The authors acknowledge the Walloon Region of Belgium within the framework of the SENSOTEM and NANOTIC research projects as well as the National Funds For the Scientific Research (F.R.S.-F.N.R.S) for their financial support.

1. Protein Sample Preparation
<ol>
	<li>Produce and purify the hybrid protein BlaP-cAb-Lys3 as reported in our previous study15. Store the protein in 50 mM phosphate buffer pH 7.4 with the following composition: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4&#160;&#160;and 0.24 g of KH2PO4 dissolved in 800 mL of distilled water Fix the pH of the solution to 7.4 before adjusting the final volume of the solution to 1 L. Filter sterilize the protein solutio...

<ol>
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In this work we present a method to functionalize a nanobody using the BlaP &#946;-lactamase as a carrier protein and we show that we can successfully implement the resulting hybrid protein in a potentiometric sensor assay. The main innovation aspect of our work compared to other biosensor assays is the covalent coupling of the antibody part to the enzymatic activity that generates the electrical signal. This so-called protein insertion technology presents advantages and limitations that will be the main focus of this se...

Biosensors are analytical devices that combine a bio-molecular interaction with physical or chemical signaling devices referred to as transducers1. The recorded signals can then be interpreted and converted to monitor the interactions between the immobilized and free partners. Most of the biosensors involve the use of an antibody to detect analytes such as hormones or different pathogen markers2. Different sensor formats can be used and include mass-based, magnetic, optical or electrochemical biosensors. The latter are among the most commonly used sensors, and function by converting a binding event into an electrical signal. The performances and sensitivities of all antibody-based biosensors are strongly dependent on essentially two parameters: i) the quality of the antibody and ii) the properties of the system used to generate the signal2.
Antibodies are high-molecular mass dimeric proteins (150&#8211;160 kDa) that are composed of two heavy chains and two light chains. The interaction between the light and heavy chains is mostly stabilized by hydrophobic interactions as well as a conserved disulfide bond. Each chain includes a variable domain that interacts with the antigen essentially via three hypervariable regions named Complementary Determining Regions (CDR1-2-3). Despite numerous advances in the field, the large-scale expression of full-length antibodies with low-cost expression systems (e.g., E. coli) often leads to the production of unstable and aggregated proteins. This is why various antibody fragments have been engineered such as single-chain variable fragments3 (ScFvs &#8776; 25 kDa). They consist of the variable domains of respectively one heavy and one light chains that are covalently linked by a synthetic amino acid sequence. However, these fragments often display a poor stability and have the tendency to aggregate, since they expose a large portion of their hydrophobic regions to the solvent4. In this context, single chain camelid antibody fragments, referred to as nanobodies or VHHs, seem to be excellent alternatives to ScFvs. These domains correspond to the variable domains of camelid single-chain antibodies. In contrast to conventional antibodies, camelid antibodies are devoid of light chains and only contain two heavy chains5. Therefore, nanobodies are the smallest monomeric antibody fragments (12 kDa) able to bind to an antigen with an affinity similar to that of conventional antibodies6. In addition, they present improved stability and solubility compared to other full-length antibodies or antibody fragments. Finally, their small sizes and their extended CDR3 loops allow them to recognize cryptic epitopes and bind to enzyme active sites7,8. Nowadays, these domains are receiving considerable attention and have been combined to the biosensor technology. For example, Huang et al. have developed a nanobody-based biosensor for the detection and quantification of human prostate-specific antigen (PSA)9.
As mentioned-above, an important parameter in biosensor assays is the efficiency of the system used to generate the electric signal. For this reason, enzyme-based electrochemical biosensors have attracted ever-increasing attention and have been used widely for various applications such as health care, food safety, and environmental monitoring. These biosensors rely on the catalytic hydrolysis of a substrate by an enzyme to generate the electrical signal. In this context, &#946;-lactamases were shown to be more specific, more sensitive and easier to implement experimentally than many other enzymes such as alkaline phosphatase or horseradish peroxidase10. &#946;-lactamases are enzymes that are responsible for bacterial resistance to &#946;-lactam antibiotics by hydrolyzing them. They are monomeric, very stable, efficient, and of small size. Moreover, domain/peptide insertions into &#946;-lactamases generate bi-functional chimeric proteins that were shown to be efficient tools to study protein-ligand interactions. Indeed, recent studies have shown that insertion of antibody variable fragments into the TEM1 &#946;-lactamase results in a chimeric protein that remains able to bind with high affinity to its target antigen. Interestingly, the antigen binding was shown to induce allosteric regulation of TEM1 catalytic activity11,12. Furthermore, we showed in several studies that protein domain insertion into a permissive loop of the Bacillus licheniformis BlaP &#946;-lactamase generates functional chimeric proteins that are well suited to monitor protein-ligand interactions13,14. We recently inserted a nanobody, named cAb-Lys3, into this permissive insertion site of BlaP15. This nanobody was shown to bind to hen-egg-white lysozyme (HEWL) and to inhibit its enzymatic activity16. We showed that the generated hybrid protein, named BlaP-cAb-Lys3, retained a high specificity / affinity against HEWL while the &#946;-lactamase activity remained unchanged. Then we successfully combined the hybrid &#946;-lactamase technology to an electrochemical biosensor and showed that the amount of generated electric signal was dependent of the interaction between BlaP-cAb-Lys3 and HEWL immobilized on an electrode. Indeed, hydrolysis of &#946;-lactam antibiotics by BlaP induces a proton release that can be converted into a quantitative electric signal. This combination of the hybrid &#946;-lactamase technology with an electrochemical biosensor is fast, sensitive, quantitative, and allows real-time measurement of the generated signal. This methodology is described herein.

