Bioelectrochemical platforms to study and detect emerging pathogens.

The ongoing SARS-CoV-2 pandemic has emphasized the importance of technologies to rapidly detect emerging pathogens and understand their interactions with hosts. Platforms based on the combination of biological recognition and electrochemical signal transduction, generally termed bioelectrochemical platforms, offer unique opportunities to both sense and study pathogens. Improved bio-based materials have enabled enhanced control over the biotic-abiotic interface in these systems. These improvements have generated platforms with the capability to elucidate biological function rather than simply detect targets. This advantage is a key feature of recent bioelectrochemical platforms applied to infectious disease. Here, we describe developments in materials for bioelectrochemical platforms to study and detect emerging pathogens. The incorporation of host membrane material into electrochemical devices has provided unparalleled insights into the interaction between viruses and host cells, and new capture methods have enabled the specific detection of bacterial pathogens, such as those that cause secondary infections with SARS-CoV-2. As these devices continue to improve through the merging of hi-tech materials and biomaterials, the scalability and commercial viability of these devices will similarly improve.

Creation of a low cost, low light bioluminescence sensor for real time biological nitrate sensing in marine environments.

The concentration of nitrate (NO3-) in Narragansett Bay has been shown to undergo considerable temporal and spatial variation. However, the dynamics of this flux has never been monitored on a fine-scale (<100 m, < 1 d) or in real-time. Whole-cell bio-reporters are promising candidates for low cost environmental sensing of bioavailable nutrients. Yet difficulties remain in creating sensors for long term deployment in the marine environment. This paper describes the creation and validation of a low-cost sensor using a self-bioluminescent strain of the cyanobacteria Synechococcus elongatus pcc 7942 for the direct measurement of bioavailable nitrate. Nitrate bioavailability was measured by monitoring light emission from a luxAB based promotor fusion to glnA using a light to frequency sensor and single board microcontroller. Sensor designs are presented in this manuscript with specific focus on storage, cell viability, and compatibility with the marine environment. Sensors were able to consistently assess nitrate standards as low as 1 ppm (16.3 µM). Using a wavelet denoising approach to reduce white noise and hardware noise, nitrate detection of standards as low as 0.037 ppm (0.65 µM) was achieved. Good sensitivity and low cost make these sensors ideal candidates for continuous monitoring of biological nitrates in estuarine systems.

The response of Synechococcus sp. PCC 7002 to micro-/nano polyethylene particles - Investigation of a key anthropogenic stressor.

Microplastics or plastic particles less than 5 mm in size are a ubiquitous and damaging pollutant in the marine environment. However, the interactions between these plastic particles and marine microorganisms are just starting to be understood. The objective of this study was to measure the responses of a characteristic marine organism (Synechococcus sp. PCC 7002) to an anthropogenic stressor (polyethelene nanoparticles and microparticles) using molecular techniques. This investigation showed that polyethylene microparticles and nanoparticles have genetic, enzymatic and morphological effects on Synechococcus sp. PCC 7002. An RT-PCR analysis showed increases in the expression of esterase and hydrolase genes at 5 days of exposure to polyethylene nanoparticles and at 10 days of exposure to polyethylene microparticles. A qualitative enzymatic assay also showed esterase activity in nanoparticle exposed samples. Cryo-scanning electron microscopy was used to assess morphological changes in exopolymer formation resulting from exposure to polyethylene microparticles and nanoparticles. The data from this paper suggests that microplastic and nanoplastics could be key microbial stressors and should be investigated in further detail.

Interaction of Cyanobacteria with Nanometer and Micron Sized Polystyrene Particles in Marine and Fresh Water.

Microplastics and nanoplastics are emerging pollutants, widespread both in marine and in freshwater environments. Cyanobacteria are also ubiquitous in water and play a vital role in natural ecosystems, using photosynthesis to produce oxygen. Using photography, fluorescence microscopy and cryogenic and scanning electron microscopy (cryo-SEM, SEM) we investigated the physicochemical response of one of the most predominant seawater cyanobacteria (Synechococcus elongatus, PCC 7002) and freshwater cyanobacteria (S. elongatus Nageli PCC 7942) when exposed to 10 µm diameter polystyrene (microPS) and 100 nm diameter polystyrene (nanoPS) particles. Marine and freshwater cyanobacteria formed aggregates with the nanoPS, bound together by extracellular polymeric substances (EPS), and these aggregates sedimented. The aggregates were larger, and the sedimentation was more rapid for the marine system. Aggregate morphologies were qualitatively different for the microPS samples, with the bacteria linking up a small number of particles, all held together by EPS. There was no sedimentation in these samples. The cyanobacteria remained alive after exposure to the particles. The particle size- and salt concentration-dependent response of cyanobacteria to these anthropogenic stressors is an important factor to consider for a proper understanding of the fate of the particles as well as the bacteria.

Rapid electrophoretic recovery of DNA from dried blood spots.

Large-scale genetic screening of neonatal dried blood spots for episomal DNA has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of dried blood spots remains one of the most time consuming, costly, and labor-intensive parts of DNA analysis. In the present study, we developed and optimized a rapid methodology using only 50 V and heat to extract episomal DNA from dried blood spots prepared from diagnostic cord blood samples. This electric field DNA extraction is the first methodology to use an electric field to extract episomal DNA from a dried blood spot. This 25-minute procedure has one of the lowest times for the extraction of episomal DNA found within the literature and this novel procedure not only negates the need for costly treatment and wash steps, but reduces the time of manual procedures by more than 30 min while retaining the 75-80% of the yield. Combined with real-time PCR, this novel method of electric field extraction not only provides an effective tool for the large scale genetic analysis of neonates, but a key step forward in the simplification and standardization of diagnostic testing.

Lipase degradation of plasticized polyvinyl chloride endotracheal tube surfaces to create nanoscale features.

Polyvinyl chloride (PVC) endotracheal tubes (ETTs) nanoetched with a fungal lipase have been shown to reduce bacterial growth and biofilm formation and could be an inexpensive solution to the complex problem of ventilator-associated pneumonia (VAP). Although bacterial growth and colonization on these nanoetched materials have been well characterized, little is known about the mechanism by which the fungal lipase degrades the PVC and, thus, alters its properties to minimize bacteria functions. This study used X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to better describe the surface chemistry of both unetched and lipase nanoetched PVC ETT. ATR-FTIR analysis of the unetched and treated surfaces showed a similar presence of a plasticizer. This was confirmed by XPS analysis, which showed an increase of carbon and the presence of oxygen on both unetched and nanoetched surfaces. A quantitative comparison of the FTIR spectra revealed significant correlations (Pearson's correlation, R=0.997 [R2=0.994, P<0.001]) between the unetched and nanomodified PVC ETT spectra, demonstrating similar surface chemistry. This analysis showed no shifting or widening of the bands in the spectra and no significant changes in the intensity of the infrared peaks due to the degradation of the plasticizer by the fungal lipase. In contrast, results from this study did demonstrate significantly increased nanoscale surface features on the lipase etched compared to non-etched PVC ETTs. This led to a change in surface energetics, which altered ion adsorption to the ETTs. Thus, these results showed that PVC surfaces nanoetched with a 0.1% lipase solution for 48 hours have no significant change on surface chemistry but do significantly increase nanoscale surface roughness and alters ion adsorption, which suggests that the unique properties of these materials, including their previously reported ability to decrease bacterial adhesion and growth, are due to the changes in the degree of the nanoscale roughness, not changes in their surface chemistry.

Decreased Pseudomonas aeruginosa biofilm formation on nanomodified endotracheal tubes: a dynamic lung model.

Ventilator-associated pneumonia (VAP) is a serious complication of mechanical ventilation that has been shown to be associated with increased mortality rates and medical costs in the pediatric intensive care unit. Currently, there is no cost-effective solution to the problems posed by VAP. Endotracheal tubes (ETTs) that are resistant to bacterial colonization and that inhibit biofilm formation could provide a novel solution to the problems posed by VAP. The objective of this in vitro study was to evaluate differences in the growth of Pseudomonas aeruginosa on unmodified polyvinyl chloride (PVC) ETTs and on ETTs etched with a fungal lipase, Rhizopus arrhizus, to create nanoscale surface features. These differences were evaluated using an in vitro model of the pediatric airway to simulate a ventilated patient in the pediatric intensive care unit. Each experiment was run for 24 hours and was supported by computational models of the ETT. Dynamic conditions within the ETT had an impact on the location of bacterial growth within the tube. These conditions also quantitatively affected bacterial growth especially within the areas of tube curvature. Most importantly, experiments in the in vitro model revealed a 2.7 log reduction in the number (colony forming units/mL) of P. aeruginosa on the nanoroughened ETTs compared to the untreated PVC ETTs after 24 hours. This reduction in total colony forming units/mL along the x-axis of the tube was similar to previous studies completed for Staphylococcus aureus. Thus, this dynamic study showed that lipase etching can create surface features of nanoscale roughness on PVC ETTs that decrease bacterial attachment of P. aeruginosa without the use of antibiotics and may provide clinicians with an effective and inexpensive tool to combat VAP.

Decreased Staphylococcus aureus biofilm formation on nanomodified endotracheal tubes: a dynamic airway model.

Ventilator-associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation for over 24 hours increases the risk of VAP and is associated with high morbidity, mortality, and medical costs. Cost-effective endotracheal tubes (ETTs) that are resistant to bacterial infections could help prevent this problem. The objective of this study was to determine differences in the growth of Staphylococcus aureus on nanomodified and unmodified polyvinyl chloride (PVC) ETTs under dynamic airway conditions simulating a ventilated patient. PVC ETTs were modified to have nanometer surface features by soaking them in Rhizopus arrhisus, a fungal lipase. Twenty-four-hour experiments (supported by computational models) showed that airflow conditions within the ETT influenced both the location and the concentration of bacterial growth on the ETTs, especially within areas of tube curvature. More importantly, experiments revealed a 1.5 log reduction in the total number of S. aureus on the novel nanomodified ETTs compared with the conventional ETTs after 24 hours of airflow. This dynamic study showed that lipase etching can create nanorough surface features on PVC ETTs that suppress S. aureus growth, and thus may provide clinicians with an effective and inexpensive tool to combat VAP.

Nanotechnology: pediatric applications.

Ventilator-associated pneumonia (VAP) is a serious and costly clinical problem affecting pediatrics today. This device-related infection is thought to be directly linked to the colonization of the endotracheal tube (ETT) during long-term mechanical ventilation. Because of unspecific radiographic and clinical signs, VAP is especially difficult to diagnose in the pediatric population. Treatment with antibiotics is often ineffective, and VAP is associated with high morbidity, mortality, and medical costs. The use of nanomodified coatings on ETT may provide an effective strategy to prevent biofilm formation and ETT colonization. Nanoparticles such as selenium and iron oxide have been shown to penetrate into the biofilm reaching the protected cells antibiotics often miss. Moreover, nanoetching techniques can modify the topography of the ETT surface interfering with bacterial adhesion. This review seeks to examine the antimicrobial properties of both nanoparticles and nanomodified surfaces and to characterize their effectiveness at reducing bacterial colonization on ETT.