Integrating scFv into xMAP Assays for the Detection of Marine Toxins

Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication;environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv). Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to "preserve" monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated

A Fully-Flexible Solution-Processed Autonomous Glucose Indicator

We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM-45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries

Multilayer Epitaxial Graphene on Silicon Carbide: A Stable Working Electrode for Seawater Samples Spiked with Environmental Contaminants

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis

Integrating Single Domain Antibodies into Field-Deployable Rapid Assays

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19

Array Biosensor for Toxin Detection: Continued Advances

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system

Effect of Linker Length on Cell Capture by Poly(ethylene glycol)-Immobilized Antimicrobial Peptides

Development of antimicrobial peptide (AMP)-functionalized materials has renewed interest in using poly­(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategiessuch as changing tether lengthmay result in large differences in the surface properties of the immobilized AMPs that are not easily predictable

Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

Abstract Background A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity. Results An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15–17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies. Conclusions SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola