonstrate that the nanocellulose-graphene-nanoplatinum material is an excellent paper-based platform for development of electrochemical biosensors targeting small molecules or whole cells for use in point of care biosensing

Salmonella enterica biofilm-mediated dispersal by nitric oxide donors in association with cellulose nanocrystal hydrogels

Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies

Pentachlorophenol Induction of the Pseudomonas aeruginosa mexAB-oprM Efflux Operon: Involvement of Repressors NalC and MexR and the Antirepressor ArmR

Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM - its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels - suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature

Non-invasive characterization of real time biofilm analyte flux

The use of tools (such as sensors) which are capable of real time analysis of compounds in temporally and spatially dynamic environmental conditions is one of the most important areas of research within environmental engineering. Recent technological advancements in electrochemistry, data acquisition, and optics have made possible the development of highly selective/sensitive sensing tools with high temporal spatial resolution. In addition to ongoing research aimed at developing new and enhanced sensors (e.g., increased sensitivity, enhanced analyte selectivity, reduced response time, and novel microfabrication approaches), work over the last few decades has also advanced sensor utility through new sensing modalities that extend and enhance the data recorded by the sensor. When combined with recent advancements in nanoscale technology, these tools are commonly utilized in a variety of applications and sensing modalities (e.g., lab-on-chip devices and chemical assays). One of the most complex and dynamic areas of research in environmental science and engineering is the design of bioprocessors for treating complex wastestreams. Application of tools which are at the forefront of the scientific frontier for characterizing phenomena within these bioprocesses provides invaluable information on non steady state phenomena. One of the most commonly studied microorganisms in bioprocesses (due to its rate limiting role in the nitrogen cycle) is the obligate aerobic chemoautotroph Nitrosomonas europaea. Inhibition studies and fundamental analysis of metabolomics are important in efficient bioprocess design, as N. europaea lack the catabolic flexibility of other bacteria; and are generally more susceptible to decreased kinetic rates (or even cell lysis). Thus, much research is conducted for N. europaea within self-assembled matrices (biofilms); which are significantly different than their planktonic counterpart; demonstrating unique resistance to toxicity, varying growth kinetics, and community dynamics. The ecological and chemical dynamics within N. europaea biofilms are extremely important to the field of environmental engineering, and characterization of physiological phenomena requires in vivo analysis using non-invasive techniques. The use of a novel sensing modality established in other biological disciplines (e.g., biomedicine, plant physiology) to investigate dynamic analyte flux in environmental biofilms is presented (special focus is on N. europaea biofilms). The major challenges associated with use of this technique are addressed, and successful use of the technique for analyzing biologically active transport is demonstrated. Results from this research will be used to enhance fundamental understanding of complex phenomena within biofilms, improve biofilm modeling, develop real time monitoring devices, and improve in situ remediation of complex compounds

Rapid and label-free Listeria monocytogenes detection based on stimuli-responsive alginate-platinum thiomer nanobrushes

In this work, we demonstrate the development of a rapid and label-free electrochemical biosensor to detect Listeria monocytogenes using a novel stimulus–response thiomer nanobrush material. Nanobrushes were developed via one-step simultaneous co-deposition of nanoplatinum (Pt) and alginate thiomers (ALG-thiomer). ALG-thiomer/Pt nanobrush platform significantly increased the average electroactive surface area of electrodes by 7 folds and maintained the actuation properties (pH-stimulated osmotic swelling) of the alginate. Dielectric behavior during brush actuation was characterized with positively, neutral, and negatively charged redox probes above and below the isoelectric point of alginate, indicating ALG-thiomer surface charge plays an important role in signal acquisition. The ALG-thiomer platform was biofunctionalized with an aptamer selective for the internalin A protein on Listeria for biosensing applications. Aptamer loading was optimized and various cell capture strategies were investigated (brush extended versus collapsed). Maximum cell capture occurs when the ALG-thiomer/aptamer is in the extended conformation (pH > 3.5), followed by impedance measurement in the collapsed conformation (pH < 3.5). Low concentrations of bacteria (5 CFU mL−1) were sensed from a complex food matrix (chicken broth) and selectivity testing against other Gram-positive bacteria (Staphylococcus aureus) indicate the aptamer affinity is maintained, even at these pH values. The new hybrid soft material is among the most efficient and fastest (17 min) for L. monocytogenes biosensing to date, and does not require sample pretreatment, constituting a promising new material platform for sensing small molecules or cells.This article is published as Oliveira, Daniela A., Eric S. McLamore, and Carmen L. Gomes. "Rapid and label-free Listeria monocytogenes detection based on stimuli-responsive alginate-platinum thiomer nanobrushes." Scientific Reports 12, no. 1 (2022): 21413. DOI: 10.1038/s41598-022-25753-7, Copyright 2022 The Author(s). Attribution 4.0 International (CC BY 4.0). Posted with permission

