由美国农业部研究员计划和美国农业部作物种质资源委员会部分提供了资助这项研究。

1。从马铃薯块茎淀粉的直链淀粉测定<ol><li>皮切新鲜块茎切成小方块。 </li><li>将马铃薯立方体小的棕色袋子，并在-80过夜存放°C。 </li><li>转移土豆立方体尼龙袋和冷冻干燥。 </li><li>冷冻干燥后，用研钵和杵或Wiley磨磨土豆样品成粉末。 </li><li>在50ml管中，加入20〜30毫克的冻干，地面块茎的样品。 </li><li>制备45％的水溶液（重量/体积）高氯酸（混合24.4毫升的60％高氯酸和25.6毫升的超纯水。 </li><li>加入500μl的45％（W / V）高氯酸和摇晃或转动它来驱散淀粉颗粒。 </li><li>后在室温下4分钟的温育期间，将16毫升的超纯水到溶液中，并通过涡旋混合。 </li><li>后非水溶性物质已结算到管的底部（7-10分钟后），转移40微升的溶液到微量滴定板的（避免任何颗粒的移液）。 </li><li>加入50微升的碘溶液（2克碘化钾+1 g口2 900毫升的超纯水）和移液混合样本。 </li><li>读取吸光度在550nm处，并立即620纳米。 </li><li>每个样本的直链淀粉/支链淀粉比例（620 nm/550 nm的吸光度）与从直链淀粉和支链淀粉的溶液在一定浓度范围内的产生的标准曲线进行比较后确定的直链淀粉百分比。此过程将在下面说明。读出的空白（碘溶液和高氯酸）以及测试样品。使用空白数据的分析。 </li></ol> 2。直链淀粉/支链淀粉曲线<ol><li>在单独的管权衡直链淀粉12.5毫克和12.5毫克支链淀粉。 </li><li>加至各管中加入5ml 45％（重量/体积）的高氯酸和完全溶解。 </li><li>对每一种溶液（直链淀粉和支链淀粉）的，带至50ml用超纯水的最终体积。 </li><li>混合6.25毫升直链淀粉的股票从上一步18.75毫升超纯水。这将使6.25毫克/毫升的直链淀粉的解决方案。 </li><li>重复上述步骤，支链淀粉的解决方案。 </li><li>使用从步骤2.4和2.5中直链淀粉和支链淀粉标准溶液，制备直链淀粉百分比标准，是0-100％直链淀粉（100-0％的支链淀粉），以5毫升，以创建在10％的时间间隔的标准曲线的最终体积。 例如，为了准备: 0％直链淀粉/ 100％支链淀粉标准，吸管5毫升支链淀粉的解决方案。 10％的直链淀粉/ 90％支链淀粉标准，结合0.5毫升直链淀粉和4.5毫升支链淀粉的解决方案。 20％的直链淀粉/ 80％支链淀粉标准，结合1.0毫升直链淀粉和4.0毫升支链淀粉的解决方案。 30％的直链淀粉/ 70％支链淀粉标准，结合1.5毫升直链淀粉和3.5ml支链淀粉的解决方案。 40％的直链淀粉/ 60％支链淀粉标准，结合2.0毫升直链淀粉和2.0ml的支链淀粉的解决方案。 50％的直链淀粉/ 50％支链淀粉标准，结合2.5毫升直链淀粉和2.5ml支链淀粉的解决方案。 60％的直链淀粉/ 40％的支链淀粉标准，结合3.0毫升直链淀粉和2.0ml的支链淀粉，等等）。 </li><li>转移40微升每标准混合物中，在微量滴定板。包括一个井用40微升45％（重量/体积），高氯酸作为空白。 </li><li>加50微升碘溶液（2克KI +1 g口2在900ml的超纯水）和通过移液混合各样品（包括空白）。 </li><li>读取吸光度在550nm处，并立即为620nm，并计算出直链淀粉/支链淀粉比例对于每个浓度。 </li></ol>

<ol>
	<li>Composition Hoover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+structure,+and+physicochemical+properties+of+tuber+and+root+starches:+a+review.">molecular structure, and physicochemical properties of tuber and root starches: a review.</a> Carbohydr. 45 (3), 253-267 (2001).</li><li>Bertoft, E., Blennow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Potato+Starch.">Structure of Potato Starch.</a> Advances in Potato Chemistry and Technology. , 83-98 (2009).</li><li>Campbell, M. R., Yeager, H., Abdubek, N., Pollak, L., Glover, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+amylose+screening+in+maize+starches+from+exotic+backgrounds.">Comparison of methods for amylose screening in maize starches from exotic backgrounds.</a> Cereal Chem. 79, 317-321 (2002).</li><li>Zhu, T., Jackson, D. S., Wehling, R. L., Geera, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+amylose+determination+methods+and+the+development+of+a+dual+wavelength+iodine+binding+technique.">Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique.</a> Cereal Chem. 85, 51-58 (2008).</li><li>Hovenkamp-Hermelink, J., Devries, J., Adamse, P., Jacobsen, E., Witholt, B., Feenstra, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+the+amylose+amylopectin+ratio+in+small+amounts+of+tuber+and+leaf+tissue+of+the+potato.">Rapid estimation of the amylose amylopectin ratio in small amounts of tuber and leaf tissue of the potato.</a> Potato Res. 31, 241-246 (1988).</li><li>Shannon, J. C., Garwood, D. L., Boyer, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Physiology+of+Starch+Development.">Genetics and Physiology of Starch Development.</a> Starch. , 23-82 (2009).</li><li>Haase, N. U., Plate, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+potato+starch+in+relation+to+varieties+and+environmental+factors.">Properties of potato starch in relation to varieties and environmental factors.</a> St&#228;rke. 48 (5), 167-171 (1996).</li><li>Johnston, F. B., Urbas, B., Khanzada, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+storage+on+the+size+distribution+and+amylose/amylopectin+ratio+in+potato+starch+granules.">Effect of storage on the size distribution and amylose/amylopectin ratio in potato starch granules.</a> Am. Pot. J. 45, 315-321 (1968).</li><li>Gibson, T. S., Solah, V. A., McCleary, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+to+measure+amylose+in+cereal+starches+and+flours+with+concanavalin+A.">A procedure to measure amylose in cereal starches and flours with concanavalin A.</a> J. Cereal Sci. 25 (2), 111-119 (1997).</li><li>Sun, S., Matheson, N., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+amylose+content+of+starches+after+precipitation+of+amylopectin+by+concanavalin+A.">Estimation of amylose content of starches after precipitation of amylopectin by concanavalin A.</a> Starch. 42, 302-305 (1990).</li></ol>

我们什么都没有透露。

马铃薯块茎中直链淀粉含量一般为20-35％7,8。如果大部分值超出了这个范围，考虑这些可能的错误:1）原始数据被用来代替空白数据的直链淀粉/支链淀粉标准曲线的建设; 2）原始数据被用来代替空白数据的直链淀粉的测定/支链淀粉比例从马铃薯样品，或3）用于分析水不够纯净。我们注意到，通过使用蒸馏水和不超纯水在分析中，所获得的值分别为不可再现的。一个简单的方法来验证碘液?...

约80％的马铃薯块茎鲜重的是水，几乎所有的剩余的干物质是淀粉1。大多数淀粉（70％）的组成的支链淀粉，而其余部分是直链淀粉。直链淀粉和支链淀粉的比例是影响淀粉的物理性能的最重要的属性。直链淀粉是直链α1-4葡萄糖链，而支链淀粉是直链α1-4链与α1-6分支2。方法如碘结合，差示扫描量热法（DSC），高效体积排阻色谱法（HPSEC）和刀豆蛋白A相互作用已经制定了在不同的作物种类3,4淀粉的决心。每个协议需要特殊的技能和装备，使其难以扩大量化各种样品的时间。我们在这里介绍的方法是从Hovenkamp-Hermelink 等人的 5修饰的协议，它是一种双波长碘结合方法基于染色淀粉粒。该方法的相对其他的优点，包括从生淀粉的直链淀粉测定无需纯化和使用的双波长系统中，以增加该方法4,6的精度。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Reagents</td>
 </tr>
 <tr>
 <td>Amylose </td>
 <td>Sigma</td>
 <td>859656</td>
 <td></td>
 </tr>
 <tr>
 <td>Amylopectin </td>
 <td>Sigma</td>
 <td>A8515</td>
 <td></td>
 </tr>
 <tr>
 <td>Perchloric Acid 60%</td>
 <td>Sigma</td>
 <td>311413</td>
 <td>Very hazardous to skin and eyes</td>
 </tr>
 <tr>
 <td>Iodine</td>
 <td>Sigma</td>
 <td>23214TD</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium iodide</td>
 <td>Sigma</td>
 <td>08625JE</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Plate Reader</td>
 <td>Bio Tek</td>
 <td>ELx800</td>
 <td></td>
 </tr>
 </tbody>
</table>

rapid high throughput amylose determination in freeze dried potato tuber samples

这个协议描述了通过一个依赖于碘和葡萄糖分子中淀粉的链之间的复合物的形成放比色法高。碘形成络合物与两个直链淀粉和支链淀粉中长链。加入碘的淀粉样品后，直链淀粉和支链淀粉的最大吸收出现在620和550nm处分别。直链淀粉/支链淀粉比例可以从620和550 nm处的吸光度值之比来估计并比较它们的标准曲线，其中特定的已知浓度作图的吸收值。这种高通量，廉价的方法可靠，重复性好，让土豆克隆的大量人口的评价。

从不同的直链淀粉/支链淀粉浓度溶液的空白数据构建的标准曲线显示了近似的吸光度比值介于0.7〜1.6（ 表1）。从这些数据的线性回归趋势线以后用于推断冷冻干燥马铃薯样品中直链淀粉含量（ 图1）。 该厂生产环境对在马铃薯块茎7的直链淀粉含量有一定的影响。在两个独立的外勤地点复制直链淀粉含量测定表明马铃薯块茎中低变化（ <strong...

Watch this Scientific Journal Video about Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples at JoVE.com

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

快速高通量直链淀粉测定冻干马铃薯块茎样品

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

rapid-high-throughput-amylose-determination-freeze-dried-potato-tuber

Research

JoVE Journal

Chemistry

13.9K 次观看.  University of Wisconsin - Madison. 这个协议描述了通过一个依赖于碘和葡萄糖分子中淀粉的链之间的复合物的形成放比色法高。碘形成络合物与两个直链淀粉和支链淀粉中长链。加入碘的淀粉样品后，直链淀粉和支链淀粉的最大吸收出现在620和550nm处分别。直链淀粉/支链淀粉比例可以从620和550 nm处的吸光度值之比来估计并比较它们的标准曲线，其中特定的已知浓度作图的吸收值。这种高通量?...

视频: Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

快速比色测定RNA和DNA生物分子样品进行定性区分

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

科研

化学

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞<em&gt;体外</em

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...