We express our gratitude to the children and families of St Louis Children&#39;s Hospital who participated in this study. We acknowledge the unique efforts of Ms. Stacy Postma and Ms. Janet Sokolich during the breath collection. This work is supported by the St. Louis Children&#39;s Hospital Foundation.

The study has been approved by the Institutional Review Board of Washington University School of Medicine (#201709030). Informed consent was obtained from a parent or legal guardian prior to inclusion in the study. Photographs in Figure 2 reproduced with written informed parental consent.
1. Breath sampler assembly
<ol>
	<li>Using disposable gloves, attach a cardboard mouthpiece to the breath sampler, as shown in Supplemental Figure 1. Attach a short length of large diameter tubing to the other extreme of the breath sampler, as shown in Supplemental Figure 1. Use new tubing for each patient.</li>
	<li>Attach the breath connector to the bag valve via the tubing. See Figure 1 for photo of the breath sampler and bag connected.</li>
	<li>Turn the knurled thumbscrew on the side of the inlet fitting counterclockwise to unlock the valve and push the stem of the bag valve down, to open the inlet fitting for sampling.</li>
	<li>Lock the valve open, by turning the knurled thumbscrew on the side of the inlet fitting clockwise.</li>
	<li>Confirm that the blue valve on the breath sampler is open (parallel with the connector).</li>
	<li>Write patient ID, date, and time on the label of the polymer bag.</li>
	<li>Condition TD sorbent tube prior to breath collection using recommended procedures (available from individual manufacturers). Cap and store thermal desorption tubes at 4&#8211;5 &#176;C prior to breath collection to minimize artifacts.</li>
</ol>
2. Breath collection
<ol>
	<li>Perform a demonstration of breath exhalation to the child by using a breath sampler (without a bag). Explain to the child that they should breathe out like they would do when &#8220;blowing up a balloon&#8221; and continue breathing out as far as they can comfortably. Put the cardboard mouthpiece between your lips and exhale as far as you can.</li>
	<li>Provide the child with a new breath sampler attached to a bag and ask them to exhale as in the demonstration, as illustrated in Figure 2.</li>
	<li>Close the blue valve on the breath sampler device as soon as the child has finished breathing out. Reopen the valve as needed prior to additional exhalations.</li>
	<li>Repeat step 2.2 and 2.3 until at least 1 L of breath has been collected. For a healthy child this may take 2 exhalations, and for a sick or younger child 2&#8211;4 exhalations. 1 L of breath is the minimum analytical requirement. Note on the bag label how many breaths were collected from the patient. See Supplemental Figure 2 for a photo of bag containing different volumes of breath.</li>
	<li>Before detaching the bag from the breath sampler, make sure to loosen the knurled thumbscrew on the side of the inlet fitting by turning it anticlockwise and push the stem of the valve up to close the inlet fitting. See Supplemental Figure 3 for photo of the bag valve in the open and closed position.</li>
	<li>Lock the bag valve closed by turning the knurled thumbscrew on the side of the inlet fitting clockwise.</li>
	<li>Detach the bag from the breath sampler.</li>
	<li>Dispose of the mouthpiece and put aside the breath sampler for cleaning prior to use with a different patient.</li>
</ol>
3. Breath transfer to thermal desorption tubes
<ol>
	<li>Remove the TD tube from the refrigerator. Remove the long-term storage caps of the sorbent tube, using the manufacturer-provided tube capping/uncapping tool.</li>
	<li>Attach the grooved end of the TD sorbent tube to the sampling bag using tubing. Note that tube orientation is important, as TD tubes are designed to have air flowing in one direction only, starting from the grooved end. Note that the transfer of breath from bag to TD should be done within 1 hour of breath collection.</li>
	<li>Insert the other end of the TD tube into the tubing, which is connected to a pump.</li>
	<li>Turn the pump on and set to run at 100 mL/min for 10 min.</li>
	<li>Open the valve on the bag by turning anticlockwise the knurled thumbscrew on the side of the inlet fitting and push the stem of the valve down to open the inlet fitting. This is illustrated in Supplemental Figure 4, which demonstrates breath transfer into a TD sorbent tube using a pump.</li>
	<li>Start the pump, which will stop after 10 min of collection.</li>
	<li>Remove the TD sorbent tube and tighten the caps onto both ends using the tube capping/uncapping tool. Long-term storage caps must be firmly tightened in order to ensure a leak-tight seal.</li>
	<li>Place a sticker on the end of one cap to indicate the tube has been used. On the sticker, indicate patient study identification (ID) number and date.</li>
	<li>Place tube in a small re-sealable plastic bag. Store sorbent tube at 4&#8211;5 &#176;C. Press the rest of the breath out of the bag and discard bag. Record patient study ID, TD tube serial number, collection day, time of breath collection, time of breath transfer, and food intake (time of food intake prior to breath collection and meals consumed).</li>
</ol>
4. Ambient air collection
<ol>
	<li>Collect the ambient air samples in patient&#8217;s environment immediately after breath collection.
	<ol>
		<li>Attach the bag to the pump outlet port by using tubing, as illustrated in Supplemental Figure 5.</li>
		<li>Push the stem of the bag valve down to open the inlet fitting for sampling.</li>
		<li>Lock the valve open by turning clockwise the knurled thumbscrew on the side of the inlet fitting.</li>
	</ol>
	</li>
	<li>Turn the pump on and set to run at 100 mL/min for 12 min. The pump will collect 1,200 mL of ambient air.</li>
	<li>After the requested volume has been collected, loosen the knurled thumbscrew on the side of the inlet fitting by turning it anticlockwise and push the stem of the valve up to close the inlet fitting.</li>
	<li>Lock the bag valve closed by turning clockwise the knurled thumbscrew on the side of the inlet fitting.</li>
	<li>Detach the bag from the pump.</li>
	<li>Follow the same steps as in section 3. The only difference is that ambient air VOCs will be transferred, not those from breath.</li>
</ol>
5. Sample and data analysis
NOTE: Conditions for analysis of breath and air samples have been described previously9.
<ol>
	<li>Analyze collected data and detect compounds in the chromatograms. Use typical software programs to find and identify all compounds detected by the instrument (Figure 3A). For example, use a deconvolution feature to identify compounds. Filter data using retention window size factor of 80, mass heights filters &#8805;100 counts, and compound absolute area filter &#8805;500 counts.</li>
	<li>Use chemical standards to identity compounds in breath and air samples. Extract the base ion peak area of compounds of interest, such as isoprene and &#946;-pinene (Figure 4), and compare the levels of volatiles in breath and air.</li>
</ol>

<ol>
	<li>Ahmed, W. M., Lawal, O., Nilsen, T. M., Goodacre, R., Fowler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhaled+volatile+organic+compounds+of+infection:+a+systematic+review.">Exhaled volatile organic compounds of infection: a systematic review.</a> ACS Infectious Diseases. 3 (10), 695-710 (2017).</li><li>Berna, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+breath+specimens+for+biomarkers+of+Plasmodium+falciparum+infection.">Analysis of breath specimens for biomarkers of Plasmodium falciparum infection.</a> Journal of Infectious Diseases. 212 (7), 1120-1128 (2015).</li><li>Lawal, O., Ahmed, W. M., Nijsen, T. M. E., Goodacre, R., Fowler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhaled+breath+analysis:+a+review+of+'breath-taking'+methods+for+off-line+analysis.">Exhaled breath analysis: a review of 'breath-taking' methods for off-line analysis.</a> Metabolomics. 13 (10), (2017).</li><li>Kang, S., Thomas, C. L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+long+may+a+breath+sample+be+stored+for+at-80+degrees+C?+A+study+of+the+stability+of+volatile+organic+compounds+trapped+onto+a+mixed+Tenax:Carbograph+trap+adsorbent+bed+from+exhaled+breath.">How long may a breath sample be stored for at-80 degrees C? A study of the stability of volatile organic compounds trapped onto a mixed Tenax:Carbograph trap adsorbent bed from exhaled breath.</a> Journal of Breath Research. 10 (2), (2016).</li><li>Basanta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+metabolomic+analysis+of+breath+using+differential+mobility+spectrometry+in+patients+with+chronic+obstructive+pulmonary+disease+and+healthy+smokers.">Non-invasive metabolomic analysis of breath using differential mobility spectrometry in patients with chronic obstructive pulmonary disease and healthy smokers.</a> Analyst. 135 (2), 315-320 (2010).</li><li>Mochalski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+breath+levels+of+selected+volatile+organic+compounds+in+healthy+volunteers.">Blood and breath levels of selected volatile organic compounds in healthy volunteers.</a> Analyst. 138 (7), 2134-2145 (2013).</li><li>Mochalski, P., Wzorek, B., Sliwka, I., Amann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+different+polymer+bags+for+storage+of+volatile+sulphur+compounds+relevant+to+breath+analysis.">Suitability of different polymer bags for storage of volatile sulphur compounds relevant to breath analysis.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 877 (3), 189-196 (2009).</li><li>Mochalski, P., King, J., Unterkofler, K., Amann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+selected+volatile+breath+constituents+in+Tedlar,+Kynar+and+Flexfilm+sampling+bags.">Stability of selected volatile breath constituents in Tedlar, Kynar and Flexfilm sampling bags.</a> Analyst. 138 (5), 1405-1418 (2013).</li><li>Schaber, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breathprinting+reveals+malaria-associated+biomarkers+and+mosquito+attractants.">Breathprinting reveals malaria-associated biomarkers and mosquito attractants.</a> Journal of Infectious Diseases. 217 (10), 1553-1560 (2018).</li><li>Herbig, J., Beauchamp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+in+the+analysis+of+breath+gas+volatiles.">Towards standardization in the analysis of breath gas volatiles.</a> Journal of Breath Research. 8 (3), (2014).</li><li>Phillips, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+volatile+organic+compounds+in+the+breath+of+normal+humans.">Variation in volatile organic compounds in the breath of normal humans.</a> Journal of Chromatography B. 729 (1-2), 75-88 (1999).</li><li>Eckel, S. P., Baumbach, J., Hauschild, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+importance+of+statistics+in+breath+analysis-hope+or+curse?.">On the importance of statistics in breath analysis-hope or curse?.</a> Journal of Breath Research. 8 (1), (2014).</li></ol>

The authors have nothing to disclose.

Despite considerable progress in breath research over the last decade, standardized practices for the sampling and analysis of breath gas volatiles remain undefined10. A primary reason for this lack of standardization has been the diversity of breath collection methods, which have direct impact on the resulting chemical diversity present in any given exhaled breath sample. Breath exhalate contains an extensive range of volatile organic compounds at highly varied concentrations6</...

Biomarkers can yield valuable information about normal and pathological biological processes that may contribute to clinically identifiable disease. Recently, there has been increasing interest in the evaluation of breath volatiles as biomarkers for a variety of disease states, including infection, metabolic disorders, and cancer 1. Exhaled breath contains quantifiable levels of volatile organic compounds (VOCs), semi-volatile organic compounds, and microbially derived material (e.g., nucleic acids from bacteria and viruses). The central goal of exhaled breath analysis is to gain insight into the status of a medical condition and/or environmental exposures non-invasively. There are various methods for collecting and analyzing exhaled breath, depending on the constituents of interest. Currently there is no standardized exhaled breath collection method, which complicates comparative analysis of results across studies. Standardizing breath collection procedures is essential, as the sampling procedure itself has a considerable effect on the downstream results of breath analyses.
In many studies, late respiratory breath sampling is employed2,3. This sampling involves discarding the initial portion of exhaled breath (&#34;dead space&#34;), in order to preferentially capture the air at the end of the breath cycle. The advantage of this strategy is that it minimizes the levels of exogenous VOC (e.g., environmental VOCs), while enriching for endogenous, patient-specific VOCs. This method excludes the first few seconds of exhalation from an individual before collecting the breath sample. Other investigators have employed a pressure sensor to activate sampling during a predefined phase of expiration4,5. Because pressure sensors require complex engineering, this alternative method requires a dedicated and relatively costly sampling device.
Pediatric breath sampling can be particularly challenging. A key concern is that young children may be unable to cooperate with protocols for voluntary exhalation of &#34;dead space&#34; air. For this reason, it is easier to obtain mixed respiratory breath from children. However, a major caveat with mixed respiratory breath samples is the risk of environmental and material contamination. Therefore, the feasibility of pediatric collection is a driving concern in the field.
In addition, to collection methods, storage of breath samples can also influence sample quality. The high humidity in breath exhalate and the ultra-low concentrations (parts-per-trillion) of volatile organic breath compounds make breath samples particularly susceptible to problems related to storage6,7. Despite the great potential of real-time techniques like proton transfer reaction-mass spectrometry (PTR-MS), GC-MS remains the gold standard for the analysis of breath samples. Since GC-MS analysis of breath samples is an offline technique, it is coupled with pre-concentration methods such as thermal desorption (TD) tubes, solid phase micro-extraction, and needle trap devices. Prior to pre-concentration, breath samples need to be temporarily stored in polymer bags8. Polymer bags are popular because of their moderate price, relatively good durability, and reusability. While bags may be reused, time and effort are required to ensure efficient cleaning7,8. Each specific bag type also requires empirically determined and standardized procedures for quality control, reusability, and recovery.
TD tubes are widely used for breath pre-concentration because they capture a large number of volatiles and can be customized. The absorbent materials used for packing TD tubes may be adapted to particular applications and particular target volatiles of interest. TD tubes substantially improve the convenience of breath biomarker studies, especially at remote field sites, because TD tubes safely store breath volatiles for at least two weeks and are easy to transport3.
