Bioanalytical Tools Research Articles

The use of tomato aminoaldehyde dehydrogenase 1 for the detection of aldehydes in fruit distillates

Plant NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases. They participate in the metabolism of polyamines or osmoprotectants. The enzymes are characterized by their broad substrate specificity covering ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The isoenzyme 1 from tomato (Solanum lycopersicum; SlAMADH1) oxidizes aliphatic aldehydes very efficiently and converts also furfural, its derivatives or benzaldehyde, which are present at low concentrations in alcoholic distillates such as fruit brandy. In this work, SlAMADH1 was examined as a bioanalytical tool for their detection. These aldehydes arise from fermentation processes or thermal degradation of sugars and their presence is related to health complications after consumption including nausea, emesis, sweating, decrease in blood pressure, hangover headache, among others. Sixteen samples of slivovitz (plum brandy) from local producers in Moravia, Czech Republic, were analyzed for their aldehyde content using a spectrophotometric activity assay with SlAMADH1. In all cases, there were oxidative responses observed when monitoring NADH production in the enzymatic reaction. Aldehydes in the distillate samples were also subjected to a standard determination using reversed-phase HPLC with spectrophotometric and tandem mass spectrometric detection after a derivatization with 2,4-dinitrophenylhydrazine. Results obtained by both methods were found to correlate well for a majority of the analyzed samples. The possible applicability of SlAMADH1 for the evaluation of aldehyde content in food and beverages has now been demonstrated.

The assessment of WWTP performance: Towards a jigsaw puzzle evaluation?

A chemical and bio-analytical protocol is proposed as a holistic monitoring framework for the assessment of WWTPs (Wastewater Treatment Plants) performance. This combination of tests consists of: i) an analysis of emerging contaminants, to be added to the established physico-chemical parameters in order to understand the causes of (new) pollution phenomena and ii) some of the bio-analytical tools most widely applied in the field of wastewater research, which provide information on groups of chemicals with a common mode of toxic action (baseline toxicity, estrogenicity and mutagenicity/genotoxicity, selected as the most representative for human health). The negative effects of the discharge can thus be highlighted directly and used to assess the global environmental impact of WWTPs. As a validation, this multi-tiered approach was applied to a full-scale WWTP (150,000 p.e.), where different measurements were carried out: EDCs (Endocrine Disrupting Compounds) detection; algal growth inhibition, bioluminescence inhibition and acute toxicity test (for baseline toxicity); an E-Screen-like assay (for estrogenic activity); Ames, Allium cepa and Comet tests (for mutagenic/genotoxic activity). As a result, the WWTP showed good performance for all these issues, displaying its ability to enhance effluent quality, except for residual mutagenic behaviour, probably due to the by-products generated by the tertiary ozonation.

Linking in Vitro Effects and Detected Organic Micropollutants in Surface Water Using Mixture-Toxicity Modeling.

Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-κB) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

A tiered, integrated biological and chemical monitoring framework for contaminants of emerging concern in aquatic ecosystems.

The chemical-specific risk-based paradigm that informs monitoring and assessment of environmental contaminants does not apply well to the many thousands of new chemicals that are being introduced into ambient receiving waters. We propose a tiered framework that incorporates bioanalytical screening tools and diagnostic nontargeted chemical analysis to more effectively monitor for contaminants of emerging concern (CECs). The framework is based on a comprehensive battery of in vitro bioassays to first screen for a broad spectrum of CECs and nontargeted analytical methods to identify bioactive contaminants missed by the currently favored targeted analyses. Water quality managers in California have embraced this strategy with plans to further develop and test this framework in regional and statewide pilot studies on waterbodies that receive discharge from municipal wastewater treatment plants and stormwater runoff. In addition to directly informing decisions, the data obtained using this framework can be used to construct and validate models that better predict CEC occurrence and toxicity. The adaptive interplay among screening results, diagnostic assessment and predictive modeling will allow managers to make decisions based on the most current and relevant information, instead of extrapolating from parameters with questionable linkage to CEC impacts. Integr Environ Assess Manag 2016;12:540-547. © 2015 SETAC.

Chemical derivatization in LC-MS bioanalysis: current & future challenges.

Chemical derivatization is an important tool in bioanalysis. Because of the extensive amount of background information available for classic and established derivatization techniques, we suggest using these techniques initially to solve bioanalytical issues. In this paper, we will present current technical challenges that require modified derivatization and related analytical techniques. In real laboratory circumstances, sometimes classic derivatization techniques lead to liquid chromatographic problems induced by excess hydrophobicity and chemical complexity of derivatives. With regard to derivatization techniques, another concern is procedural complexity. We will present some recent technique modifications that can be used to overcome these difficul ties. In addition, we will present some unexplored fields related to derivatization. We will describe the combined use of derivatization with microflow LC–MS and introduce the idea of new chemical tagging techniques.

A zero-step functionalization on paper-based biosensing platform for covalent biomolecule immobilization

As a potential platform for point-of-care clinical analyses and environment monitoring, paper biosensors have received considerable attention. In many cases, the conjugation of biomolecules onto paper surface is crucial for increasing the functionalities of paper-based bioanalytical devices. Until now, it is sometimes argued as in the literature that finding a surface chemistry for biomolecule covalent grafting to paper still remains a challenge. Here the study shows that at least to a certain extent some aspects of the argument involved is questionable, by demonstrating that paper without any modification could be utilized for the covalent conjugation of enzymes and serves as a tool for bioanalysis. Moreover, the detailed analysis of biomolecule immobilization strategies on paper through polysaccharide-coating chemistry has been offered as a contrast. We believe that the proposed method could provide a valuable perspective for paper-based biosensors.

Numerical Simulations of the Digital Microfluidic Manipulation of Single Microparticles.

Single-cell analysis techniques have been developed as a valuable bioanalytical tool for elucidating cellular heterogeneity at genomic, proteomic, and cellular levels. Cell manipulation is an indispensable process for single-cell analysis. Digital microfluidics (DMF) is an important platform for conducting cell manipulation and single-cell analysis in a high-throughput fashion. However, the manipulation of single cells in DMF has not been quantitatively studied so far. In this article, we investigate the interaction of a single microparticle with a liquid droplet on a flat substrate using numerical simulations. The droplet is driven by capillary force generated from the wettability gradient of the substrate. Considering the Brownian motion of microparticles, we utilize many-body dissipative particle dynamics (MDPD), an off-lattice mesoscopic simulation technique, in this numerical study. The manipulation processes (including pickup, transport, and drop-off) of a single microparticle with a liquid droplet are simulated. Parametric studies are conducted to investigate the effects on the manipulation processes from the droplet size, wettability gradient, wetting properties of the microparticle, and particle-substrate friction coefficients. The numerical results show that the pickup, transport, and drop-off processes can be precisely controlled by these parameters. On the basis of the numerical results, a trap-free delivery of a hydrophobic microparticle to a destination on the substrate is demonstrated in the numerical simulations. The numerical results not only provide a fundamental understanding of interactions among the microparticle, the droplet, and the substrate but also demonstrate a new technique for the trap-free immobilization of single hydrophobic microparticles in the DMF design. Finally, our numerical method also provides a powerful design and optimization tool for the manipulation of microparticles in DMF systems.

Recent Advances in the Methodology and Application for the Metabolism of Phytochemical Compounds-An Update Covering the Period of 2009-2014.

The research of drug metabolism is essential for successful drug discovery. Phytochemicals, particularly those from herbal medicines, have the potential to affect diseases such as cancer, heart disease, diabetes, and high blood pressure, etc., showing great advantages in disease prevention and personalized treatment. In the past decade, phytochemical metabolism played a major role in the discovery of valuable leading compounds of new drugs. However, the metabolic studies of phytochemical compounds are extremely challenging due to numerous analogs with similar structures, complex endogenous matrices with multi-components in biofluids and cell or tissue extracts. For the feasibility of monitoring phytochemical metabolism, suitable bioanalytical tools and strategies have to be developed and validated. Here we critically review the literature from 2009 to 2014, and present generalized strategy for phytochemical metabolism in biological matrices, including the methodology of metabolism, sample preparation methods and detection techniques. In addition, the present paper covers the diverse aspects of metabolism studies on phenolic acids, flavonoids, anthraquinones, alkaloids, terpenoids, coumarins, lignans and steroids in medicinal plants. Finally, prospects for further research are also considered.

The Efficacy of Ozone/BAC Treatment on Non-Steroidal Anti-Inflammatory Drug Removal from Drinking Water and Surface Water

Three non-steroidal anti-inflammatory drugs (NSAIDs)—ketoprofen, naproxen and piroxicam—in both deionized (DI) water and surface lake water (SW) (Tallahassee, FL), were exposed to varying ozone treatment regimes or H2O2/O3 advanced oxidation on the laboratory bench. Recently used biofilm-supporting granular activated carbon (BAC) was sampled from a municipal drinking water treatment facility (Tampa, FL, USA), and employed to determine the bio-availability of chemical intermediates formed in ozonated water. Advanced chemical analysis was used to identify oxidation by-products formed and combined with a bioanalytical tool to assess non-specific toxicity (Microtox assay). All 3 target pharmaceuticals were efficiently removed by different processes, with a lower NSAIDs removal yield observed in lake water compared to DI water experiments. The removal yields of ketoprofen, naproxen, and piroxicam improved with increasing ozone dose, H2O2/O3 ratio and empty bed contact time (EBCT) with BAC. Ozonation with BAC filtration had a positive impact by reducing the initial ozone dose required to achieve > 90% removal of all 3 pharmaceuticals (when an initial ozone dose < 1 mg L-1 was combined with EBCT < 15 min). The toxicity evolution of the treated samples was monitored by Microtox bioassay. Ozone doses higher than 2 mg L-1 for 2 min contact time were optimal to reach the lower water samples toxicity with NSAIDs removal yields ranging from 95.5 to 99.0% in DI water and from 77 to 90% in SW. Also, higher ozone doses were not shown to remove the residual toxicity. In contrast, the BAC filtration hardly decreases the sample toxicity when an EBCT of 15 min was chosen despite a NSAIDs removal yield equal or higher than 90% in SW.

Future water quality monitoring — Adapting tools to deal with mixtures of pollutants in water resource management

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.

A method for determining the actual rate of orientation switching of DNA self-assembled monolayers using optical and electrochemical frequency response analysis.

Electrostatic control of the orientation of fluorophore-labeled DNA strands immobilized on an electrode surface has been shown to be an effective bioanalytical tool. Modulation techniques and later time-resolved measurements were used to evaluate the kinetics of the switching between lying and standing DNA conformations. These measurements, however, are the result of a convolution between the DNA "switching" response time and the other frequency limited responses in the measurement. In this work, a method for analyzing the response of a potential driven DNA sensor is presented by calculating the potential effectively dropped across the electrode interface (using electrochemical impedance spectroscopy) as opposed to the potential applied to the electrochemical cell. This effectively deconvolutes the effect of the charging time on the observed frequency response. The corrected response shows that DNA is able to switch conformation faster than previously reported using modulation techniques. This approach will ensure accurate measurements independent of the electrochemical system, removing the uncertainty in the analysis of the switching response, enabling comparison between samples and measurement systems.

Micro- and Nano-Chamber Array Chips for a Single Molecule Analysis

This paper reports the design, fabrication, and application of micro- and nanoscale chamber array chips toward a single nucleus assay as a new experimental format for research in biology. Micro- and nanoscale chamber array on a chip becomes a powerful new tool for bioanalysis since it could stochastically capture and measure biomolecules at a single molecule level. Our fundamental interests are how reaction-space size affects an enzyme activity and how small space we could extrapolate enzyme kinetics in small space from old enzyme kinetic data in bulk. So we have measured a single β-galactosidase activity in various sizes of micro and nanochambers, from 5 μm to 500 nm cubic, and found that the activity gradually decreased according to its chamber size. Because the specific surface area increases according to the decrease of the chamber volume, non-specific adsorption might be a factor to suppress the activity. However, several experiments including surface coating and repetition of the enzyme capturing elucidated that non-specific adsorption is not a major factor affecting the activity. In the field of gene delivery systems, it is still unknown that virus vector showed much higher transportation efficiency of plasmid DNA from cytoplasm to nucleus in contrast to non-virus vectors. To improve gene transportation efficiency, which directly lead to increase transfection efficiency, gene transportation mechanism through nucleus membrane should be cleared. But unfortunately there are no experimental protocol to quantitatively evaluate the transportation efficiency through the nucleus membrane. So, in this study, nuclei were extracted from cells by gentle detergent treatments and captured into a PDMS or PMMA microchamber at a single nucleus level. After the confirmation of capturing a single nucleus into a microchamber, nucleotides and nuclear membrane stained nuclei, transportation of mRNA by molecular beacon were observed.

