NASBA Coupled to Paper Microfluidics for RNA Detection.

The analysis of RNA sequences is crucial to obtain invaluable insights into disease prognosis. Reliable and rapid diagnostic solutions at the site of sample collection contribute toward optimal delivery of medical treatment. For this reason, the development of more sensitive and portable RNA detection techniques are expected to advance current point-of-care (POC) diagnostic capabilities. Advancements of POC diagnostic technologies will also contribute to counter the spread of emerging viruses. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most commonly used technique to identify etiological organisms of infections. However, the need for thermocycler and fluorescent measurement renders RT-PCR less suitable for POC applications. Here, we provide a step-by-step protocol of Nucleic Acid Sequence-Based Amplification (NASBA), a robust isothermal RNA amplification technique, coupled with a portable paper microfluidics detection format.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

A novel RNA detection technique for point-of-care identification of pathogens.

Despite significant progress in recent years to improve capabilities to diagnose infections at point-of-care (POC), there are still technical hurdles that need to be overcome to ensure proper medical interventions. Current microbial POC tests involve polymerase chain reaction (PCR) or sandwich immunoassay (IA) based detection formats. PCR is highly sensitive but requires complex instrumentation, whereas lateral flow (LF) based IA tests are handheld but lack sensitivity. We present here a portable and sensitive technique by integrating an isothermal RNA amplification approach with IA detection format. The technique comprises i) Nucleic Acid Sequence Based isothermal Amplification (NASBA), ii) amplicon tagging with hapten labeled probes, iii) capturing the amplicon and iv) formation of a sandwich complex with an antibody (Ab) that selectively recognizes the DNA-RNA duplex. The results can be extended to develop an automated, portable and highly sensitive diagnostic platform suitable for POC applications.

Review of Biomarkers and Analytical Methods for Organophosphate Pesticides and Applicability to Nerve Agents.

INTRODUCTION: Recent malicious use of chemical warfare agents (CWAs) is a reminder of their severity and ongoing threat. One of the main categories of CWAs is the organophosphate (OP) nerve agents. Presently, there is an urgent need to identify and evaluate OP nerve agent biomarkers that can facilitate identification of exposed individuals post-CWA incident. While exposures to OP nerve agents may be scenario-specific, the public is commonly exposed to OP compounds through the ubiquitous use of OP pesticides, which are chemically related to nerve agents. Therefore, a systematic literature review and methodological quality assessment were conducted for OP pesticide biomarker studies to serve as a baseline to assess if these approaches may be adapted to OP nerve agent exposures. MATERIALS AND METHODS: We conducted a systematic literature review to identify biomarkers of OP pesticide exposures. English language studies of any design that reported primary data on biomarkers for exposures in nonhuman primates or adult human study participants were eligible for inclusion. Using standard criteria for assessing the completeness of reported analytical methods, the quality of study methods was critically evaluated. RESULTS: A total of 1,044 studies of biomarkers of OP pesticide exposure were identified, of which 75 articles satisfied the inclusion and exclusion criteria. These studies described 143 different analyte/sample matrix combinations: 99 host-based biomarkers, 28 metabolites, 12 pesticides, and 4 adducts. The most commonly reported biomarkers were dialkyl phosphate urinary metabolites (22 studies), blood acetylcholinesterase, and plasma butyrylcholinesterase (26 studies each). None of the assessed quality review criteria were fully addressed by all identified studies, with almost all criteria scoring less than 50%. CONCLUSION: Cholinesterase activity may have utility for identifying individuals with exposures surpassing a given threshold of OP nerve agent, but further investigation of how acetylcholinesterase and butyrylcholinesterase levels correlate with observed patient symptoms may be required to ensure accuracy of results. As CWAs and nerve agents are more readily used, more standardized reporting of biomarker measurements are needed to develop new approaches for OP nerve agent biomarkers.