In an effort to standardize pediatric breath collection for biomarker discovery, here we describe a simple method to collect breath from young children. To illustrate the representative results of the implemented protocols, de-identified data are presented from an on-going cohort of children (age 8-17) undergoing evaluation for nonalcoholic fatty acid liver disease (NAFLD). Full results and analysis of this study will be reported in a later publication. In this work, we report a sub-set of data to demonstrate application of our protocol. In brief, children are instructed to exhale normally via mouthpiece into a polymer bag, as if &#34;blowing a balloon.&#34; The process is repeated 2-4 times until 1 L of breath is collected. The sample is then transferred into a TD tube and stored at 5 &#176;C prior to GC-MS analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:1303px;" width="1301">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:243px;">Breath bag&#160;</td>
			<td style="width:171px;">SKC</td>
			<td style="width:119px;">237-03</td>
			<td style="width:771px;">These are 3 L bags</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cardboard mouthpiece&#160;</td>
			<td style="width:171px;">A-M systems</td>
			<td style="width:119px;">161902</td>
			<td>0.86&#34; OD, 2.00&#34; L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Large diameter tubing</td>
			<td style="width:171px;">Cole Parmer</td>
			<td style="width:119px;">95802-11</td>
			<td>Silicone Tubing, 1/4&#34;ID x 5/16&#34;OD,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long-term storage caps&#160;</td>
			<td style="width:171px;">Markes International</td>
			<td>C-CF010</td>
			<td>Brass storage cap &#188;&#34; &#38; PTFE ferrule, pk 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male adapter</td>
			<td style="width:171px;">Charlotte Pipe</td>
			<td style="width:119px;">2109</td>
			<td>Part 1/3 of breath connector (1/2&#34; Universal part No. 436-005)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male adapter (made from Teflon)</td>
			<td style="width:171px;">In-house built</td>
			<td></td>
			<td>Part 3/3 of breath connector (1/4&#34; ID x 1/2&#34; MIP). This part was specially machined from rods made from virgin Teflon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump</td>
			<td style="width:171px;">SKC</td>
			<td>220-1000TC-C</td>
			<td>Pocket PumpTouch with Charger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small diameter tubing&#160;</td>
			<td style="width:171px;">Supelco</td>
			<td style="width:119px;">20533</td>
			<td style="width:771px;">Teflon tubing&#160; L &#215; O.D. &#215; I.D. 25 ft &#215; 1/4 in. (6.35 mm) &#215; 0.228 in. (5.8 mm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal desorption tubes&#160;</td>
			<td style="width:171px;">Markes International</td>
			<td style="width:119px;">C2-CAXX-5314</td>
			<td>Tube, inert, TnxTA/Sulficarb, cond/cap, pk 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube capping/uncapping tool</td>
			<td style="width:171px;">Markes International</td>
			<td>C-CPLOK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two-way ball valve connector&#160;</td>
			<td style="width:171px;">Homewerks Worldwide</td>
			<td style="width:119px;">VBV-P40-E3B</td>
			<td>Part 2/3 of breath connector (1/2&#34;)</td>
		</tr>
	</tbody>
</table>

breath collection from children for disease biomarker discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

In our study, breath samples were collected from 10 children (8-17 years old) undergoing evaluation at St. Louis Children&#39;s Hospital. Breath samples and ambient air samples (n = 10) were collected as described above. Samples were analyzed using gas chromatography quadrupole time-of-flight mass spectrometry (GC-QToF-MS) and thermal desorption, as previously described9. After removal of background contaminants, the implemented protocols yielded an averag...

Watch this Scientific Journal Video about Breath Collection from Children for Disease Biomarker Discovery at JoVE.com

Breath Collection from Children for Disease Biomarker Discovery

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

breath-collection-from-children-for-disease-biomarker-discovery

Research

JoVE Journal

Chemistry

6.8K Views.  Washington University School of Medicine. This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Video: Breath Collection from Children for Disease Biomarker Discovery

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 &#956;M for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced ...