作者承认为使研究成为可能沙特资助的奖学金（I821）。作者感谢显威刘博士在克兰菲尔德大学的TEM分析专家的支持。

1.油包水乳液的制备<ol><li>称重硬脂酸棕榈酸甘油酯（120毫克），肉豆蔻酸异丙酯（60毫克）中，d-α生育酚聚乙二醇琥珀酸酯（30毫克）和大豆卵磷脂（30毫克），并把它们添加到12.5毫升有机溶剂（6.25毫升丙酮和6.25毫升乙醇）。在该温度70℃（5℃固体脂质的熔点以上）的水浴迅速溶解该混合物。 </li><li>添加任一125，250或375毫克泊洛沙姆188的在12.5 DDH 2 O的毫升实现泊洛沙姆188溶液，这是受在与上述相同的温度下加热（分别）1-3％。 </li><li>从步骤1.2至来自步骤1.1的溶液的油相溶液中添加水相溶液在400rpm以形成磁力搅拌下乳剂。搅拌该乳液以400rpm再进行4小时，使有机溶剂蒸发。 </li></ol> 2.固化和同质化<ol><li>离开ŧ他乳剂在冷室（4℃）2小时，以固化/结晶。 </li><li>为了获得NLC，若使乳液纯粹的高分散（HSD）用一均在10,000-15,000转10-40分钟。 </li></ol> 3. NLC制备优化<ol><li>采取从步骤2.2样品在10000 15000和20000转的速度1,2和3％经受HSD的表面活性剂浓度，分别为10，20，30和40分钟的时间间隔，分别</li><li>检查粒度（PS），聚分散指数（PDI），形态和超微结构20的样品。 注:产生具有最小尺寸（155纳米）和PDI（0.2）值的颗粒被确定为最佳参数。 </li></ol> 4.准备DAC加载NLC的（NLC-DAC） <ol><li>如在水浴将混合物在70℃溶解之前加入DAC（70毫克）的在步骤1.1中所述制备的油相溶液。 </li><li> P如在步骤1.2和1％的表面活性剂描述repare水相溶液，并加入该溶液至该准备在步骤4.1中滴加在400rpm以形成磁力搅拌下的乳液。搅拌另外4小时，该乳液以蒸发有机溶剂。 </li><li>离开该乳液在冷室（4℃）2小时，以固化/如在步骤2中所述结晶，最后，若使乳液使用在步骤3中确定的最佳参数高剪切分散（HSD）。 </li></ol>

<ol>
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作者什么都没有透露。

基于脂质的纳米颗粒已被用于递送疏水性药物提供高度亲脂性载体。一个NLC是第二代固体脂质纳米结构载体，其在室温和体温固体。固体脂质掺入在NLC结果的液体脂质中一个不太完美结晶，从而增加了药物装载效率和也减少储存期间包封药物的驱逐。 为NLC合成中，最常用的方法包括油包水乳化，均质和固化/结晶21,22。均质化可以联络委员会到在水相中完全分散，同时...

达卡巴嗪（DAC）是施加通过核酸甲基化或直接DNA损伤的抗肿瘤活性，从 ​​而导致细胞周期停滞和细胞死亡1烷化剂。 作为第一线化疗剂，DAC已单独或与其它化疗药物用于治疗各种癌症2-6组合使用。它是用于治疗皮肤和转移性黑色素瘤，这是皮肤癌3,7,8的最积极的形式迄今使用的最活性剂。的响应速度，但是，只有20％在最好的，并且治疗效果往往伴随着严重的全身性副作用。 在其天然形式中，DAC是亲水性的并且是不稳定的，由于其光敏性9。临床使用的唯一可用的式目前是在悬浮液用于静脉内输注7,8所用的无菌粉末。低响应速度和高的全身毒性ř吃的药物，主要是由于其水溶性较差，因此低可用性目标在现场，和高配的非靶部位，从而限制了药物10的最大剂量。静脉入院耐药性的发展一起后快速降解和代谢限制临床应用和药物11的治疗效果。因此，迫切需要开发替代DAC制剂用于治疗恶性黑色素瘤。 含有脂质体的胶体体系，胶束或纳米颗粒已经集中研究了其在药物递送中使用如由Marilene 等人 12纳米粒子作为潜在药物载体已被吸引在过去十年中越来越多的关注，因为它们增加载药量的能力审查效率，控制药物释放，提高药物的药代动力学和生物分布，并由此计算的r-得出药全身毒性13。只有少数nanoformulations然而，迄今已对DAC递送研究，表示从相片变性药，增加药物溶解度的保护，以及改进的治疗效果10,14,15。然而，这些制剂有些还使用的是不符合成本效益的合成聚合物的纳米颗粒封装低效率受到影响。 纳米结构的脂质载体（NLC）由固体和液体脂质的混合物，已经开发了用于药物递送16,17。待包封的药物通常可溶于所述液体脂质和固体脂质相18，导致高的负荷和控制释放19。本研究旨在开发基于使用棕榈酸甘油酯和肉豆蔻酸异丙酯脂类NLC封装一个新的DAC配方。制备涉及油包水乳液，蒸发，凝固，和homogenizatioñ。该制剂已被表征为NLC尺寸，形状，超微结构，和分散度，药物包封率和药物负载20。

<table border="0" cellpadding="0" cellspacing="0" width="968">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dacarbazine (DAC)</td>
			<td style="width:467px;">Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">D2390-100MG</td>
			<td style="width:167px;">drug used for uploading</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glyceryl palmitostearate&#160;</td>
			<td>Gattefoss&#233; (Saint_Priest_c&#233;dex, France)</td>
			<td style="width:119px;">85251-77-0</td>
			<td>solid lipid&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">d-&#945;- Tocopherol polyethylene glycol succinate (TPGS)</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">57668</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poloxamer 188</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">15759-1KG</td>
			<td>liqiud phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">650501-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">459836-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soybean lecithin (SL)</td>
			<td>Cuisine Innovation (Dijon, France)</td>
			<td style="width:119px;">SLL1402</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Double-distilled water was collected in our laboratory from</td>
			<td>Millipore-Q Gradient A10 ultra-pure water system (Millipore, France)</td>
			<td style="width:119px;">SAS - 67120&#160;</td>
			<td>aqueous phase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T 25 digital ULTRA-TURRAX</td>
			<td style="width:467px;">IKA</td>
			<td style="width:119px;">3725000</td>
			<td>as high shear disperser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hotplate Magnetic Stirrer</td>
			<td>Scientific Support, Inc</td>
			<td style="width:119px;">1454&#160;</td>
			<td>emulsion homogenization</td>
		</tr>
	</tbody>
</table>

encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier

在临床使用达卡巴嗪（DAC）的唯一配方是静脉内输注，呈现较差治疗特性由于在水溶液中药物的低分散度。为了克服这个，包括棕榈酸甘油酯和异丙基肉豆蔻酸酯的纳米结构的脂质载体（NLC）的开发，以封装DAC。具有受控尺寸联络委员会采用以下油包水乳状液的凝固的高剪切分散（HSD）来实现的。合成参数，包括表面活性剂浓度，速度和HSD的时间进行了优化，以实现与尺寸，多分散指数为155±10nm的ζ电位最小NLC，0.2±0.01，和-43.4±2毫伏，分别。最佳工艺参数也用于DAC加载NLC的准备。装有DAC所得NLC分别具有尺寸，多分散指数为190±10nm的ζ电位，0.2±0.01，和-43.5±1.2毫伏。该药物包封EFFICIENCY和载药量分别达到98％和14％。这是使用NLC的DAC封装的第一份报告，这意味着NLC可能是一个新的潜在的候选者作为药物载体，以提高DAC的治疗特性。

国家图书馆和国家图书馆-DAC采用棕榈酸甘油酯和肉豆蔻酸异丙酯使用不同的参数的筹备工作特点是PS，PDI，形态和超微结构20。各国联络委员会的PS和PDI分别为表面活性剂浓度，HSD速度和持续时间有关。作为判断PS和联络委员会的PDI，最好的结果是用表面活性剂的1％和15,000rpm的30分钟（ 图1A，B和C）一种纯粹的分散速度，因此这...

Watch this Scientific Journal Video about Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier at JoVE.com

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

对于纳米结构的脂质载体（NLC）的合成中最常用的方法包括油包水乳化，均质和凝固。这是由凝固后施加高剪切分散，实现与希望的尺寸，提高了药物包封和药物负载效率为达卡巴嗪传递一个潜在的载体NLC在这里进行修改。

使用纳米脂质载体癌症治疗剂，达卡巴嗪封装

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

encapsulation-cancer-therapeutic-agent-dacarbazine-using

Research

JoVE Journal

Chemistry

11.0K 次观看.  Cranfield University. 对于纳米结构的脂质载体（NLC）的合成中最常用的方法包括油包水乳化，均质和凝固。这是由凝固后施加高剪切分散，实现与希望的尺寸，提高了药物包封和药物负载效率为达卡巴嗪传递一个潜在的载体NLC在这里进行修改。 

视频: Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

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 ...

科研

化学

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

比色测定的具体措施颗粒酶B蛋白水解活动:水解的Boc-丙氨酸 - 丙氨酸-ASP-S-BZL的

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

内部自组装脂质颗粒的简易制备碳纳米管稳定

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

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.

核磁共振作为鱼油补充剂的血脂的快速评价一个强大的工具

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 ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

从分子到材料: 通过卤素键合工程新的离子液体晶体

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

有机结构定向无代理合成，用于_BEA型沸石膜

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...