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Intracellular spaces are partitioned into separate compartments to ensure that numerous biochemical reactions and cellular functions take place in a spatiotemporally controlled manner. Biomacromolecules including proteins and RNAs undergo liquid-liquid phase separation and subsequent phase transition to form biological condensates with diverse material states. The material/physical properties of biological condensates are crucial for fulfilling their distinct physiological functions, and abnormal material properties can cause deleterious effects under pathological conditions. Here, we review recent studies showing the role of the material properties of biological condensates in their physiological functions. We also summarize several classic methods as well as newly emerging techniques for characterization and/or measurement of the material properties of biological condensates., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Since the specific proteins (carbohydrate antigens, ligands and interleukins) get raised up in body tissue or fluids in cancer cases, early detection of them will provide an effective treatment and survival rate. Sensitive and accurate determination of multiple cancer proteins can be engaged in chorus by simultaneous/multiplex detection in the biomedical fields. Bioassaying technology is one of the non-invasive, high-sensitive, and economical methods. Currently, extensive application of nanomaterial (biocompatible polymers, metallic and metal oxide) in bioassays resulted in ultra-high sensitive and selective diagnosis. This review article focuses on types of multiplex bioassays for delicate and specific determination of cancer proteins for diagnostic aims. It also covers two modes of multiplex bioassays as multi labeled bioassays and spatially-separated test zones (multi-electrode mode). In this review, the nanotechnological, structural, and technical perspectives in the multiplex analysis of cancer proteins were discussed. Finally, the use of different types of nanomaterials, polysaccharides, biopolymers and their advantages in signal amplification are discussed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Many agrochemicals have endocrine disrupting properties. A subset of these chemicals is characterized as "estrogenic". In this review, we describe several distinct ways that chemicals used in crop production can affect estrogen signaling. Using three agrochemicals as examples (DDT, endosulfan, and atrazine), we illustrate how screening tests such as the US EPA's EDSP Tier 1 assays can be used as a first-pass approach to evaluate agrochemicals for endocrine activity. We then apply the "Key Characteristics" approach to illustrate how chemicals like DDT can be evaluated, together with the World Health Organization's definition of an endocrine disruptor, to identify data gaps. We conclude by describing important issues that must be addressed in the evaluation and regulation of hormonally active agrochemicals including mixture effects, efforts to reduce vertebrate animal use, chemical prioritization, and improvements in hazard, exposure, and risk assessments., (Copyright © 2020 Elsevier B.V. All rights reserved.)

It is well documented that thyroid hormone (TH) action is critical for normal brain development and is mediated by both nuclear and extranuclear pathways. Given this dependence, the impact of environmental endocrine disrupting chemicals that interfere with thyroid signaling is a major concern with direct implications for children's health. However, identifying thyroid disrupting chemicals in vivo is primarily reliant on serum thyroxine (T4) measurements within greater developmental and reproductive toxicity assessments. These studies do not examine known TH-dependent phenotypes in parallel, which complicates chemical evaluation. Additionally, there exist no recommendations regarding what degree of serum T4 dysfunction is adverse, and little consideration is given to quantifying TH action within the developing brain. This review summarizes current testing strategies in rodent models and discusses new approaches for evaluating the developmental neurotoxicity of thyroid disrupting chemicals. This includes assays to identify adverse cellular effects of the brain by both immunohistochemistry and gene expression, which would compliment serum T4 measures. While additional experiments are needed to test the full utility of these approaches, incorporation of these cellular and molecular assays could enhance chemical evaluation in the regulatory arena., (Published by Elsevier B.V.)

Antibody-based therapeutic classes are evolving from monoclonal antibodies to antibody derivatives with complex structures to achieve advanced therapeutic effect. These antibody derivatives may contain multiple functional domains and are often vulnerable to in vivo biotransformation. Understanding the pharmacokinetics of these antibody derivatives requires a sophisticated bioanalytical approach to carefully characterize the whole drug and each functional domain with respect to quantity, functionality enabled by biotransformation, and corresponding immune responses. Ligand binding assays and liquid chromatography-mass spectrometry assays are predominantly used in bioanalytical support of monoclonal antibodies and are continuously used for antibody derivatives such as antibody drug conjugate and bispecific antibodies. However, they become increasingly cumbersome in coping with increased complexity of drug modality and associated biotransformation. In this mini-review, we examined the current pharmacokinetic assays in the literature for antibody drug conjugate and bispecific antibodies, and presented our view of promising bioanalytical technologies to address the distinct bioanalytical needs of complex modalities.