Oscillatory glucose flux in INS 1 pancreatic beta cells: A self-referencing microbiosensor study

Signaling and insulin secretion in beta cells have been reported to demonstrate oscillatory modes, with abnormal oscillations associated with type 2 diabetes. We investigated cellular glucose influx in beta cells with a self-referencing (SR) microbiosensor based on nanomaterials with enhanced performance. Dose-response analyses with glucose and metabolic inhibition studies were used to study oscillatory patterns and transporter kinetics. For the first time, we report a stable and regular oscillatory uptake of glucose (averaged period 2.9 +/- 0.6 min), which corresponds well with an oscillator model. This oscillatory behavior is part of the feedback control pathway involving oxygen, cytosolic Ca(2+)/ATP, and insulin secretion (periodicity approximately 3 min). Glucose stimulation experiments show that the net Michaelis-Menten constant (6.1 +/- 1.5 mM) is in between GLUT2 and GLUT9. Phloretin inhibition experiments show an EC(50) value of 28 +/- 1.6 mu M phloretin for class I GLUT proteins and a concentration of 40 +/- 0.6 mu M phloretin caused maximum inhibition with residual nonoscillating flux, suggesting that the transporters not inhibited by phloretin are likely responsible for the remaining nonoscillatory uptake, and that impaired uptake via GLUT2 may be the cause of the oscillation loss in type 2 diabetes. Transporter studies using the SR microbiosensor will contribute to diabetes research and therapy development by exploring the nature of oscillatory transport mechanisms. (C) 2010 Elsevier Inc. All rights reserved

One-Step Fabrication of Stimuli-Responsive Chitosan-Platinum Brushes for Listeria monocytogenes Detection

Bacterial contamination in food-processing facilities is a critical issue that leads to outbreaks compromising the integrity of the food supply and public health. We developed a label-free and rapid electrochemical biosensor for Listeria monocytogenes detection using a new one-step simultaneous sonoelectrodeposition of platinum and chitosan (CHI/Pt) to create a biomimetic nanostructure that actuates under pH changes. The XPS analysis shows the effective co-deposition of chitosan and platinum on the electrode surface. This deposition was optimized to enhance the electroactive surface area by 11 times compared with a bare platinum–iridium electrode (p < 0.05). Electrochemical behavior during chitosan actuation (pH-stimulated osmotic swelling) was characterized with three different redox probes (positive, neutral, and negative charge) above and below the isoelectric point of chitosan. These results showed that using a negatively charged redox probe led to the highest electroactive surface area, corroborating previous studies of stimulus–response polymers on metal electrodes. Following this material characterization, CHI/Pt brushes were functionalized with aptamers selective for L. monocytogenes capture. These aptasensors were functional at concentrations up to 106 CFU/mL with no preconcentration nor extraneous reagent addition. Selectivity was assessed in the presence of other Gram-positive bacteria (Staphylococcus aureus) and with a food product (chicken broth). Actuation led to improved L. monocytogenes detection with a low limit of detection (33 CFU/10 mL in chicken broth). The aptasensor developed herein offers a simple fabrication procedure with only one-step deposition followed by functionalization and rapid L. monocytogenes detection, with 15 min bacteria capture and 2 min sensing.This article is published as Oliveira, Daniela A., Suleiman Althawab, Eric S. McLamore, and Carmen L. Gomes. "One-step fabrication of stimuli-responsive chitosan-platinum brushes for listeria monocytogenes detection." Biosensors 11, no. 12 (2021): 511. DOI: 10.3390/bios11120511. Copyright 2021 by the authors. Attribution 4.0 International (CC BY 4.0). Posted with permission