Bioanalytical tools: half a century of application for potable reuse

In vitro bioassays are often thought as novel tools by water stakeholders. They have, however, been used for over half a century in assessment of recycled water quality.

Highlights from the 2014 Applied Pharmaceutical Analysis conference.

The 10th annual Applied Pharmaceutical Analysis (APA) conference was held from 8th to 10th September in Cambridge, MA, USA. This year's APA conference focused on three different 'workshops' over the 3 days: Regulated Bioanalysis, Biotransformation, and Discovery. There was a great amount of information discussed by a variety of experts over the 3 days. This included, among other things; speakers from the US FDA discussing statute changes and guidelines, leaders from academic laboratories discussing innovation in bioanalytical tools, and industry scientists discussing current trends in the industry. The conference afforded attendees the opportunity to learn from the speakers during their sessions. In addition, there was ample opportunity for attendees and speakers both to learn from each other through informal interactions.

Fourier Transform Infrared Spectroscopy and Imaging in Cancer Diagnosis and Characterization

Fourier Transform Infrared (FTIR) spectroscopy and imaging techniques are formidable, novel, rapid and non-destructive tools for characterization of different biological systems from molecules to membranes, cells and tissues. These techniques with multivariate analysis tools can be used in the diagnosis/follow-up of diseases including cancer, and in monitoring drug or chemical induced alterations in tissues and cells. In the current study, Attenuated Total Reflectance FTIR spectroscopy together with chemometric (cluster and principal component) analysis were utilized as a diagnostic tool for urinary bladder cancer while FTIR imaging technique was used in the clarification of sodium butyrate (NaB) induced-differentiation in colon cancer cells since butyrate has an anti-proliferative effect in colon cancer. For bladder cancer studies, bladder wash samples of bladder cancer and control groups were used directly for spectra collection. ATR-FTIR studies revealed significant alterations in lipid, protein, and nucleic acid content of bladder wash samples of cancer groups as compared to the control ones. Based on these spectral variations, bladder cancer group can be successfully differentiated from control via chemometric analysis with a higher sensitivity and specificity than many other methods used currently in bladder cancer diagnosis. In colon cancer study, aggressive CaCo2 cell lines were treated with 3mM NaB and cultured for 48h for complete differentiation. Specific band ratio analysis such as lipid/protein, glycogen/phosphate and RNA/DNA were calculated from the chemical maps of control and NaB-treated cells. Imaging results indicated the reconstruction of cancer induced changes in response to NaB treatment by modulating the saturated, unsaturated, triglyceride, protein and nucleic acid content in the treated cells. The findings of this study supported that FTIR spectroscopic and imaging techniques are valuable, label-free and sensitive bio-analytical tools for cancer diagnostics and investigating the global roles of drugs on cancer treatment.

Direct detection of microRNA based on plasmon hybridization of nanoparticle dimers.

MicroRNAs (miRNA) are important for regulating a range of biochemical pathways. Abnormal levels of miRNA in cells or secreted into biological fluids have been identified in diseases. MiRNA can therefore be potential biomarkers for early disease diagnosis; however their detection and quantification are challenging. Herein we apply the sensing platform of discrete actuatable dimers for the detection of human miR-210 (hsa-miR-210-3p). The detection signal is a spectral blue shift in the hybridized plasmon mode as monitored by single-nanostructure spectroscopy. We investigate the specificity and detection limit of the platform and quantify miR-210 levels in RNA extracts of cells cultured under different oxygen tensions. In addition we demonstrate the feasibility of detection in complex media by examining miR-210 secreted in cell media. This sensing platform may be developed as a bioanalytical tool for validating miRNA profiles of biological fluids.