The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the "binding and cleavage" (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market. Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products., (Copyright © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

PEGylated biotherapeutics can elicit anti-PEG (polyethylene glycol) immune responses in patients treated with this category of drugs. While anti-PEG antibody assays for this class of biotherapeutics have become a common element of the clinical immunogenicity testing strategy, the overall antibody incidence induced by the nanoparticle (NP) delivery system (such as ACCURINS®) has not been fully studied to date. To support the immunogenicity assessment of one of Pfizer's NP-based therapeutics, consisting of gedatolisib (GEDA) encapsulated in ACCURINS® (GEDA-NP), we developed an anti-GEDA-NP antibody (ADA) assay on the MSD platform for the detection of GEDA-NP induced ADA in human serum. The focus of our strategy was on developing a clinically relevant ADA assay and systematically addressing assay interference through rigorous assay optimization. Our efforts led to a fit-for-purpose assay for the detection of anti-GEDA-NP ADA in serum samples obtained from breast cancer patients. Results from method qualification indicated robust assay performance, as highlighted by inter and intra-assay precision within 25% CV for all controls, and reproducible response profiles across multiple runs during the assessment of assay cut points with breast cancer samples. The assay sensitivity was between 4.3 ng/mL and 123 ng/mL for surrogate positive controls of IgG and IgM isotypes, respectively. Additionally, assay interference from nonspecific matrix proteins and circulating drug was addressed, which ensured accurate assessment of ADA incidence that can be attributed to GEDA-NP.

Over the past decades, microfluidics has emerged as an increasingly important tool to perform biochemical assays for diagnosis and healthcare. The precise fluid control and molecule manipulation within microfluidics greatly contribute to developing assays with simplicity and convenience. The advantages of microfluidics, including decreased consumption of reagents and samples, lower operating and analysis time, much lower cost, and higher integration and automation over traditional systems, offer a great platform to meet the needs of point-of-care applications. In this Review, versatile strategies are outlined and recent advances in microfluidics-implemented assays are discussed from the perspective of readout, because a convenient and straightforward readout is what a biochemical assay requires and the end user desires. Functions and properties arising from each readout are reviewed and the advantages and limitations of each readout are discussed together with current challenges and future perspectives., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cellular thermal shift assay (CETSA) was introduced in 2013 to investigate drug-target engagement inside live cells and tissues. As with all thermal shift assays, the response measured by CETSA is not simply governed by ligand affinity to the investigated target protein, but the thermodynamics and kinetics of ligand binding and protein unfolding also contribute to the observed protein stabilization. This limitation is commonly neglected in current applications of the method to validate the target of small-molecule probes. Instead, there is an eagerness to make direct comparisons of CETSA measurements with functional and phenotypic readouts from cells at 37 °C. Here, we present a perspective of the early CETSA literature and put the accumulated data into a quantitative context. The analysis includes annotation of ~270 peer-reviewed papers, the majority of which do not consider the underlying biophysical basis of CETSA. We also detail what future technology developments are needed to enable CETSA-based optimization of structure-activity relationships and more appropriate comparisons of these data with functional or phenotypic responses. Finally, we describe ongoing developments in assay formats that allow for CETSA measurements at single-cell resolution, with the aspiration to allow differentiation in cellular target engagement between cells in co-cultures and more complex models, such as organoids and potentially even tissue.

Ca 2+ , Na + and K + - permeable ion channels as well as GPCRs linked to Ca 2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and G q -coupled GPCRs.

