Name: hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins
Uploaded: 2014-08-07T20:45:00
Description: Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Michael Henry, Dublin City University, venligt bistået med indsamling og analyse af data vist i figur 5. Dette arbejde blev delvist støttet af (1) George Mason University, (2) den italienske IstitutoSuperiore di Sanita «inden for rammerne af den italienske / USA samarbejdsaftale mellem amerikanske Department of Health og Human Services, George Mason University, og den italienske sundhedsministerium, (3) NIH, IMAT programmet tilskud 1R21CA137706-01 og 1R33CA173359-01 til LAL, og (4) Ceres Nanovidenskab, Inc .

Humant plasma og urin blev opsamlet fra raske frivillige donorer, med skriftligt informeret samtykke, efter George Mason University Institutional Review Board godkendte protokoller. Donorerne blev ligeligt fordelt mellem hvide mænd og kvinder i alderen mellem 25 og 42. Prøver blev analyseret enkeltvis og blev ikke medtaget. 1. Nanopartikel Forarbejdning af serum eller plasma prøver Potentielle lave rigelige biomarkører i plasma fanget i opløsning med hydrogel ...

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Klinisk relevans En serum eller plasma prøve menes at indeholde lav overflod cirkulerende proteiner og peptider, som kan give en rig kilde til information om tilstanden af ​​organismen som helhed. Trods løftet om serum proteomics, er der tre grundlæggende og alvorlige fysiologiske barrierer modarbejde biomarkør opdagelse og oversættelse til klinisk fordel 10, 11, 16, 25. 1. Vigtige diagnostiske biomarkører kan eksistere i eks...

På trods af gennemførelsen af det menneskelige genom sekventering, er ikke blevet gjort betydelige fremskridt med at indkredse biomarkører prædiktive af sygdommens tidlige stadium, eller at korrelere med terapeutisk udfald, eller prognose 1. En af grundene til denne mangel på fremskridt er, at mange potentielt vigtige biomarkører findes i en koncentration under grænsen for konventionelle massespektrometri og andre biomarkører platforme afsløring. Massespektrometri (MS) og Multiple Reaction overvågning (MRM) har en følsomhed typisk større end 50 ng / ml, mens størstedelen af ​​de analytter måles ved immunoassays i et klinisk laboratorium fald i området mellem 50 pg / ml og 10 ng / ml . Det betyder, at mange biomarkører, især i den tidlige fase af en sygdom, der ikke kan påvises ved konventionel MS og MRM 2. Desuden tilstedeværelsen af ​​høj overflod proteiner, såsom albumin og immunoglobulin i komplekse biologiske væsker ofte maske med milliard-fold excess lav forekomst, lavmolekylære proteiner og peptider 3, 4. Af denne grund er påkrævet med flere prøver forberedende skridt forud for massespektrometri sekventering og identifikation. En sådan forberedende skridt beskæftiger udtømning af høj overflod proteiner med kommercielt tilgængelige udtynding kolonner 5-8. Desværre er dette trin fører til reduktion af udbyttet af kandidat biomarkører fordi de ofte er ikke-covalent forbundet med bærerproteiner, der bliver fjernet. En anden udfordring er repræsenteret ved stabiliteten af kandidat biomarkører ex vivo, når prøverne er indsamlet. Proteiner er udsat for nedbrydning af endogene eller exogene proteaser 9. Hydrogel nanopartikler kan transcendere disse kritiske udfordringer ved at forstærke den formodede biomarkør koncentrationen til et niveau inden for området af analysen samtidig beskytte proteinet mod nedbrydning 10-13. Det er vigtigt at bemærke, thpå LMW proteiner i blodet er en blanding af små intakte proteiner samt fragmenter af store proteiner. Væv-afledte proteiner er større end 60 kDa, er for store til passivt ind i blodet gennem det vaskulære basalmembran, men de kan være repræsenteret i blod som peptider eller proteinfragmenter 14. Vores mål er at måle nye cirkulerende biomarkører, der kan være kandidater til tidlig påvisning af sygdom, patient lagdeling til terapi, og monitorering af respons på behandlingen. Vores nanopartikler er skabt til selektivt at udelukke høj overflod immunoglobuliner og albumin, samtidig med at opfange små proteiner og peptider og koncentrere dem op til 100 gange afhængig af volumen start. Vores gruppe identificeret et sæt af små organiske farvestoffer, der med succes kan virke som høj affinitet molekylære lokkemad for proteiner og peptider. Protein-dye binding menes at skyldes en kombination af hydrofob og elektrostatisk interaktionsek. De aromatiske ringe på farvestoffet interleave med proteiner via hydrofobe lommer på proteinet overflade 11. Lokkemaden, afhængig af deres kemi, udviser en særlig affinitet for udvalgte klasser af analytter. Lokkemaden konkurrere med bærerproteiner, såsom albumin, for proteiner eller peptider. De lavmolekylære proteiner / peptider blive fanget i partiklen. Højmolekylære proteiner, såsom albumin og immunoglobulin forhindres partiklen grund af sigtning kapacitet på grund af den restriktive pore i hydrogelen 11 (figur 1). Hydrogel nanopartikler syntetiseres ved udfældning polymerisation initieret af ammoniumpersulfat 11. N-isopropylacrylamid (NIPAM) er co-monomerer af acrylsyre (AAC) og allylamin (AA) og tværbindingsmiddel N, N&#39;-Methylenebisacrylamide (BIS) lov at reagere ved 70 ° C i 6 timer i fortyndede betingelser 11, 13. Den høje proteinbinding affinitet af poly (N isopropylacrylamid-co-acrylsyre) (poly (NIPAM-CO-AAC) nanoparticlesis opnås ved kovalent at inkorporere aminoholdige farvestoffer (dvs.., Sulfonatedanthraquinonetriazine farvestoffer) i nanopartikler gennem en amideringsreaktion udføres i vandige eller organiske opløsningsmidler, afhængigt af hydrofile / hydrofobe egenskaber for farvestofferne 11, 13. nukleofil substitution af amingrupperne i nanopartikel med chlorid atom af en anthraquinonetriazine farvestof udnyttes til at skabe farvestof-holdige poly (NIPAM co-Allylamin) (AA) nanopartikler 11, 12. En to-trins polymerisation proces udnyttes til at skabe hydrogel nanopartikler indeholdende et yderstof af vinylsulfonsyre (VSA) 11 13. Hydrogel nanopartikler kan anvendes til forskellige biologiske væsker, herunder fuldblod, plasma, serum, cerebrospinalvæske, sved og urin. I et skridt, i opløsning, nanoparticles udføre en hurtig (inden for minutter) beslaglæggelse og koncentration af lavmolekylære analytter 10, 11, 13, 15-18. Proteiner elueres derefter fra nanopartikler og detekteres under anvendelse af western blotting 19-21, massespektrometri 10, 11, 13, 15, 18, ​​22, 23, immunassays / ELISA 10, 11, 15, 18, ​​eller omvendt fase-protein microarray 16 24 assays. Nanopartikler funktionaliseret med kemisk agn, og præsentere en kerne eller kerne shell arkitektur, opsamling og koncentrere proteiner baseret på agn / shell fysisk-kemiske egenskaber. Forskellige farvestoffer inkorporeres i nanopartiklerne vil derfor fange forskellige undersæt af proteiner med varierende effektivitet baseret på farvestoffet affinitet, pH af opløsningen, og tilstedeværelse / fravær af konkurrerende høje rigelige proteiner 13. Endvidere vil mængden af ​​nanopartikler i forhold til mængden af ​​opløsningen påvirker proteinudbytte fra nanopartikler. Disse aspekter afhydrogel nanopartikel høst er påvist ved hjælp af tre forskellige nanopartikler lokkemidler til høst proteiner fra plasma prøver, der indeholder store mængder af protein og fra urinprøver, som typisk ikke indeholder store mængder protein. I denne protokol vi demonstrere høst og koncentrere tumornekrosefaktor-alpha (TNFa) fra plasmaprøver under anvendelse af poly (NIPAM-co-AAC), poly (NIPAM / farvestof) og kerne- shell nanopartikler (poly (NIPAM-co-VSA)) . Poly (NIPAM / farvestof) nanopartikler er vist til at koncentrere Mycobacterium arter antigen, der blev føjet til humane urinprøver, at efterligne Mycobacterium tuberculosis inficerede individer.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

Roman biomarkør opdagelse spiller en afgørende rolle i at yde mere følsom og specifik påvisning sygdom. Desværre er mange med lav hyppighed biomarkører, der findes i biologiske væsker kan ikke let afsløres med massespektrometri eller immunassays, fordi de er til stede i meget lav koncentration, er labil, og er ofte maskeret af høj overflod proteiner, såsom albumin eller immunglobulin. Bait indeholdende poly (N-isopropylacrylamid) (NIPAM) baseret nanopartikler er i stand til at overvinde disse fysiologiske barrierer. I et skridt, de er i stand til at indfange, koncentrere sig og bevare biomarkører fra kropsvæsker. Lavmolekylære analytter indtaste kernen af ​​nanopartikel og opfanges af forskellige organiske kemiske farvestoffer, der fungerer som høj affinitetsprotein lokkemad. De nanopartikler er i stand til at koncentrere proteiner af interesse ved flere størrelsesordener. Denne koncentration faktor er tilstrækkelig til at forøge protein-niveau, således at proteinerne er inden fordetektionsgrænse nuværende massespektrometre, Western blotting og immunoassays. Nanopartikler kan inkuberes med et væld af biologiske væsker, og de er i stand til i høj grad berige koncentrationen af ​​lavmolekylære proteiner og peptider med udelukkelse af albumin og andre højmolekylære proteiner. Vore data viser, at en 10.000 gange amplifikation i koncentrationen af ​​en bestemt analyt kan opnås, så massespektrometri og immunassays til påvisning af hidtil upåviselige biomarkører.

Hydrogel Nanopartikel Størrelse og Ensartethed Poly (NIPAM-AAC) partikler er blevet fremstillet med ekstremt højt udbytte og reproducerbarhed mellem og inden partier. Partiklerne har meget god kolloid stabilitet ved RT i løbet af den tid, der kræves for registrering, lagring, og eluering af proteiner (mindst 48 timer), og nanopartikel nedbør er ikke blevet observeret (figur 1) 11. Den kolloide stabilitet kan være meget vigtigt for hurtig pro...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

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

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.

Hydrogel Nanopartikel Høst af plasma eller urin til påvisning af lav tæthed Proteiner

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

Research

JoVE Journal

Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Dyrkning af humane neurale stamceller i en 3-dimensionel selvsamlende peptid Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

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

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

Design og konstruktion af Artificial ekstracellulære matrix (aECM) Proteiner fra<em&gt; Escherichia coli</em&gt; Til Hud Tissue Engineering

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

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Rod-baserede Fabrikation af tilpasses Soft Robotic pneumatisk griber Devices for Delicate Tissue manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

Målrettet plasmamembran Levering af en hydrofob Cargo indkapslet i en Liquid Crystal Nanopartikel Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

Mønster bioaktive proteiner eller peptider på Hydrogel ved hjælp af fotokemi for biologiske applikationer

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Retroductal nanopartikel injektion til Murine submandibulære kirtel

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Apparatus for Harvesting Tissue Microcolumns

Apparater til høstning væv Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Opdigte en nyre Cortex ekstracellulære Matrix-afledte Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Fotodegradable Hydrogel-grænseflader til bakteriescreening, udvælgelse og isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...