Name: the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions
Uploaded: 2018-02-01T13:00:00
Description: Watch this Scientific Journal Video about The Use of a ÃŽÂ²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

Forfatterne anerkender det vallonske Region i Belgien inden for rammerne af forskningsprojekterne, SENSOTEM og NANOTIC samt den nationale midler For den videnskabelige forskning (F.R.S.-F.N.R.S) for deres finansielle støtte.

1. protein prøveforberedelse
<ol><li>Producere og rense hybrid protein BlaP-cAb-Lys3 som rapporteret i vores tidligere undersøgelse15. Gemme protein i 50 mM fosfat buffer pH 7,4 med følgende sammensætning: 8 g NaCl, 0,2 g af KCl, 1.44 g Na2HPO4 og 0,24 g KH2PO4 opløst i 800 mL destilleret vand lave pH af løsningen på 7,4 før justering af det endelige rumfang af opløsning til 1 sterilisere L. Filter protein løsning.</li>
	<li>Forberede en høn...

<ol>
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I dette arbejde præsenterer vi en metode til at functionalize en nanobody ved hjælp af BlaP β-lactamase som en transportør protein og vi viser, at vi med succes kan gennemføre de resulterende hybrid protein i en potentiometrisk sensor assay. Den vigtigste nyskabelse aspekt af vores arbejde i forhold til andre biosensor assays er den kovalente kobling af antistof del til den enzymatiske aktivitet, der genererer det elektriske signal. Denne såkaldte protein indsættelse teknologi præsenterer fordele og begrænsninge...