Cell-based assays including both cell identification assays and cell functional assays are broadly used in life science research and for medical applications. Traditionally, cell-based assays are performed in professional laboratories. Although such an approach is reliable, it significantly limits the accessibility of cell-based assays by many researchers without specialized cell imaging facilities and does not permit on-site use at most clinics. Over recent years, growing efforts have been made to overcome this limitation by developing various portable cell imaging platforms. Herein, we provide a timely review on the recent development of portable imaging systems and their biomedical applications. We describe the respective applications of three main classes of portable imaging technologies including miniaturized microscope, lens-free imaging device and smartphone-based microscope for cell-based assays. We conclude by discussing our views on the opportunities, problems and future directions of this emerging field., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment., (Copyright © 2017. Published by Elsevier Inc.)

Planning and transfer of a new technology platform developed in an academic setting to a start-up company for medical diagnostic product development may appear daunting and costly in terms of complexity, time, and resources. In this review we outline the key steps taken and lessons learned when a technology platform developed in an academic setting was transferred to a start-up company for medical diagnostic product development in the interest of elucidating development toolkits for academic groups and small start-up companies starting on the path to commercialization and regulatory approval., (© 2017, The American Society for Clinical Pharmacology and Therapeutics.)

This chapter reviews the current knowledge and recent progress in the field of environmental, aquatic ecotoxicogenomics with a focus on transcriptomic methods. In ecotoxicogenomics the omics technologies are applied for the detection and assessment of adverse effects in the environment, and thus are to be distinguished from omics used in human toxicology [Snape et al., Aquat Toxicol 67:143-154, 2004]. Transcriptomic methods in ecotoxicology are applied to gain a mechanistic understanding of toxic effects on organisms or populations, and thus aim to bridge the gap between cause and effect. A worthwhile effect-based interpretation of stressor induced changes on the transcriptome is based on the principle of phenotypic-anchoring [Paules, Environ Health Perspect 111:A338-A339, 2003]. Thereby, changes on the transcriptomic level can only be identified as effects if they are clearly linked to a specific stressor-induced effect on the macroscopic level. By integrating those macroscopic and transcriptomic effects, conclusions on the effect-inducing type of the stressor can be drawn. Stressor-specific effects on the transcriptomic level can be identified as stressor-specific induced pathways, transcriptomic patterns, or stressors-specific genetic biomarkers. In this chapter, examples of the combined application of macroscopic and transcriptional effects for the identification of environmental stressors, such as aquatic pollutants, are given and discussed. By means of these examples, challenges on the way to a standardized application of transcriptomics in ecotoxicology are discussed. This is also done against the background of the application of transcriptomic methods in environmental regulation such as the EU regulation Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

High-quality preclinical bioassay models are essential for drug research and development. We reviewed the emerging body-on-a-chip technology, which serves as a promising model to overcome the limitations of traditional bioassay models, and introduced existing models of body-on-a-chip, their constitutional details, application for drug testing, and individual features of these models. We put special emphasis on the latest trend in this field of incorporating barrier tissue into body-on-a-chip and discussed several remaining challenges of current body-on-a-chip., (© 2015 Society for Laboratory Automation and Screening.)

The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.

Synthetic biology follows the traditional engineering paradigm of designing, building, testing and learning to create new biological systems. While such approaches have enormous potential, major challenges still exist in this field including increasing the speed at which this workflow can be performed. Here, we present recently developed microfluidic tools that can be used to automate the synthetic biology workflow with the goal of advancing the likelihood of producing desired functionalities. With the potential for programmability, automation, and robustness, the integration of microfluidics and synthetic biology has the potential to accelerate advances in areas such as bioenergy, health, and biomaterials.

Having observed a large variation in the number and type of original preclinical publications for newly registered drugs, we have explored whether longitudinal trends and/or factors specific for certain drugs or their manufacturers may explain such variation. Our analysis is based on 1954 articles related to 170 newly approved drugs. The number of preclinical publications per compound declined from a median of 10.5 in 1991 to 3 in 2011. A similar trend was observed for the number of in vivo studies in general, but not in the subset of in vivo studies in animal models of disease. The percentage of compounds with studies using isolated human cells or cell lines almost doubled over time from 37 to 72%. Number of publications did not exhibit major differences between compounds intended for human versus veterinary use, therapeutic areas, small molecules versus biologicals, or innovator versus follow-up compounds; however, some companies may publish fewer studies per compound than others. However, there were qualitative differences in the types of models being used depending on the therapeutic area; specifically, compounds for use in oncology very often used isolated cells and cell lines, often from human origin. We conclude that the large variation in number and type of reported preclinical data is not easily explained. We propose that pharmaceutical companies should consistently provide a comprehensive documentation of the preclinical data they generate as part of their development programs in the public domain to enable a better understanding of the drugs they intend to market.

Thyroglobulin (Tg) is a high molecular weight glycoprotein located mainly in thyroid follicles, where thyroid hormones are synthesized and stored. In patients with differentiated thyroid cancer of follicular origin, serum Tg levels become undetectable following total thyroidectomy and iodine-131 remnant ablation. It is a key biomarker to follow-up patients with differentiated thyroid cancer, in combination with neck ultrasound monitoring. The measurement of Tg in the wash-out of the needle used for fine needle aspiration biopsy is a valuable aid to the diagnosis of lymph node metastasis. The presence of anti-thyroglobulin antibodies affects reliability of Tg results measured in serum or plasma. Systematic investigation of such antibodies is required to validate any Tg assay. Elevated or rising levels of anti-thyroglobulin antibodies can in turn be used as a surrogate tumor marker of thyroid cancer. The development of second-generation Tg assay (automated, highly sensitive) has enabled significant advances in the management of differentiated thyroid cancer: early detection of persistent or recurrent disease and follow-up care simplified in low-risk patients. Testing of serum Tg can also be useful in evaluating other clinical situations such as congenital hypothyroidism, endemic goiter and thyrotoxicosis factitia.

Throughout the last 50 years, the paradigm for carcinogenicity assessment has depended on lifetime bioassays in rodents. Since 1997, the International Conference on Harmonisation (ICH) S1B has permitted the use of a 2-year rodent bioassay (usually in the rat) and an alternative, genetically modified mouse model to support cancer risk assessment of pharmaceuticals. Since its introduction, it has become apparent that many of the stated advantages of the 6-month Tg mouse bioassay have, in actual fact, not been realized, and the concern exists that an albeit imperfect, 2-year mouse bioassay has been replaced by a similarly imperfect 6-month equivalent. This essay argues strongly that model systems, using cancer as the end point, should be discontinued, and that the recent initiatives, from the Organization for Economic Cooperation and Development and Institute of Peace and Conflict Studies, on "mode of action," "adverse outcome pathways," and "human relevance framework" should be embraced as being risk assessments based upon the available science. The recent suggested revisions to the ICH S1 guidelines, utilizing carcinogenicity assessment documents, go some way to developing a science-based risk assessment that does not depend almost entirely on a single, imperfect, cancer-based end point in nonrelevant animal species., (© 2015 by The Author(s).)

Introduction: The comprehensive in vitro proarrhythmia assay (CiPA) is a nonclinical, mechanism-based paradigm for assessing drug proarrhythmic liability., Topics Covered: The first CiPA assay determines effects on cloned human cardiac ion channels. The second investigates whether the latter study-generated metrics engender proarrhythmic markers on a computationally reconstructed human ventricular action potential. The third evaluates conclusions from, and searches possibly missed effects by in silico analysis, in human stem cell-derived cardiomyocytes (hSC-CMs). CiPA ad hoc Expert-Working Groups have proposed patch clamp protocols for seven cardiac ion channels, a modified O'Hara-Rudy model for in silico analysis, detailed procedures for field (MEA) and action potential (VSD) measurements in hSC-CMs, and 29 reference drugs for CiPA assay testing and validation., Discussion: CiPA adoption as drug development tool for identifying electrophysiological mechanisms conferring proarrhythmic liability to candidate drugs is a complex, multi-functional task requiring significant time, reflection, and efforts to be fully achieved., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The advent of high-content screening more than a decade ago remodeled drug discovery workflows by recasting the role of cell-based approaches in target identification, primary screening, lead optimization, and toxicity. The ability to identify and quantify compound effects on multiple cellular functions allows for rapid characterization of chemical libraries. Laser scanning imaging cytometry (LSIC) is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using laser scanning excitation through a specialized F-theta scan lens. This configuration results in rapid whole well scanning and large depth of field. The recent availability of systems equipped with multiple sources of laser excitation and arrays of detectors for spectral analysis has significantly increased its applicability through enabling more fluorescent reagents and higher levels of multiplexing. LSIC is being used most extensively for phenotypic screening especially in areas such as cell health, RNA interference (RNAi) screening, and three-dimensional cell models. This review communicates advances in LSIC and how it is being applied by presenting an overview of the technology and a range of real-world case studies.

