وقد تم تمويل هذا العمل من جانب التحالف من قبل جائزة مركز NanoHealth (W81XWH-11-2-0168) وتكساس مركز للطب النانوي السرطان (1U54CA151668-01).

<ol>
	<li>Wulfkuhle, J. D., Liotta, L. A., Petricoin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+applications+for+the+early+detection+of+cancer.">Proteomic applications for the early detection of cancer.</a> Nat. Rev. Cancer. 3, 267-275 (2003).</li><li>Etzioni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+case+for+early+detection.">The case for early detection.</a> Nat. Rev. Cancer. 3, 243-252 (2003).</li><li>Hanash, S. M., Pitteri, S. J., Faca, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+plasma+proteome+for+cancer+biomarkers.">Mining the plasma proteome for cancer biomarkers.</a> Nature. 452, 571-579 (2008).</li><li>Liotta, L. A., Ferrari, M., Petricoin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+proteomics:+written+in+blood.">Clinical proteomics: written in blood.</a> Nature. 425, 905-905 (2003).</li><li>Petricoin, E. F., Belluco, C., Araujo, R. P., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+peptidome:+a+higher+dimension+of+information+content+for+cancer+biomarker+discovery.">The blood peptidome: a higher dimension of information content for cancer biomarker discovery.</a> Nat. Rev. Cancer. 6, 961-967 (2006).</li><li>Anderson, N. L., Anderson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+plasma+proteome:+history,+character,+and+diagnostic+prospects.">The human plasma proteome: history, character, and diagnostic prospects.</a> Mol. Cell Proteomics. 1, 845-867 (2002).</li><li>Gorg, A., Weiss, W., Dunn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+two-dimensional+electrophoresis+technology+for+proteomics.">Current two-dimensional electrophoresis technology for proteomics.</a> Proteomics. 4, 3665-3685 (2004).</li><li>Liu, H., Sadygov, R. G., Yates, J. R., 3rd, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+random+sampling+and+estimation+of+relative+protein+abundance+in+shotgun+proteomics.">A model for random sampling and estimation of relative protein abundance in shotgun proteomics.</a> Anal. Chem. 76, 4193-4201 (2004).</li><li>Rabilloud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+gel+electrophoresis+in+proteomics:+old,+old+fashioned,+but+it+still+climbs+up+the+mountains.">Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains.</a> Proteomics. 2, 3-10 (2002).</li><li>Hu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+of+the+nanotexture+of+mesoporous+silica+films+and+their+functionalized+derivatives+for+selectively+harvesting+low+molecular+weight+protein.">Tailoring of the nanotexture of mesoporous silica films and their functionalized derivatives for selectively harvesting low molecular weight protein.</a> ACS Nano. 4, 439-451 (2010).</li><li>Bouamrani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoporous+silica+chips+for+selective+enrichment+and+stabilization+of+low+molecular+weight+proteome.">Mesoporous silica chips for selective enrichment and stabilization of low molecular weight proteome.</a> Proteomics. 10, 496-505 (2010).</li></ol>

ليس لدينا ما يكشف.