Biosensorer er analytiske enheder, der kombinerer en bio-Molekylær interaktion med fysiske eller kemiske signaling enheder kaldes transducere1. Optaget-signaler kan derefter fortolkes og konverteres til at overvåge interaktioner mellem immobiliserede og gratis partnere. De fleste af biosensorer indebærer brug af et antistof til at opdage analysander såsom hormoner eller forskellige patogen markører2. Forskellige sensor-formater kan bruges og omfatter masse-baseret, magnetisk, optisk eller elektrokemiske biosensorer. Sidstnævnte er blandt de mest almindeligt anvendte sensorer, og fungerer ved at konvertere en bindende begivenhed til et elektrisk signal. Forestillinger og følsomhed af alle antistof-baserede biosensorer er stærkt afhængige af hovedsagelig to parametre: i) kvaliteten af antistoffet og ii) systemet bruges til at generere signal2egenskaber.
Antistoffer er høj-molekylære masse dimerisk proteiner (150-160 kDa), der består af to tunge kæder og to lys kæder. Samspillet mellem de lette og tunge kæder er for det meste stabiliseret med hydrofobe interaktioner samt en bevaret disulfid obligation. Hver kæde omfatter en variabel domæne, der interagerer med antigen hovedsagelig via tre hypervariable regioner opkaldt supplerende bestemmelse af regioner (CDR1-2-3). Trods mange fremskridt inden for feltet, de storstilede udtryk for fuld længde antistoffer med low-cost udtrykket systemer (fx E. coli) ofte fører til produktion af ustabile og aggregerede proteiner. Det er derfor forskellige antistof fragmenter er blevet udviklet som single-kæde variabel fragmenter3 (ScFvs ≈ 25 kDa). De består af de variable domæner i henholdsvis en tung og en lys kæder, der er kovalent forbundet af en syntetisk aminosyresekvens. Men disse fragmenter ofte vise en dårlig stabilitet og har tendens til at sammenlægge, eftersom de udsætter en stor del af deres hydrofobe regioner til solvent4. I denne forbindelse synes enkelt kæde camelid antistof fragmenter, kaldet nanobodies eller VHHs, at være gode alternativer til ScFvs. Disse domæner svarer til de variable domæner i camelid single-kæde antistoffer. I modsætning til konventionelle antistoffer, camelid antistoffer er blottet for lys kæder og kun indeholde to tunge kæder5. Derfor, nanobodies er de mindste monomere antistof fragmenter (12 kDa) stand til at binde sig til antigen, en affinitet svarer til konventionelle antistoffer6. Derudover nuværende de forbedret stabilitet og Opløselighed i forhold til andre fuld længde antistoffer eller antistof fragmenter. Endelig, deres små størrelser og deres udvidede CDR3 sløjfer tillade dem at genkende kryptiske epitoper og binde til enzymet aktive steder7,8. I dag er disse domæner får stor opmærksomhed og har været kombineret til biosensor teknologi. For eksempel, Huang mfl. har udviklet en nanobody-baseret biosensor for påvisningen og kvantificeringen af humane prostata-specifikt antigen (PSA)9.
Som nævnt-ovenfor er en vigtig parameter i biosensor assays effektiviteten af systemet bruges til at generere det elektriske signal. Af denne grund, enzym-baserede elektrokemiske biosensorer har tiltrukket stadig større opmærksomhed og har været udbredt til forskellige applikationer såsom sundhedspleje, fødevaresikkerhed og miljøovervågning. Disse biosensorer stole på katalytiske hydrolyse af et underlag af et enzym til at generere det elektriske signal. I denne sammenhæng viste β-lactamaser sig at være mere specifik, mere følsomme og lettere at gennemføre eksperimentelt end mange andre enzymer såsom basisk fosfatase eller peberrodsperoxidase10. Β-lactamaser er enzymer, der er ansvarlig for bakteriel resistens over for β-lactam antibiotika af hydrolyzing dem. De er monomere, meget stabile, effektive, og af ringe størrelse. Derudover generere domæne/peptid indsættelser i β-lactamaser bi-funktionelle kimære proteiner, der viste sig at være effektive redskaber til at undersøge protein-ligand interaktioner. Ja, nyere undersøgelser har vist at indsættelse af antistof variable fragmenter i TEM1 β-lactamase resultater i en kimære protein, der fortsat er stand til at binde med høj affinitet til dens mål antigen. Interessant, blev antigen-bindende vist sig at fremkalde allosteriske regulering af TEM1 katalytiske aktivitet11,12. Endvidere viste vi i flere undersøgelser at protein domæne indsættelse i en eftergivende loop af Bacillus licheniformis BlaP β-lactamase genererer funktionelle kimære proteiner, der er velegnet til at overvåge protein-ligand interaktioner13 ,14. Vi har for nylig indsat en nanobody, opkaldt cAb-Lys3, i denne eftergivende indsættelsesstedet BlaP15. Denne nanobody blev vist til at binde til høne-æggehvide lysozym (HEWL) og til at hæmme dets enzymatiske aktivitet16. Vi viste at det genererede hybrid protein, opkaldt BlaP-cAb-Lys3, bevaret en høj specificitet / affinitet mod HEWL mens β-lactamase aktivitet forblev uændret. Så vi med held kombinerede bastard β-lactamase teknologi til en elektrokemisk biosensor og viste, at mængden af genererede elektrisk signal var afhængig af samspillet mellem BlaP-cAb-Lys3 og HEWL immobiliseret på en elektrode. Faktisk, hydrolyse af β-lactam antibiotika ved BlaP inducerer en proton udgivelse, der kan omdannes til en kvantitativ elektrisk signal. Denne kombination af β-lactamase Hybridteknologien med en elektrokemisk biosensor er hurtig, følsomme, kvantitative, og giver mulighed for real-time måling af den genererede signal. Denne metode er beskrevet heri.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Reagents</td>
			<td style="width:215px;"></td>
			<td style="width:128px;"></td>
			<td style="width:615px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td>Sigma-Aldricht</td>
			<td>V000225&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td>Sigma-Aldricht</td>
			<td>1551128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldricht</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8211;HCl</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldricht</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>NIST2186II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Sigma-Aldricht</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alanine</td>
			<td>Sigma-Aldricht</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HClO4</td>
			<td>Fluka</td>
			<td>34288</td>
			<td>1M HClO4 solution, distributor : Sigma-Aldricht</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">casein hydrolysate</td>
			<td>Sigma-Aldricht</td>
			<td>22090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzylpenicillin sodium</td>
			<td>Sigma-Aldricht</td>
			<td>B0900000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">hen egg white lysozyme</td>
			<td>Roche</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">heptane</td>
			<td>Sigma-Aldricht</td>
			<td>246654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">methanol</td>
			<td>Sigma-Aldricht</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium hydroxide solution</td>
			<td>Sigma-Aldricht</td>
			<td>380539</td>
			<td>28% NH3&#160;in H2O, purified by double-distillation (concentrated?)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laboratory consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657165</td>
			<td>CELLSTAR 6-Well Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>WTW</td>
			<td>1AA110</td>
			<td>Lab pH meter inoLab pH 7110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vacuum and filtration system</td>
			<td>Nalgene</td>
			<td>NALG300-4100</td>
			<td>Filter holders with receiver, distributor : VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">potentiometric sensor chips</td>
			<td></td>
			<td></td>
			<td>manufactured by Yunus and colleagues (ref 16)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGSTAT30 Autolab</td>
			<td>Metrohm Autolab</td>
			<td></td>
			<td>discontinued, succesor Autolab PGSTAT302N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital multimeter, METRAHit 22M</td>
			<td>Gossen Metrawatt</td>
			<td></td>
			<td>discontinued, successor Metrahit Base</td>
		</tr>
	</tbody>
</table>

the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions

Biosensorer bliver stadig vigtigere og mere gennemført i forskellige felter som patogen detektion, Molekylær diagnose, miljøovervågning og mad sikkerhed kontrol. I denne sammenhæng brugte vi β-lactamaser som effektiv reporter enzymer i flere protein-protein interaktion undersøgelser. Derudover opfordrer deres evne til at acceptere indsætninger af peptider eller struktureret proteiner/domæner kraftigt til brug af disse enzymer til at generere kimære proteiner. I en nylig undersøgelse indsat vi et enkelt domæne antistof fragment i Bacillus licheniformis BlaP β-lactamase. Disse små domæner, også kaldet nanobodies, er defineret som de antigen-bindende domæner af enkelt kæde antistoffer fra camelids. Som fælles dobbelt kæde antistoffer viser de høje tilhørsforhold og særlige forhold til deres mål. Den resulterende kimære protein udstillet en høj affinitet mod sit mål samtidig bevare β-lactamase aktivitet. Dette tyder på, at nanobody og β-lactamase fraspaltning forbliver funktionel. I det foreliggende arbejde rapporterer vi en detaljeret protokol, der kombinerer vores hybrid β-lactamase system til biosensor teknologi. Specifikke bindingen af nanobody til sit mål kan blive opdaget takket være en conductimetric måling af protoner frigivet ved den katalytiske aktivitet af enzymet.

Design og konstruktion af den kimære protein BlaP-cAb-Lys3
Figur 1 repræsenterer indsættelse af cAb-Lys3 i en eftergivende loop af BalP klasse A β-lactamase fra Bacillus licheniformis. Indsættelse blev udført mellem rester Asp198 og Lys199. En thrombin kavalergang websted blev indført på hver side af førerhuset-Lys3. Celler forvandlet med en konstitutiv udtryk plasmid kodning BlaP-cAb-Lys3 kimære protein var i st...

Watch this Scientific Journal Video about The Use of a ÃŽÂ²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

The Use of a ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

I dette arbejde rapportere vi en ny metode til at undersøge protein-protein interaktioner ved hjælp af en conductimetric biosensor baseret på β-lactamase Hybridteknologien. Denne metode bygger på frigivelse af protoner ved hydrolyse af β-lactamer.

Brug af en β-lactamase-baseret Conductimetric Biosensor Assay til påvisning Biomolekylær interaktioner

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Research

JoVE Journal

Bioengineering

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Bestemmelse af transport Rate af Xenobiotics og nanomaterialer over placenta ved hjælp af<em&gt; Ex vivo</em&gt; Menneskelige placentaperfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Brug af Label-fri Optiske Biosensors Detect Modulation af kalium kanaler af G-protein koblede receptorer

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Anvendelse af et High-throughput<em&gt; In vitro</em&gt; Mikrofluidsystem at udvikle Oral Multi-arter biofilm

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Brug af en rotte Model til at studere ventrale Abdominal Brok Reparation

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

PIP-on-a-chip: En etiketfri undersøgelse af protein-phosphoinositid-interaktioner

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Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Tør Film Photoresist-baserede elektrokemisk mikrofluid Biosensor Platform: Enhed fabrikation, On-chip Assay forberedelse og drift

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Udvikling af en elektrokemisk DNA Biosensor at opdage en fødevarebårne patogenet

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...