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			<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

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

Design and engineering of the chimeric protein BlaP-cAb-Lys3
Figure 1 represents the insertion of cAb-Lys3 into a permissive loop of the BalP class A &#946;-lactamase from Bacillus licheniformis. The insertion was performed between residues Asp198 and Lys199. A thrombin cleavage site was introduced on each side of cAb-Lys3. Cells transformed with a constitutive expression plasmid encoding the BlaP-cAb-Lys3 chimeric protein...

Watch this Scientific Journal Video about The Use of a Î²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

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

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

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

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

The overall goal of this Conductimetric Biosensor Assay is to detect biomolecular interactions, using the hybrid beta-lactamase technology. We describe a method to detect antibody antigen interactions, using a chimeric beta-lactamase protein. The principle of this method relies on detecting binding by measuring the proton release of the beta-lactamase activity.The main advantage of this technique over a conventional immunosensors is the use of a chimeric beta-lactamase, makes cheaper and faster sensory assays by using a hybrid protein to detect the analytes Demonstrating the procedure will be Mathieu Dondelinger, a graduate researcher. Begin this procedure with protein sample and solution preparation as described in the text protocol. Prepare the reusable electrode by dipping the tips into wells of a 96-well plate containing 300 microliters per well of electrode preparation solution for two minutes with gentle mixing at room temperature.Repeat this step three times. Then, rinse the electrodes by dipping the tips into wells of a 96-well plate containing 300 microliters per well of distilled water for two minutes with gentle mixing at room temperature. To regenerate the electrodes, dip the tips into wells of a 96-well plate containing 300 microliters per well of regeneration solution overnight at four degrees Celsius or one hour at room temperature.Then, perform three washes of the electrodes by dipping the tips into wells of 96-well plate containing 300 microliters per well of phosphate-buffered saline. Perform each wash for two minutes with gentle mixing at room temperature. Coat hen-egg-white lysozyme onto the polyaniline surface of the electrode by depositing a 15-microliter drop of 40 micrograms per milliliter of HEWL prepared in phosphate buffer onto the electrode surface.Incubate the electrode one hour at room temperature. Next, perform three washes of the electrodes with phosphate buffer by dipping the electrode parts of the sensor chips into a well of a 96-well plate containing 300 microliters per well of phosphate buffer. Perform each wash for two minutes with gentle mixing at room temperature.Saturate the electrodes by adding a 30-microliter drop of the blocking solution onto the electrode's surface. Incubate for one hour at room temperature. Then, wash three times as before.Dilute the BlaP-cAb-Lys3 or BlaP solutions to 20 micrograms per milliliter in binding buffer. Then, apply a 15-microliter drop of this diluted solution onto the electrodes. Incubate the electrodes for one hour at room temperature.After the antigen nanobody reaction, wash the electrodes three times with washing solution. Then, wash the electrodes of the sensor in detection buffer by dipping the electrodes into a well of a 96-well plate containing 300 microliters per well of detection buffer. And incubate briefly for one minute.For detection, connect