Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in ...these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge—the efficient recombinant production of horseradish peroxidase enzymes.
MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are ...currently detected by northern blotting, real‐time PCR, and microarray techniques. These expensive, complicated, and time‐consuming techniques are not feasible for on‐site miR determination. In this study, amperometric magnetobiosensors involving RNA‐binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19‐based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR‐21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast‐tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug‐discovery programs.
A throw‐away idea: An amperometric magnetosensor based on a specific RNA probe (red strands) and protein p19 as biorecognition elements was developed as a disposable diagnostic tool for the determination of miR‐21 (green strands) in cancer cells and tissues. Labeling of the biotinylated RNA hybrid with streptavidin conjugated to horseradish peroxidase (Strep–HRP) enabled electrochemical detection on a screen‐printed carbon electrode (SPCE).
Ebola outbreak-2014 (mainly Zaire strain related Ebola virus) has been declared most widely spread deadly persistent epidemic due to unavailability of rapid diagnostic, detection, and therapeutics. ...Ebola virus disease (EVD), a severe viral hemorrhagic fever syndrome caused by Ebola virus (EBOV) is transmitted by direct contact with the body fluids of infected person and objects contaminated with virus or infected animals. World Health Organization (WHO) has declared EVD epidemic as public health emergency of international concern with severe global economic burden. At fatal EBOV infection stage, patients usually die before the antibody response. Currently, rapid blood tests to diagnose EBOV infection include the antigen or antibodies capture using ELISA and RNA detection using RT/Q-PCR within 3–10 days after the onset of symptoms. Moreover, few nanotechnology-based colorimetric and paper-based immunoassay methods have been recently reported to detect Ebola virus. Unfortunately, these methods are limited to laboratory only. As state-of-the art (SoA) diagnostics time to confirm Ebola infection, varies from 6h to about 3 days, it causes delay in therapeutic approaches. Thus developing a cost-effective, rapid, sensitive, and selective sensor to detect EVD at point-of-care (POC) is certainly worth exploring to establish rapid diagnostics to decide therapeutics. This review highlights SoA of Ebola diagnostics and also a call to develop rapid, selective and sensitive POC detection of EBOV for global health care. We propose that adopting miniaturized electrochemical EBOV immunosensing can detect virus level at pM concentration within ∼40min compared to 3 days of ELISA test at nM levels.
•EVD is deadly persistent epidemic due to unavailability of rapid diagnosis and therapeutics.•Unfortunately, available EVD detection tools are limited to laboratory only.•Thus developing efficient e sensor to detect EVD at point-of-care is needed.•For rapid detection, we propose electrochemical Ebola virus sensing as possible solution.•These sensors can detect virus level at pM concentration within ∼40min.
Cellphone-based devices for bioanalytical sciences Vashist, Sandeep Kumar; Mudanyali, Onur; Schneider, E. Marion ...
Analytical and bioanalytical chemistry,
05/2014, Letnik:
406, Številka:
14
Journal Article
Recenzirano
Odprti dostop
During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, ...cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commercially-available in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities.
Figure
The universal Rapid Diagnostic Test (RDT) reader developed at UCLA. It can read various lateral flow assays for point-of-care and telemedicine applications
Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical ...sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g.
TERT
) were, on average, more than twofold higher than those of other assays (e.g.
ERV3
). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity.
Figure
Comparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA
Although many African countries have achieved high levels of HIV diagnosis, funding constraints have necessitated greater focus on more efficient testing approaches. We compared the impact and ...cost-effectiveness of several potential new testing strategies in South Africa, and assessed the prospects of achieving the UNAIDS target of 95% of HIV-positive adults diagnosed by 2030. We developed a mathematical model to evaluate the potential impact of home-based testing, mobile testing, assisted partner notification, testing in schools and workplaces, and testing of female sex workers (FSWs), men who have sex with men (MSM), family planning clinic attenders and partners of pregnant women. In the absence of new testing strategies, the diagnosed fraction is expected to increase from 90.6% in 2020 to 93.8% by 2030. Home-based testing combined with self-testing would have the greatest impact, increasing the fraction diagnosed to 96.5% by 2030, and would be highly cost-effective compared to currently funded HIV interventions, with a cost per life year saved (LYS) of $394. Testing in FSWs and assisted partner notification would be cost-saving; the cost per LYS would also be low in the case of testing MSM ($20/LYS) and self-testing by partners of pregnant women ($130/LYS).
Detection of bacteria in bloodstream infections and their antibiotic susceptibility patterns is critical to guide therapeutic decision-making for optimal patient care. Current culture-based assays ...are too slow (>48 h), leading to excessive up-front use of broad-spectrum antibiotics and/or incorrect antibiotic choices due to resistant bacteria, each with deleterious consequences for patient care and public health. To approach this problem, we describe a method to rapidly isolate bacteria from whole blood using inertial microfluidics and directly determine pathogen identity and antibiotic susceptibility with hybridization-based RNA detection. Using the principle of Dean flow fractionation, bacteria are separated from host blood cells in a label-free separation method with efficient recovery of even low abundance bacteria. Ribosomal RNA detection can then be applied for direct identification of low abundance pathogens (~100 per mL) from blood without culturing or enzymatic amplification. Messenger RNA detection of antibiotic-responsive transcripts after brief drug exposure permits rapid susceptibility determination from bacteria with minimal culturing (~10(5) per mL). This unique coupling of microfluidic cell separation with RNA-based molecular detection techniques represents significant progress towards faster diagnostics (~8 hours) to guide antibiotic therapy.
Highlights • Engineered protein switches are increasingly used in molecular diagnostics. • Engineered protein switches can be used to sense and actuate cellular functions. • Empirical design rules to ...construct tailor-engineered protein switches are emerging. • Future synthetic signaling circuits will see networks of engineered protein switches.
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