This review describes the principle and applications of bioluminescent enzymatic toxicity bioassays. This type of assays uses bacterial coupled enzyme systems: NADH:FMN-oxidoreductase and luciferase to replace living organisms in developing cost-competitive biosensors for environmental, medical and industrial applications. These biosensors instantly signal chemical and biological hazards and allow for detecting a great amount of toxic compounds with advantages associated with fast results, high sensitivity, simplicity, low cost and safety of the procedure.

Chemiluminescence-based bioassays have become increasingly important in clinical, pharmaceutical, environmental, and food safety fields owing to their high sensitivity, wide linear range, and simple instrumentation. During the past decade, it has been found that metal nanoparticles can initiate various liquid-phase chemiluminescence reactions as catalysts, reductants, energy acceptors, and nanosized reaction platforms owing to their unique optical, catalytic, and surface properties and chemical reactivity, which are very important for chemiluminescence bioassays based on metal nanoparticles as nanoprobes or a nanointerface. In this article, we summarize recent progress in metal-nanoparticle-initiated liquid-phase chemiluminescence, including reaction systems, mechanisms, and their applications in chemiluminescence-based bioassays, especially for immunoassays, DNA assays, aptamer-based assays, high-performance liquid chromatography or capillary electrophoresis analysis, and flow injection analysis.

It was discovered at the beginning of this Century that living bacteria-and specifically the extremophile Pseudomonas syzgii-could be captured inside growing crystals of pure water-corroding semiconductors-specifically germanium-and thereby initiated pursuit of truly functional "biochip-based" biosensors. This observation was first made at the inside ultraviolet-illuminated walls of ultrapure water-flowing semiconductor fabrication facilities (fabs) and has since been, not as perfectly, replicated in simpler flow cell systems for chip manufacture, described here. Recognizing the potential importance of these adducts as optical switches, for example, or probes of metabolic events, the influences of the fabs and their components on the crystal nucleation and growth phenomena now identified are reviewed and discussed with regard to further research needs. For example, optical beams of current photonic circuits can be more easily modulated by integral embedded cells into electrical signals on semiconductors. Such research responds to a recently published Grand Challenge in ceramic science, designing and synthesizing oxide electronics, surfaces, interfaces and nanoscale structures that can be tuned by biological stimuli, to reveal phenomena not otherwise possible with conventional semiconductor electronics. This short review addresses only the fabrication facilities' features at the time of first production of these potential biochips.

Mouse bioassays have been a mainstay for detecting harmful concentrations of marine algal toxins in shellfish for over 70 years. Routine monitoring involves intraperitoneal injection of shellfish extracts into mice; shellfish contaminated with algal toxins are thus identified by mortality in exposed mice. With the advent of alternative test methods to detect and quantify specific algal toxins has come increasing criticism of enduring use of mouse bioassays for shellfish safety testing. However, the complete replacement of shellfish safety mouse bioassays by chemical, antibody-based, and functional assays has been and will continue to be a gradual process for various reasons, including skills availability and instrument costs for chromatography-based toxin monitoring. Mouse bioassays for shellfish safety testing do not comply with modern standards for laboratory animal welfare, specifically the requirement in published official methods for death as a test outcome. Mouse bioassays for algal biotoxins in shellfish, as well as fundamental algal toxin research endeavors using in vivo models, are amenable to revision and refinement from a humane endpoints perspective. Regulated hypothermia may be a useful and easily acquired nonlethal toxicological endpoint; objective determination of neuromuscular blockade may allow algal neurotoxin testing and research to enter the domain of humane endpoints evaluation. Relinquishing reliance on subjective test endpoints, including death, will likely also deliver collateral improvements in assay variability and sensitivity.

Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons and stem cell-derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is hyperactivated in most cancers and represents a plausible therapeutic target. In the absence of STAT3-selective small-molecule inhibitors, we sought to develop pSTAT3/1 high-content imaging (HCS) assays to screen for selective inhibitors of STAT3 pathway activation in head and neck squamous cell carcinomas (HNSCC) tumor cell lines. Based on the expression of the interleukin-6 (IL-6)Rα and gp130 subunits of the IL-6 receptor complex and STAT3, we selected the Cal33 HNSCC cell line as our model. After developing image acquisition and analysis procedures, we rigorously investigated the cytokine activation responses to optimize the dynamic ranges of both assays and demonstrated that the pan-Janus kinase inhibitor pyridone 6 nonselectively inhibited pSTAT3 and pSTAT1 activation with 50% inhibition concentrations of 7.19 ± 4.08 and 16.38 ± 8.45 nM, respectively. The optimized pSTAT3 HCS assay performed very well in a pilot screen of 1,726 compounds from the Library of Pharmacologically Active Compounds and the National Institutes of Health clinical collection sets, and we identified 51 inhibitors of IL-6-induced pSTAT3 activation. However, only three of the primary HCS actives selectively inhibited STAT3 compared with STAT1. Our follow-up studies indicated that the nonselective inhibition of cytokine induced pSTAT3 and pSTAT1 activation by G-alpha stimulatory subunit-coupled G-protein-coupled receptor agonists, and forskolin was likely due to cyclic adenosine monophosphate-mediated up-regulation of suppressors of cytokine signaling 3. Azelastine, an H1 receptor antagonist approved for the treatment of seasonal allergic rhinitis, nonallergic vasomotor rhinitis, and ocular conjunctivitis, was subsequently confirmed as a selective inhibitor of IL-6-induced pSTAT3 activation that also reduced the growth of HNSCC cell lines. These data illustrate the power of a chemical biology approach to lead generation that utilizes fully developed and optimized HCS assays as phenotypic screens to interrogate specific signaling pathways.

Ionic liquids (ILs) are novel solvents that display a number of unique properties, such as negligible vapor pressure, thermal stability (even at high temperatures), favorable viscosity, and miscibility with water and organic solvents. These properties make them attractive alternatives to environmentally unfriendly solvents that produce volatile organic compounds. In this article, a critical review of state-of-the-art developments in the use of ILs for the separation and preconcentration of bioanalytes in biological samples is presented. Special attention is paid to the determination of various organic and inorganic analytes--including contaminants (e.g., pesticides, nicotine, opioids, gold, arsenic, lead, etc.) and functional biomolecules (e.g., testosterone, vitamin B12, hemoglobin)--in urine, blood, saliva, hair, and nail samples. A brief introduction to modern microextraction techniques based on ILs, such as dispersive liquid-liquid microextraction (DLLME) and single-drop microextraction (SDME), is provided. A comparison of IL-based methods in terms of their limits of detection and environmental compatibilities is also made. Finally, critical issues and challenges that have arisen from the use of ILs in separation and preconcentration techniques are also discussed.

Fluorine, chlorine, bromine, and iodine have been studied in biological samples and other related matrices owing to the need to understand the biochemical effects in living organisms. In this review, the works published in last 20 years are covered, and the main topics related to sample preparation methods and analytical techniques commonly used for fluorine, chlorine, bromine, and iodine determination in biological samples, food, drugs, and plants used as food or with medical applications are discussed. The commonest sample preparation methods, as extraction and decomposition using combustion and pyrohydrolysis, are reviewed, as well as spectrometric and electroanalytical techniques, spectrophotometry, total reflection X-ray fluorescence, neutron activation analysis, and separation systems using chromatography and electrophoresis. On this aspect, the main analytical challenges and drawbacks are highlighted. A discussion related to the availability of certified reference materials for evaluation of accuracy is also included, as well as a discussion of the official methods used as references for the determination of halogens in the samples covered in this review.