the digital multimeter to the computer. Then, plug the sensor chip to the digital multimeter via the copper circuitry part. Initiate the sensor response by applying a 50-microliter drop of four millimolar benzylpenicillin solution on the positive-labeled electrodes.Apply a 50-microliter drop of the detection solution on the negative-labeled electrodes to serve as the negative control. Monitor the conductometric values with the connected digital multimeter for 30 minutes. Visualize the data in realtime directly on the screen, or export the digital data as a text file.These raw data can be plotted in order to create a graphical output that represents the measurements of the conductivity changes between the reference and sample electrodes against time. To construct the chimeric protein, the class A beta-lactamase BlaP was used as the carrier protein. Here, the catalytic residues are presented in red.The insertion site located in a solvent-exposed loop is indicated in yellow. The camelid antibody fragment named cAb-Lys3 was inserted into BlaP. The resulting chimeric protein, BlaP-cAb-Lyse3, is able to bind HEWL while the beta-lactamase activity is retained.HEWL was immobilized onto a polyaniline coded electrode before loading BlaP-cAb-Lys3. The release of protons upon benzylpenicillin hydrolysis by the immobilized chimeric protein induces conductivity changes that can be interpreted by the user. The sensors contain eight chips organized as four pairs.Each chip includes three electrodes, one polyaniline-coated working electrode, one reference electrode, and one counter electrode. For each pair, the chip labeled negative corresponds to a negative control, whereas the chip labeled positive corresponds to the tested sample. Shown here are conductance measurements showing the specific interaction of BlaP-cAb-Lys3 to HEWL.The conductivity changes result from the proton release induced by antibiotic hydrolysis by the immobilized chimeric beta-lactamase. No binding could be detected for the negative control, native beta-lactamase BlaP. Once mastered, this technique can be done in 1/2 a day if it performed properly and the experimental conditions are optimized.Generally, individuals new to this method will struggle because the insertion of an antibody fragment into BlaP is challenging. While attempting this procedure, it is important to remember that the quality of the generated data will depend on the functionality of the chimeric beta-lactamase. Following this procedure, other methods that allow the quantification of the detected interaction can be performed in order to determine the equilibrium association constant of this interaction.After its development, this technique paved the way for researchers in the field of immune biosensor to explore molecular interactions in various samples ranging from living organisms to different environments. The implications of this technique extend toward the detection of numerous biomolecules since the chimeric beta-lactamase technology is versatile tool to functionalize any antibody or a protein fragment in the mains. Therefore, it could be used to develop cheap and fast diagnostic tools in numerous fields, such as human and animals health.

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8.8K vues.  University of Liège. The overall goal of this Conductimetric Biosensor Assay is to detect biomolecular interactions, using the hybrid beta-lactamase technology. We describe a method to detect antibody antigen interactions, using a chimeric beta-lactamase protein. The principle of this method relies on detecting binding by measuring the proton release of the beta-lactamase activity.The main advantage of this technique over a conventional immunosensors is the use of a chimeric beta-lactamase, makes cheaper a...