We would like to thank comments from two anonymous reviewers as well members of the Poulain Lab for insightful discussion on the development of the anaerobic biosensor. An Early Researcher Award from the Province of Ontario and a Discovery Grant and an accelerator supplement from the Natural Sciences and Engineering Research Council of Canada to A.J.P. funded this study.

The authors have nothing to disclose.

Microbial biosensors are useful tools to identify mechanisms by which metals are interacting with microbes. Here, we describe a method that can anaerobically quantify HgII and CdII bioavailability to a Gram-negative host cell (E. coli) and give a quantifiable result within a few hours. One of the major strengths of this protocol is that it allows extensive control of metal speciation in the exposure medium by avoiding strong binding ligands or components that may lead to metal precipitation. Metal speciation has been modelled and tested in this exposure medium using PHREEQC17, however other metal speciation software may be deployed. In the case of no added ligands, Hg speciation is expected to be present as 97% Hg(OH)2 and 3% Hg(NH3)22+, while Cd speciation will be present as 59% Cd-&#946;-Glycerophosphate, 25% Cd2+, and 16% CdSO4. Using a simple thermodynamic modelling software, the user can design exposure media and test for the bioavailability of the metal of interest. In addition, the biosensor host cell (E. coli NEB5&#945;) is viable over a wide range of pH (5-8.5) and NaCl concentration (0-0.55 M)17.
Hg methylation is an anaerobic process, and the protocol outlined in this study does not have a requirement for oxygen, allowing for more accurate description of anaerobic metabolism on metal bioavailability. This is important because the presence of oxygen alters gene expression profiles48,49&#160;and hence, potential transport pathways; therefore this method presents an advantage over currently exising aerobic alternatives. The biosensing construct presented here can potentially be used with other anaerobic hosts that may be more relevant for mercury methylation (e.g., Geobacter, Desulfovibrio), but maybe less tractable than E. coli. One current limitation of the approach presented here is that our limit of detection has not yet reached pM levels, contrary to existing aerobic systems4,19,37. It is however important to note that to achieve these low detection limits several steps need to be taken44: i) ligand addition is required to ensure that Hg remains in solution and does not adsorb onto the microbial cell wall (Hg will be irreversible bound to cell surface thiols preventing its bioavailability25,27; see the threshold response for Hg in Figure 3A), ii) modifications to cell density, or iii) modify the genetic construct to include transport proteins of the mer-operon (namely merT and merP), increasing Hg flux inside the cell50,51. These modifications would be beneficial in detecting low concentrations of Hg, but not necessarily ideal when assessing environmentally relevant situations. Whereas previous cadmium biosensors primarily exist as a &#34;proof of principle&#34;, they were designed in complex media that do not allow the investigator to assess the role of speciation on bioavailability41,42,43,44.
The biosensor is an incredibly useful tool in determining mechanisms in which metal species are bioavailable. Because the host organism is not a Hg-methylator, it may only be used to develop a model for how Hg may enter Gram-negative bacteria and not a definitive rationale for how Hg-methylators acquire Hg. Other methods exist for determining Hg bioavailability, such as methylation, as an outcome of uptake or the use of a mass balance approach10,15,20,45,46. That being said, the method presented here offers the advantage of quasi real time bioavailability data in viable cells. We do not recommend that this method be used to quantify total Hg or Cd levels in an environmental matrix. Despite the proposed use of biosensors to determine metal concentrations in the environment36, many more readily available standard methods are available such as ICP-MS, FAAS (for Cd analysis) or Cold vapor atomic absorption spectroscopy (for Hg analysis). The biosensor can however be used to determine if a given environmental matrix has the potential to enhance or hamper bioavailability; this is achieved by performing standard additions.
The pH of the exposure media may be altered to anywhere within the range of 5 and 8.5, provided the MOPS Free acid (buffer) is exchanged with an alternate free acid of a buffer with the appropriate pKa (please see list of appropriate buffers (Ferreira et al. (2015)47) and adjusted with KOH to the appropriate pH when making the exposure media. In addition, the method is not limited to Hg and Cd, but could be extended to other metals using other transcription regulators.
The exposure assay may be modified to explore the influence of other electron acceptors such as O2 and fumarate on Hg or Cd bioavailability. Minor modifications to the method to utilize both O2 and fumarate as electron acceptors are available upon request.
In summary we would like to emphasize the following points: I) It is imperative that the concentrations of Cd or Hg stocks are known in step 2, as these will be used to calibrate the biosensor. II) On the exposure day, the growth of cells must be stopped at an OD600 of 0.6 (&#177; 0.1) and that care is taken when resuspending the cell cultures, as the biosensor is calibrated to this cell density. III) Lastly, it is important that the exposure medium is made meticulously on the exposure day. To ensure the success of the protocol, multiple cultures should be grown simultaneously (to circumvent the possibility of growth failure) and the growth medium should be remade weekly (to circumvent the metastability of the media and possible contamination). It should also be noted that biological replicates (multiple cell cultures) express variability when it comes to signal production. Although the fluorescent responses may vary from culture to culture, the fluorescent trends in response to a given variable should remain the same throughout numerous biological replicates.

Mercury (Hg) is a global pollutant and its bioavailability to Hg methylating microbes is the first step towards its biomagnification through food webs and its possible neurotoxic effects in human and wildlife1. It is currently thought that microbial Hg methylation is an intracellular process that requires: i) the species of Hg to be bioavailable2,3,4,5,6,7 and ii) for the cell to be physiologically capable of methylating Hg8,9,10. Cadmium (Cd) bioaccumulates in organisms, but does not biomagnifies in foodwebs and is widely used in industrial and commercial processes that commonly cause acute exposures in people and the environment11. Although microbes play several key roles in the fate of Hg in the environment, most studies on Cd geochemistry and ecotoxicology focus on microbial eukaryotes12. Consumption of agricultural crops (e.g., rice) is the main source of direct exposure to cadmium; in this case, the bioavailability of Cd to microbes of the rhizosphere directly influences the amount plants can accumulate13.
Hg transport pathways are complex and possibly involve an active transport step14. When a transporter is involved, recent work suggested that HgII uses a ZnII or MnII transporter7,15,16,17. Whereas CdII is hypothesized to be accidentally transported into the cytosol through divalent metal transport pathways (particularly MnII or ZnII), mechanisms of CdII transport inside the cells remain speculative, and no Cd-specific transport pathway has been identified13,18. Regardless of the nature of the transporters involved, three mechanistic factors ultimately determine the ability of metals to enter a cell: i) the metal speciation in solution2,6,15,16,17,19,20,21,22,23, ii) the biophysiochemical properties of the cell membrane17,24,25,26,27,28,29,30,31, and iii) the ability for the metal to access a transport site7,32. Cd and Hg are unlikely to exists as free ions under microbial physiologically relevant conditions due to their high affinity for Dissolved Organic Matter (DOM), chelating contaminants (e.g., EDTA), or reduced sulfur moieties33,34,35 (CdII can exist as a free ion or form ion-pairs in the absence of these ligands). There is a lack of efficient methods in determining how these metal species are bioavailable under conditions relevant to their fate in the environment. For instance, Hg is methylated under anaerobic conditions14, and both cadmium and Hg are soft metals (or class B cations), requiring that their speciation be investigated under conditions that maintain the integrity of reduced sulfur groups.
Microbial biosensors are bacterial cells that emit a quantifiable signal in response to the intracellular concentrations of a metal, in this case Hg or Cd. As such, they are ideal tools to understand how metals enter a cell36, provided that exposure conditions are carefully controlled for. Hg biosensors typically contain gene fusions between the regulatory circuitry of the mer-operon (including genes encoding for the transcription regulator MerRas well as the operator and promoter regions of the operon), and reporting genes (e.g., lux, gfp, lac genes). When mercury is present in the cytoplasm, it will bind to MerR, resulting in transcription of the reporting genes and subsequent signal production19,37. Specific Cd biosensors are usually designed using the cadC, cadAC, zntA or zntR encoded transcription regulators38, but it is worth noting that MerR has a lower, yet quantifiable affinity to Cd5. Due to aerobic restriction of most commonly used luminescent or fluorescent reporter proteins, until recently microbial biosensors remained unable to offer insights into the biotransformation of metals under anoxic conditions. This makes anaerobic detection of metals bioavailability very difficult over a range of conditions relevant to their environmental fate, specifically in the presence of redox sensitive ligands (e.g., sulfide and thiols)4,5,39.
To alleviate the methodological hurdle of live imaging in the absence of oxygen, Drepper et al. (2007) have developed a flavin-based fluorescent protein (FbFp), based on light oxygen voltage domain of SB2 protein from P. putida. This protein family is able to fluoresce in the absence of oxygen40. Building on the work of Drepper et al., our lab designed an anaerobic biosensor allowing for the study of Hg bioavailability under oxic and anoxic conditions and over a wide range of salinity 17. In the current paper, we describe how to prepare and use the biosensor to test environmental variables&#39; influence on Hg or Cd bioavailability. Although we developed the biosensor for HgII, we chose to perform experiments with CdII as a means to draw the reader&#39;s attention to the fact that biosensors may also respond to multiple stressors that are likely to co-occur in environmental matrices; in this case CdII was investigated because it is known to bind to the transcriptional regulator MerR5. Here, we show representative calibration and functional linear ranges with respect to either metal. We also give an example when the biosensor&#39;s results are conclusive (MgII and MnII on Hg bioavailability) and inconclusive (ZnII on Hg bioavailability).

<table><tbody><tr><td>7 ml standard vial, rounded interior</td><td>Delta Scientific</td><td>200-007-20</td><td>34 recommended</td></tr><tr><td>Vial tray, 21 mm openings</td><td>Delta Scientific</td><td>730-2001</td><td>4 for (4 x 8 grid)</td></tr><tr><td>24 mm Closure</td><td>Delta Scientific</td><td>600-024-01</td><td>32 recommended</td></tr><tr><td>LID CORNER NOTCH BLK STR CS/50</td><td>Corning</td><td>Corning 3931</td><td></td></tr><tr><td>Corning 96- well clear-bottom nonbinding surface microplate</td><td>Corning</td><td>Corning 3651</td><td></td></tr><tr><td>Anaerobic Chamber (glovebox)</td><td></td><td></td><td>The air in the anaerobic chamber should be (97 % N&lt;sub&gt;2 &lt;/sub&gt;&amp;plusmn; 2 % and 3 % H&lt;sub&gt;2&lt;/sub&gt; &amp;plusmn; 2 %)</td></tr><tr><td>Palladium catalyst</td><td></td><td></td><td>Converts O&lt;sub&gt;2 &lt;/sub&gt;to H&lt;sub&gt;2&lt;/sub&gt;O in the anaerobic chamber. Not required but recommended.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td></td></tr><tr><td>450 (&amp;plusmn;10) nM filter for the microplate reader</td><td></td><td></td><td></td></tr><tr><td>500 (&amp;plusmn;10) nM filter for the microplate reader</td><td></td><td></td><td></td></tr><tr><td>Anaerobic culture vial (balch tubes) + rubber stoppers</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>Modified for anaerobic culture tubes</td></tr><tr><td>MA-3000 (mercury analyzer)</td><td></td><td></td><td></td></tr><tr><td>pH probe</td><td></td><td></td><td>Any pH probe will work</td></tr><tr><td>50 mL conical sterile polypropylene centrifuge tubes.</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m polyethersulfone syringe filter + syringe</td><td></td><td></td><td></td></tr><tr><td>Sterile/clean glass bottles</td><td></td><td></td><td>For growth media and standards</td></tr><tr><td>Sterile/clean plastic or PTFE bottles</td><td></td><td></td><td>For alkali solutions (NaOH/KOH)</td></tr><tr><td>Reagents powders: Na2MoO4, Na2SeO4, H3BO3, NaOH, KOH, MnSO4, ZnSO4, CoCl2, NiCl2, ampicillin sodium salt, Difco M9 Minimal Salts, Glucose, MgSO4, Thiamine HCl, NaNO3, l-leucine, l-isoleucine valine, EDTA sodium salt, FeSO4, Sodium Beta-Gylcerophosphate, Mops free acid, (NH4)2SO4, Hg(NO3)2, CdCl2</td><td></td><td></td><td></td></tr><tr><td>Sterile Milli-Q water</td><td></td><td></td><td>Autoclaved or filter sterilized is fine.</td></tr><tr><td>Lysogeny Broth</td><td></td><td></td><td></td></tr><tr><td>Analytical grade H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr></tbody></table>

an anaerobic biosensor assay for the detection of mercury and cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Once the fluorescence peaks have been quantified according to step 5.3, the result of the fluorescence peaks can be graphed according to the variable concentration illustrating how that variable affects the relative bioavailability of either Hg or Cd. For example, the calibration curve of fluorescence over [HgII] from Figure 2 will yield the inducible data presented in Figure 3A. For HgII calibration, the curve will always contain 3 components for the Hg-inducible strain; a threshold response of about 1-2 nM HgII before fluorescence signal production is linearly proportional to [HgII], the linear range where 5 nM HgII will reliably always be in the center of that range, and a plateau where increasing [HgII] will no longer increase fluorescence signal. No change in signal production on the constitutive strain shows that toxicity from [HgII] does not affect signal production. For CdII in Figure 3B, there are always 2 components for the inducible strain; a linear range where 200-300 nM CdII will reliably always be in the center of the linear range and a plateau. A decrease in fluorescence signal with increasing [CdII] shows that higher Cd concentrations are toxic to the cells and can explain a decrease in the inducible fluorescence production after the plateau at 1000 nM CdII. Therefore, when testing Hg or Cd bioavailability with respect to an environmental variable, we suggest using 5 nM for HgII and 300 nM for CdII.
In some instances, signal production can be properly attributed to Hg or Cd bioavailability, but in other cases, signal production can be affected by variation in the physiological state of the biosensor cell host (e.g., the metal of interest or environmental conditions tested are toxic). In Figure 4A, 5 nM Hg bioavailability was tested over a gradient of Zn (0-10 &#181;M). In both Hg-inducible and constitutive strains, there is a similar decrease in signal with increasing Zn concentrations. Therefore, one cannot discriminate whether the signal results from lowered bioavailability or is a result of Zn toxicity. In Figure 4B and 4C, 5nM Hg bioavailability was tested over a gradient of MgII (0-10 mM) and MnII (0-10 &#181;M). Increasing MgII and MnII concentrations decreased the fluorescence signal of the inducible strain. On the other hand, the constitutive strain did not show a decrease in fluorescence with increasing MgII and MnII concentrations (MgII and MnII are beneficial for the cells in the production of the FbFp, as demonstrated by an increase in the fluorescence signal). This demonstrates that the cells are viable and the fluorescence decrease of the Hg-inducible strain results from a decrease in Hg bioavailability. This data emphasizes how important it is for all biosensor assays to also provide constitutive measurements of overall cell fitness.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58324/58324fig3.jpg" /> 
Figure 3: Linear ranges of the biosensor with Mercury and Cadmium. Maximum fluorescence measured as relative fluorescence units (RFU) &#177; 1 Standard Deviation emitted by E. coli NEB5&#945; harboring the pUC57merR-Pp (Hg-Inducible) and pUC19Balch-Pp (Constitutive) with the addition of A) HgII (0-15 nM) and B) CdII (0-1,000 nM) under anaerobic conditions. Fluorescence was the average of 3 technical replicates at 37 &#730;C. <a href="https://www.jove.com/files/ftp_upload/58324/58324fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58324/58324fig4.jpg" /> 
Figure 4: Example of an inconclusive result with Zinc and a conclusive result with Magnesium and Manganese. Maximum fluorescence measured as relative fluorescence units (RFU) &#177; 1 Standard Deviation emitted by E. coli NEB5&#945; harboring the pUC57merR-Pp (Hg-Inducible) and pUC19Balch-Pp (Constitutive) with the addition of A) ZnII (0-10 &#181;M), B) MgII (0-10 mM), and C) MnII (0-10 &#181;M) under anaerobic conditions. [HgII] was set to 5 nM for all treatments and fluorescence was the average of 3 technical replicates at 37 &#730;C. <a href="https://www.jove.com/files/ftp_upload/58324/58324fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium at JoVE.com

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

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Research

JoVE Journal

Environment

10.1K Views.  University of Ottawa. Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Video: An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Engineering

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Chemistry

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Bioengineering

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Biology

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Summary

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An Aptamer-based Sensor for Unchelated Gadolinium(III)

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography