Direct detection and quantification of microRNAs

The recent discovery of the potent regulatory nature of microRNAs (miRNAs), a relatively new class of approximately 22 nucleotide RNAs, has made them a primary focus in today’s biochemical and medical research. The relationship between miRNA expression patterns and the onset of cancer, as well as other diseases, has glimpsed the potential of miRNAs as disease biomarkers or drug targets, making them a primary research focus. Their promising future in medicine is hinged upon improving our scientific understanding of their intricate regulatory mechanisms. In the realm of analytical chemistry, the main challenge associated with miRNA is its detection. Their extremely small size and low cellular concentration poses many challenges for achieving reliable results. Current reviews in this area have focused on adaptations to microarray, PCR, and Northern blotting procedures to make them suitable for miRNA detection. While these are extremely powerful methods and accepted as the current standards, they are typically very laborious, semi-quantitative, and often require expensive imaging equipment and/or radioactive/toxic labels. This review aims to highlight emerging techniques in miRNA detection and quantification that exhibit superior flexibility and adaptability as well as matched or increased sensitivity in comparison to the current standards. Specifically, this review will cover colorimetric, fluorescence, bioluminescence, enzyme, and electrochemical based methods, which drastically reduce procedural complexity and overall expense of operation thereby increasing the accessibility of this field of research. The methods are presented and discussed as to their improvements over current standard methods as well as their potential complications preventing acceptance as standard procedures. These new methods have addressed the many of the problems associated with miRNA detection through the employment of enzyme-based signal amplification, enhanced hybridization conditions using PNA capture probes, highly sensitive and flexible forms of spectroscopy, and extremely responsive electrocatalytic nanosystems, among other approaches

Aequorin and Obelin Mutants with Differing Wavelengths and Bioluminescence

The invention relates to aequorin and obelin mutants whose emission is shifted with respect to wild type. The shift in emission is accomplished using a combination of mutations of amino acids within aequorin or obelin that affect bioluminescence; use of different types of chromophores, i.e., coelenterazines with variable emission characteristics; and modifications of the photoprotein with fluorophores that will allow for emission of light at longer wavelengths as a result of energy transfer. Additionally, an assay employing aequorin mutants to test for HIV-1 protease inhibitors is disclosed

Methods and Kit for Determination of Prostacyclin in Plasma

A solid-phase immunoassay for 6-keto-Prostaglandin F1α, the stable hydrolysis product of prostacyclin (Prostaglandin I2) is disclosed. Prostacyclin, a potent vasodilator with anti-platelet and anti-proliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-Prostaglandin F1α can be directly correlated with levels of prostacyclin. Therefore, 6-keto-Prostaglandin F1α has become the indicator of choice to measure prostacyclin levels. The single step immunoassay for 6-keto-Prostaglandin F1α uses the bioluminescent protein, aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-Prostaglandin F1α and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-Prostaglandin F1α towards its antibody and the bioluminescent properties of aequorin are retained in the conjugate. The concentration of 6-keto-Prostaglandin F1α after extraction from plasma shows good correlation with the concentration of 6-keto-Prostaglandin F1α obtained without prior extraction of the same plasma sample. The assay allows the measurement of 6-keto-Prostaglandin F1α directly in plasma without any pre-treatment of the samples, which results in a much simpler method with a faster assay time

Stimuli-Responsive Hydrogel Microdomes Integrated with Genetically Engineered Proteins for High-Throughput Screening of Pharmaceuticals

A hydrogel microdome that can swell in response to a stimuli or target molecule is formed by polymerizing a mixture comprising a monomer capable of forming a hydrogel with a biopolymer. An array of hydrogel microdomes can be formed on a substrate by microspotting the mixture and polymerizing. The array can be used for high-throughput screening of analytes as well as for use as an actuator and biosensor using the swelling property of the hydrogel

Systems and Methods for Diagnosis and Monitoring of Bacteria-Related Conditions

The presently-disclosed subject matter provides systems, methods, and kits for diagnosing and/or monitoring a bacteria-related condition of interest in a subject by providing a cell sensing system, each system containing a reporter molecule capable of detecting binding of a quorum sensing molecule and capable of generating a detectable signal

Comparative Study of the Performance of Two Different Luciferases for the Analysis of Fumonisin B1 in Wheat Samples

The development of two different immunoassays for the determination of fumonisin B1 in wheat samples is reported. A previously described mimopeptide for fumonisin B1 (FB1) was used to produce fusion proteins in combination with two different luciferases: Gaussia luciferase (GLuc) and NanoLuc luciferase (NLuc). The production, expression and the development of two immunoassays based on these fusion proteins (A2- GLuc and A2-NLuc) is detailed. The assay showing the best performance, A2-NLuc, with a limit of detection of 0.61 ngmL 1 and a dynamic range from 1.9 to 95 ngmL 1 , was employed for the analysis of spiked wheat samples, a reference matrix material, as well as naturally contaminated wheat samples. The recoveries obtained in the spiked samples were acceptable, between 81.5 and 109%, with relative standard deviations lower than 14%. The analysis of naturally contaminated wheat was validated by a liquid chromatography coupled to tandem mass detection method

Exploring bioanalytical applications of assisted protein reassembly

Reassembly of protein from its peptide fragments is a technique that can have many applications in the bioanalytical field. Typically, a reporter protein fragmented into its two peptides is employed as a label in this study. This fragments of peptide can reassemble yielding an active functional reporter. This reassembly of the protein can be assisted by non-covalently interacting peptides or proteins, which are attached to the fragmented reporter. This technique has been employed in several applications including study of protein-protein interactions, antibody screening, immunoassays, and high-throughput screening. This review focuses on different reporters employed in the study of reassembly of proteins and applications of this strategy in bioanalysis

Affinity Chromatography

Using exposition, graphics, and commercial videos, this module teaches the history, theory, and application of affinity chromatography in the characterization of proteins, nucleic acids, and other biochemical/biomedical systems. Problems and application examples support the tutorial material. The Theory sections also presents a helpful bibliography of further reading material

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis