-
Protein Science : a Publication of the... Aug 2021Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay...
Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k , and the rate constant of productive binding, k , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.
Topics: Alkaline Phosphatase; Biological Assay; Cell-Free System; Escherichia coli Proteins; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 33955095
DOI: 10.1002/pro.4102 -
Scientific Reports Nov 2022This study aims to investigate bacteria for biodegradation of oil pollutants from oily industrial wastewater to be used as bioremediation tools and to determine the...
This study aims to investigate bacteria for biodegradation of oil pollutants from oily industrial wastewater to be used as bioremediation tools and to determine the characterization of bioremediation bioassays. A screening bioassay was carried out using six exogenous environmental bacterial strains to degrade oily pollution, which indicated promising clearance of the oily wastewater. Two strains, namely Enterobacter cloacae 279-56 (R4) and Pseudomonas otitis MCC10330 (R19), could successfully eliminate oil content and reasonable removal of the organic load. Results showed that the two promising bacterial candidates (R4 and R19) were selected according to the preliminary screening of the six tested bacteria considered the most efficient for all the tested parameters. The highest Removal Efficiency (Removal Efficiency resulted in Residual levels of total dissolved solids (TDS), biochemical oxygen demand, chemical oxygen demand, and Oil content in the treated oily wastewater effluents are 1940, 171, 131, and 84 mg/l respectively where these results are not within safe discharge limits, except for TDS. Hence, the bioremediation assays were carried out using the mixed culture since it was the most efficient strain for degrading all tested parameters.
Topics: Bacteria; Biodegradation, Environmental; Biological Assay; Wastewater; Water Pollutants, Chemical
PubMed: 36344545
DOI: 10.1038/s41598-022-23480-7 -
Molecules (Basel, Switzerland) Feb 2019Lipases are enzymes responsible for the conversion of triglycerides and other esterified substrates, they are involved in the basic metabolism of a wide number of... (Review)
Review
Lipases are enzymes responsible for the conversion of triglycerides and other esterified substrates, they are involved in the basic metabolism of a wide number of organisms, from a simple microorganism and to mammals. They also have broad applicability in many fields from which industrial biotechnology, the production of cleaning agents, and pharmacy are the most important. The use of lipases in analytical chemistry where it can serve as a part of biosensors or bioassays is an application of growing interest and has become another important use. This review is focused on the description of lipases chemistry, their current applications and the methods for their assay measurement. Examples of bioassays and biosensors, including their physical and chemical principles, performance for specific substrates, and discussion of their relevance, are given in this work.
Topics: Biological Assay; Biosensing Techniques; Catalysis; Electrochemical Techniques; Enzyme Activation; Hydrolysis; Lipase
PubMed: 30744203
DOI: 10.3390/molecules24030616 -
Analytical and Bioanalytical Chemistry Aug 2021Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases....
Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme) by replacing the target recognition and substrate sequences within Aptazyme. This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue.
Topics: Biological Assay; Biosensing Techniques; DNA; DNA, Catalytic; Humans; Nanotechnology; Nucleic Acids; Reproducibility of Results; Thrombin
PubMed: 34184101
DOI: 10.1007/s00216-021-03458-6 -
Analytical and Bioanalytical Chemistry Mar 2011In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated... (Review)
Review
In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets-pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.
Topics: Animals; Biological Assay; Complex Mixtures; Humans; Mass Spectrometry; Proteins
PubMed: 21107824
DOI: 10.1007/s00216-010-4406-0 -
Biotechnology Advances 2024Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both... (Review)
Review
Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.
Topics: Anti-Infective Agents; Biological Products; Biological Assay
PubMed: 38185432
DOI: 10.1016/j.biotechadv.2024.108307 -
Biomedicine & Pharmacotherapy =... Sep 2021DNA methylation is an important epigenetic alteration that results from the covalent transfer of a methyl group to the fifth carbon of a cytosine residue in CpG... (Review)
Review
Application of lateral flow and microfluidic bio-assay and biosensing towards identification of DNA-methylation and cancer detection: Recent progress and challenges in biomedicine.
DNA methylation is an important epigenetic alteration that results from the covalent transfer of a methyl group to the fifth carbon of a cytosine residue in CpG dinucleotides by DNA methyltransferase. This modification mostly happens in the promoter region and the first exon of most genes and suppresses gene expression. Therefore, aberrant DNA methylation cause tumor progression, metastasis, and resistance to current anti-cancer therapies. So, the detection of DNA methylation is an important issue in diagnosis and therapy of most diseases. Conventional methods for the assay of DNA methylation and activity of DNA methyltransferases are time consuming. So, we need to multiplex operations and expensive instrumentation. To overcome the limitations of conventional methods, new methods such as microfluidic platforms and lateral flow tests have been developed to evaluate DNA methylation. The microfluidic tests are based on optical and electrical biosensing. These tests able us to can analyze DNA methylation with high efficiency and sensitivity without the need for expensive equipment and skilled people. Lateral flow strip tests are another type of rapid, simple, and sensitive test with advanced technology used to assess DNA methylation. Lateral flow strip tests are based on optical biosensors. This review attempts to evaluate new methods for assessing DNA extraction, DNA methylation and DNA methyltransferase activity as well as recent developments in microfluidic technology application for bisulfite treatment and restriction enzyme (bisulfite free), and lateral flow relying on their application in the field of recognition of DNA methylation in blood and body fluids. Also, the advantages and disadvantages of each test are reviewed. Finally, future prospects for the development of the microfluidics biodevices for the detection of DNA methylation is briefly discussed.
Topics: Animals; Biological Assay; Biomedical Research; Biosensing Techniques; DNA Methylation; Epigenesis, Genetic; Humans; Microfluidics; Neoplasms
PubMed: 34175816
DOI: 10.1016/j.biopha.2021.111845 -
Environment International Mar 2023Microalgal bioassays are widely applied to evaluate the potential toxicity of various persistent toxic substances in environmental samples due to multiple advantages,... (Review)
Review
Microalgal bioassays are widely applied to evaluate the potential toxicity of various persistent toxic substances in environmental samples due to multiple advantages, including high sensitivity, short test duration, and cost-effectiveness. Microalgal bioassay is gradually developing in method, and the scope of application to environmental samples is also expanding. Here, we reviewed the published literature on microalgal bioassays for environmental assessments, focusing on types of samples, sample preparation methods, and endpoints, and highlighted key scientific advancements. Bibliographic analysis was performed with the keywords 'microalgae' and 'toxicity' or 'bioassay', and 'microalgal toxicity'; 89 research articles were selected and reviewed. Traditionally, most studies implementing microalgal bioassays focused on water samples (44%) with passive samplers (38%). Studies using the direct exposure method (41%) of injecting microalgae into sampled water mainly evaluated toxic effects by growth inhibition (63%). Recently, various automated sampling techniques, in situ bioanalytical methods with multiple endpoints, and targeted and non-targeted chemical analyses have been applied. More research is needed to identify causative toxicants affecting microalgae and to quantify the cause-effect relationships. This study provides the first comprehensive overview of recent advances in microalgal bioassays performed with environmental samples, suggesting future research directions based on current understanding and limitations.
Topics: Microalgae; Biological Assay; Water
PubMed: 36905773
DOI: 10.1016/j.envint.2023.107869 -
Advances in Biochemical... 2014Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment,... (Review)
Review
Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.
Topics: Animals; Biological Assay; Eukaryotic Cells; Humans; Luminescent Measurements; Organic Chemicals; Risk; Toxicity Tests
PubMed: 25084996
DOI: 10.1007/978-3-662-43385-0_4 -
Frontiers in Bioscience (Landmark... Jun 2018Autoantibodies to the thyrotropin hormone receptor (TSH-R) are directly responsible for the hyperthyroidism in Graves' disease and mediate orbital manifestations in... (Meta-Analysis)
Meta-Analysis Review
Autoantibodies to the thyrotropin hormone receptor (TSH-R) are directly responsible for the hyperthyroidism in Graves' disease and mediate orbital manifestations in Graves' orbitopathy (otherwise known as thyroid eye disease). These autoantibodies are heterogeneous in their function and collectively referred to as TRAbs. Measurement of TRAbs is clinically important for diagnosis of a variety of conditions and different commercial assays with high sensitivity and specificity are available for diagnostic purposes. This review provides overwhelming evidence that the TRAbs detected in binding assays by mainly the automated electrochemical luminescence immunoassays (ECLIA) do not distinguish TRAbs that stimulate the TSH-R (called TSIs or TSAbs) and TRAbs that just inhibit the binding of TSH without stimulating the TSH-R (called TBAbs). However, TSAbs and TBAbs have divergent pathogenic roles, and depending which fraction predominates cause different clinical symptoms and engender different therapeutic regimen. Therefore, diagnostic distinction of TSAbs and TBAbs is of paramount clinical importance. To date, only bioassays such as the Mc4 TSH-R bioassay (Thyretain, Quidel) and the Bridge assay (Immulite 2000, Siemens) can measure TSAbs, with only the former being able to distinguish between TSAbs and TBAbs. On this note, it is strongly recommended to only use the term TSI or TSAb when reporting the results of bioassays, whereas the results of automated TRAb binding assays should be reported as TRAbs (of undetermined functional significance). This review aims to present a technical and analytical account of leading commercial diagnostic methods of anti-TSH-R antibodies, a metaanalysis of their clinical performance and a perspective for the use of cell based TSH-R bioassays in the clinical diagnostics of Graves' disease.
Topics: Autoantibodies; Biological Assay; Biomarkers; Graves Disease; Graves Ophthalmopathy; Humans; Receptors, Thyrotropin; Sensitivity and Specificity
PubMed: 29772543
DOI: 10.2741/4687