أدلة متزايدة على أن انخفاض الوزن الجزيئي منطقة بروتيوم الدورة الدموية هو مصدر غني من المؤشرات الحيوية التشخيصية للكشف المبكر عن المرض. في هذه التكنولوجيا، وقدمنا ​​سلسلة من رقائق السيليكا mesoporous مع مختلف أحجام المسام، والهياكل المسام والتعديلات لتخصيب انتقائية الببتيدات والبروتينات منخفضة الوزن الجزيئي. لتقييم استقرار البروتين، وحضنت رقائق MPS مع مصل الدم البشري، تجفف بعد غسلها، وتخزينها ل3wk في درجة حرارة الغرفة. وكانت أنماط بروتين / الببتيد التي تم الحصول عليها مقارنة مع تلك من المصل مجزأة طازجة (الشكل 6A)، وهو ما أكدته نتائج التحليل الإحصائي وأظهرت 6B في الشكل. ويقدر تباين إشارات ذروة تقاس السيرة الذاتية في المتوسط ​​12.7٪ لمصل الخام وعلى 14.2٪ للعينات مجزأة. يمكن للاختلافات هامشية يكون نتيجة لتقلبات داخلية وثيقة MALDI واقترح أن سولم N-رقاقة المعالجة والتخزين لا تحدث أي تغيير كبير في ملامح البروتين MS. وقد أجريت نفس التجربة على السيليكون غير مسامية. كان المصل المجفف عافيتها بعد التخزين على سطح السيليكون مجزأة على رقائق MPS قبل تحليل MALDI. حصلت على الملف MS الفقراء أظهرت ميزة استقرار سطح mesoporous. في المقارنة مع آليات افترض سابقا، ونحن نفترض أنه تم الحفاظ على الأنواع LMW المحاصرين داخل nanopores من التدهور من خلال استبعاد حجم البروتياز، أو عن طريق تثبيط نشاط بروتين من الفراغية بها في مكان ضيق من nanopores. يمكن للطريقة القائمة على رقاقة MPS أن تكون أداة قوية في الببتيد وLMW التنميط بروتين معقد من الدراسة السوائل البيولوجية. رقائق MPS هي غير مكلفة لتصنيع والسماح للإنتاج الارتقاء بها لتحقيق المعالجة في وقت واحد من عدد كبير من العينات، وتوفير ميزات مفيدة للفحص استكشافي والبيولوجيةاكتشاف علامة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> Tetraethoxysilane </td><td> سيغما الدريخ </td><td> 131903 </td><td> 98٪ </td></tr><tr><td> تدور المغطي </td><td> البيرة العلم </td><td> أوروبا الوسطى والشرقية 200X </td><td></td></tr><tr><td> البلازما آشر </td><td> كما يشار إلى مارس </td><td> AP-600 </td><td></td></tr><tr><td> الطيفية Ellipsometer </td><td> JA Woollam شركة </td><td> M-2000DI </td><td></td></tr><tr><td> MALDI-TOF </td><td> النظم البيولوجية التطبيقية </td><td> يسترسل-DE-STR </td><td></td></tr><tr><td> α-cyano-4-hydroxycinnamic حامض </td><td> سيغما الدريخ شركة </td><td> C8982 </td><td> مصفوفة لMALDI-TOF </td></tr><tr><td> عبر ،5-3-4-dimethoxy هيدروكسيcinnamic حامض </td><td> سيغما الدريخ شركة </td><td> 85429 </td><td> مصفوفة لMALDI-TOF </td></tr><tr><td> تحريك صفيحة ساخنة </td><td> الحرارية العلمية </td><td> 11-475-30Q </td><td></td></tr><tr><td> CultureWell حجرات ساترة </td><td> سيغما الدريخ </td><td> GBL103350 </td><td> 3 مم بقطر. × عمق 1 مم، 3-10 ميكرولتر، عقيم </td></tr><tr><td> Pluronic F 127 </td><td> باسف </td><td></td><td> PEO 106 PPO 70-PEO 106 </td></tr><tr><td> Pluronic L121 </td><td> باسف </td><td></td><td> PEO 5-70-PPO PEO 5 </td></tr><tr><td> Pluronic P123 </td><td> باسف </td><td></td><td> PEO 20-70-PPO PEO 20 </td></tr></tbody></table> الجدول رقم 1. الخصائص الفيزيائية الكيميائية وتركيزات تصميم (الوزن الجزيئي وايزووالكهرباء نقطة) من الببتيد ومعايير اختيار بروتين.

low molecular weight protein enrichment on mesoporous silica thin films for biomarker discovery

تحديد المؤشرات الحيوية تعميم يحمل إمكانات كبيرة للنهج غير الغازية في التشخيص المبكر والتشخيص، وكذلك لرصد كفاءة العلاج. 1-3 وتداول منخفض بروتيوم الوزن الجزيئي (LMWP) يتكون من بروتينات صغيرة تسلط من الأنسجة والخلايا أو ارتبط شظايا الببتيد المستمدة من تدهور بروتين أكبر من البروتينات، ويعانون من هذه الحالة المرضية لدى المرضى، ويعكس على الأرجح في حالة المرض. 4،5 وعلى الرغم من هذه التطبيقات السريرية المحتملة، واستخدام الطيف الكتلي (MS) لمحة عن LMWP من السوائل البيولوجية وقد ثبت أن تكون صعبة للغاية نظرا لمدى ديناميكية كبيرة من تركيزات البروتين والببتيد في مصل الدم. 6 بدون عينة قبل المعالجة، وبعض من البروتينات أكثر وفرة عالية تحجب كشف منخفضة وفرة الأنواع في المصل / بلازما. البروتين الحالي القائم على النهج، مثل ثنائي الأبعاد بولكرلميد هلام شرمectrophoresis (2D-PAGE) والبروتيوميات بندقية الأساليب كثيفة العمالة، والإنتاجية المنخفضة وملاءمة العرض محدود بالنسبة إلى التطبيقات السريرية. 7-9 وبالتالي، هناك حاجة إلى استراتيجية أكثر فعالية لعزل LMWP من الدم، والسماح للفحص إنتاجية عالية من العينات السريرية . هنا، نقدم سريعة وفعالة وموثوق بها متعددة تجزئة النظام القائم على رقائق السيليكا mesoporous خصيصا لاستهداف وإثراء LMWP. ملفقة 10،11 Mesoporous السيليكا (MPS) الأغشية الرقيقة مع ميزات الانضباطي في النانومترية الحجم باستخدام القالب كوبوليمر مسار triblock . باستخدام القوالب البوليمر المختلفة وتركيزات البوليمر في حل السلائف، تم تحديد مختلف توزيعات حجم المسام، والهياكل المسام، والاتصال والخصائص السطحية وتطبيق انتقائي للانتعاش من البروتينات كتلة منخفضة. إعراب الانتقائي للالببتيدات المخصب إلى أقسام فرعية مختلفة وفقا لخصائصها الفيزيائية والكيميائية سوف ENهانس كفاءة استرداد والكشف عن وفرة الأنواع منخفضة. بالاشتراك مع مطياف الكتلة والتحليل الإحصائي، أثبتنا العلاقة بين الخصائص nanophase من الأفلام السيليكا mesoporous رقيقة وخصوصية وفعالية من انخفاض الكتلة الحصاد بروتيوم. عرض نتائج الكشف عن هذه الوثيقة من إمكانات تكنولوجيا النانو القائم على توفير بديل قوي على الأساليب التقليدية للحصاد LMWP من السوائل البيولوجية المعقدة. وبسبب القدرة على ضبط خصائص المواد، والقدرة على الإنتاج منخفضة التكلفة، وبساطة وسرعة جمع العينات، ومتطلبات عينة تقلص إلى حد كبير للتحليل، وهذا سوف يؤثر بشكل كبير رواية تكنولوجيا النانو في مجال البحث العلامات البيولوجية البروتين والبروتين السريرية التقييم.

Watch this Scientific Journal Video about Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery at JoVE.com

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

قمنا بتطوير تكنولوجيا تقوم على فيلم السيليكا mesoporous رقيقة لاستعادة انتقائي من البروتينات منخفضة الوزن الجزيئي والببتيدات من مصل الدم البشري. تم ضبطها بدقة الخصائص الفيزيائية والكيميائية للرقائق لدينا mesoporous لتوفير التحكم ملموسا في مجال تخصيب الببتيد وبالتالي لمحة بروتيوم المصل لأغراض التشخيص.

بروتين منخفض الوزن الجزيئي تخصيب اليورانيوم على أفلام السيليكا Mesoporous رقيقة لاكتشاف العلامات البيولوجية

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

low-molecular-weight-protein-enrichment-on-mesoporous-silica-thin

Research

JoVE Journal

Bioengineering

13.3K Views.  The Methodist Hospital Research Institute. قمنا بتطوير تكنولوجيا تقوم على فيلم السيليكا mesoporous رقيقة لاستعادة انتقائي من البروتينات منخفضة الوزن الجزيئي والببتيدات من مصل الدم البشري. تم ضبطها بدقة الخصائص الفيزيائية والكيميائية للرقائق لدينا mesoporous لتوفير التحكم ملموسا في مجال تخصيب الببتيد وبالتالي ?...

Video: Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

مخصص الصينية النامية الهامستر بروتين الخلية المبيض في استضافة فحوصات باستخدام التكنولوجيا الصوتية Microparticle الغشائية

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

منخفضة التكلفة المرحلة الميكروسكوب البرد الخفيفة التصنيع الخفيف مربوط / مجهرية إلكترون

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

التوليف ، والجمعية ، وتوصيف أحادي الطبقة المحمية الجسيمات النانوية الذهبية للأفلام كهربية البروتين أحادي الطبقة

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

[ميكروأرس] البوليمر لاكتشاف إنتاجية عالية من المواد الحيوية

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

كشف البصري<em&gt; E. القولونية</em&gt; البكتيريا عن طريق أجهزة الاستشعار السيليكون Mesoporous

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

هيدروجيل الجسيمات النانوية حصاد البلازما أو البول للكشف عن وفرة البروتينات منخفضة

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

من السريع إلى التصوير الإسفار الجزيئية قانون إنتشار على أغشية الخلايا الحية في المجهر التجاري

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

تصميم وبناء الاصطناعي خارج الخلية مصفوفة (aECM) والبروتينات من<em&gt; الإشريكية القولونية</em&gt; للبشرة هندسة الأنسجة

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

ميكروفلويديك Genipin ترسب تقنية للثقافة الموسعة من Micropatterned الأوعية الدموية العضلات الرقيقة

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...