Proteomic profiling by nanomaterials-based matrix-assisted laser desorption/ionization mass spectrometry for high-resolution data and novel protein information directly from biological samples.

Qualitative and quantitative analyses of global proteome samples derived from biocomplex mixtures are very important to understand the cellular functions in cell biology. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based proteomics has recently become one of the most informative and attractive core technologies in proteomics. Particularly, nanomaterials-based MALDI mass spectrometric methods are quickly becoming a critical miniaturized bioanalytical tool for detecting and discerning proteins from biocomplex samples. These MALDI-developed strategies allow high-throughput identification of proteins from highly complex mixtures including accurate mass measurement of peptides derived from total proteome digests and peptides/proteins separations from various samples. The nanomaterials-integrated MALDI-MS technologies in protein arrays hold much promise for interrogating the diverse and immense proteome in cell biology. As a result, nanomaterials-assisted MALDI-MS-based proteomic workflow, including sample preparation, information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins and their analysis, should make the technology more easily available to a broad community and turn it into a powerful methodology for bioanalysts.

Surface acoustic wave biosensor as a functional quality method in pharmaceutics

With the recent successes of biopharmaceuticals, new bioanalytical tools are needed for the functional quality characterization of new biological entities and biosimilars towards marketing authorization approval and during the product life. Biosensors are emerging technologies in the pharmaceutical field in drug discovery, development and Quality Control (QC) stages; however, they are not yet included in any pharmacopoeia. Surface acoustic wave (SAW) biosensors allow the user to detect and quantify binding events, giving information not only on affinity (KD) and kinetics (kon and koff), but also on viscoelastic effects. We present here an analytical quality by design (aQbD) based approach using somatropin (rhGH, recombinant human growth hormone) and derivatives as model biotechnological drugs and an antibody interaction partner to evaluate the functional quality. Using techniques described in ICH Q2, Q5 and Q8–11, we evaluated the performance of this SAW biosensor technique from a pharmaceutical quality point of view. CMAs (Critical Method Attributes) such as sensitivity, selectivity and variability are evaluated as a function of different experimental variables using DoEs (design of experiments). We suggest qualification tests as well as SSTs (system suitability tests), as required by the pharmaceutical competent authorities and recommend the inclusion of biosensor techniques into the different pharmacopoeia and pharmaceutical guidelines.

Chemical and bioanalytical assessment of coal seam gas associated water

Environmental context Water associated with coal seam gas is generally of poor quality and thus its management and potential further usage is a subject of concern. In a comprehensive study involving chemical and bioanalytical assessments of coal seam gas associated water, we found that less than 5% of the biological effects could be explained by chemical analysis. The use of bioanalytical tools to complement chemical analysis is recommended for monitoring the quality of water associated with coal seam gas. Abstract A comprehensive study was undertaken involving chemical (inorganic and organic) and bioanalytical assessments of coal seam gas associated water (CSGW) in Queensland, Australia. CSGW is a by-product of the gas extraction process and is generally considered as water of poor quality. CSGW is disposed of by release to surface water, reinjected to groundwater or beneficially reused. In this study, groundwater samples were collected from private wells tapping into the Walloon Coal Measures, the same coal aquifer exploited for coal seam gas production in the Surat Basin. The inorganic characteristics of these water samples were almost identical to the CSGW from the nearby gas field, with high sodium, bicarbonate and chloride concentrations but low calcium, magnesium and negligible sulfate concentrations. As for organic compounds, low levels of polyaromatic hydrocarbons (PAHs) were detected in the water samples, and neither phenols nor volatile organic compounds were found. Five of the fourteen bioassays tested gave positive responses (arylhydrocarbon-receptor gene activation, estrogenic endocrine activity, oxidative stress response, interference with cytokine production and non-specific toxicity), whereas the other nine assays showed no genotoxicity, protein damage or activation of hormone receptors other than the estrogen receptor. The observed effects were benchmarked against known water sources and were similar to secondary treated wastewater effluent, stormwater and surface water. As mixture toxicity modelling demonstrated, the detected PAHs explained less than 5% of the observed biological effects. These results showed that bioanalytical assessment can open new avenues for research into the potential environmental and health risk from CSGW.