Name: &beta;-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions
Uploaded: 2023-05-31T11:07:02
Description: Source:&#160; Vandevenne, M., et al. The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions. J. Vis. Exp. ...

1. Protein Sample Preparation
<ol>
	<li>Produce and purify the hybrid protein BlaP-cAb-Lys3 as reported in our previous study. 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; 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 solution.</li>
	<li>Prepare a hen egg white lysozyme (HEWL) stock solution. Dissolve 100 mg (40,000 units/mg) of commercially purchased HEWL in 10 mL of phosphate buffer saline (PBS see step 2.1.1). Sterilize the protein solution by filtration using filters with a 0.22 &#956;m cutoff.</li>
</ol>
2. Biosensor Assays
<ol>
	<li>Solution and buffer preparation
	<ol>
		<li>Prepare 50 mM PBS by dissolving 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of distilled water. Adjust to pH 7.4 with 1 N HCl or 1 N NaOH before adjusting the volume of the solution to 1 L. Filter-sterilize and store at 4 &#176;C.</li>
		<li>Prepare a saturation/blocking solution by dissolving 3 g of casein hydrolysate in 100 mL of PBS prepared as described above (see step 2.1.1). Filter sterilize and store at 4 &#176;C.</li>
		<li>Prepare a binding solution by dissolving 1 g of casein hydrolysate in 100 mL of PBS prepared as described above (see step 2.1.1). Filter sterilize and store at 4 &#176;C.</li>
		<li>Prepare a washing solution (0.1% Tween - PBS) by adding 100 &#956;L of Tween 20 (100%) in 100 mL of PBS prepared as described above (see step 2.1.1). Store at 4 &#176;C.</li>
		<li>Prepare an electrode preparation solution (1% Triton X-100 - PBS) by adding 1 mL of Triton X-100 (100%) in 100 mL of PBS prepared as described above (see step 2.1.1). Store at 4 &#176;C.</li>
		<li>Prepare an electrode regeneration solution (3.5 M KCl) by dissolving 26 g of KCl in distilled water to a final volume of 100 mL. Filter sterilized and store at 4 &#176;C.</li>
		<li>Prepare a 5 mM NaCl solution by dissolving 0.29 g of NaCl in distilled water to a final volume of 1 L. Filter sterilize and store at 4 &#176;C. Then, prepare a detection solution (4 mM benzylpenicillin) by dissolving 26.7 mg of benzylpenicillin in 20 mL of 5 mM NaCl solution. Filter sterilize and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Sensor preparation and regeneration 
	Note: The polyaniline-coated sensor chips were developed and kindly provided by Dr. P. Bogaerts, Dr. S. Yunus, and Prof. Y. Glupczynski&#8217;s (Catholic University of Louvain-la-Neuve - CHU Mont-Godinne). The description of the sensor as well as the polyaniline electro-polymerization protocols used to synthesize these sensors are detailed in their previous work. Briefly, this system uses reusable sensors of eight individual chips that were manufactured by classical printed circuit board (PCB) techniques. Individual chips are composed of three electrode round spots. The top one is the working electrode on which polyaniline was electro-synthesized. The middle one is the reference electrode and the bottom electrode constitutes the counter electrode. Both, the reference and the counter electrodes are functionalized using solid Ag/AgCl amalgam on top of the carbon layer.
	<ol>
		<li>Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 &#181;L/well of electrode preparation solution (1% Triton X-100 - PBS, see step 2.1.5.). Perform each wash for 2 min with gentle mixing at room temperature.</li>
		<li>Rinse the electrodes by dipping the tips into wells of a 96-well plate containing 300 &#181;L/well of distilled water for 2 min with gentle mixing at room temperature.</li>
		<li>Regenerate the electrodes by dipping the tips into wells of a 96-well plate containing 300 &#181;L/well of regeneration solution (3.5 M KCl, see step 2.1.6) overnight at 4 &#176;C or 1 h at room temperature.</li>
		<li>Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 &#181;L/well of phosphate buffer saline (see step 2.1.1.). Perform each wash for 2 minutes with gentle mixing at room temperature.</li>
	</ol>
	</li>
	<li>Binding assay performed on the sensor
	<ol>
		<li>Coat HEWL onto the PANI (polyaniline) surface of the electrode by depositing a 15 &#181;L drop of 40 &#181;g/mL HEWL prepared in PBS onto the electrode surface. Incubate overnight at 4 &#176;C or 1 hour at room temperature.</li>
		<li>Perform three washes of the electrodes with phosphate buffer saline (see step 2.1.1) by dipping the electrode parts of the sensor chips into wells of a 96-well plate containing 300 &#181;L/well of phosphate buffer saline. Perform each wash for 2 min with gentle mixing at room temperature.</li>
		<li>Saturate the electrodes by adding a 50 &#181;L drop of the blocking solution (see step 2.1.2) onto the electrode surface. Incubate for 1 h at room temperature. Then wash three times as described in the previous step (see step 2.3.2).</li>
		<li>Dilute the BlaP-cAb-Lys3 solution to 20 &#181;g/mL in binding solution (see step 2.1.3) and apply a 15 &#181;L drop of this diluted solution onto the electrodes. Incubate for 10 min at room temperature. After the antigen-nanobody reaction, wash three times as described in the previous step using the wash solution (see step 2.1.4). Then rinse the electrode once with PBS (see step 2.1.1).</li>
		<li>For detection, plug the sensor chip via the copper-circuitry part into a digital multimeter. Then, initiate the sensor response by applying a 50 &#181;L drop of detection solution (see step 2.1.7) onto the positive electrodes and applying a 50 &#181;L drop of NaCl 5 mM solution onto the negative electrodes (see step 2.1.7). Incubate for 30 min at room temperature. Monitor the conductance with a digital multimeter. 
		Note: The multimeter was provided by Dr. P. Bogaerts, Dr. S. Yunus, and Prof. Y. Glupczynski&#8217;s (Catholic University of Louvain-la-Neuve - CHU Mont-Godinne. This potentiostat is computer-controlled via a USB port and analyzes the eight different chips of the sensor simultaneously. The software created by Yunus and colleagues creates a real-time plot that represents the measurements of the conductance difference between the reference and sample electrodes against time.</li>
	</ol>
	</li>
</ol>

&beta;-lactamase-based conductimetric biosensor assay for protein-protein interactions

Source:&#160; Vandevenne, M., et al. <a href="https://app.jove.com/v/55414">The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions.</a> J. Vis. Exp. (2018)
This video demonstrates a conductimetric biosensor assay to study the interaction of an antibody with a target antigen immobilized on a transducer chip. The biosensor is engineered to have a bifunctional chimeric protein &#8212; a &#946;-lactamase enzyme with a functional catalytic site fused to a nanobody for binding to the immobilized target antigen. Upon binding, the catalytic hydrolysis of an antibiotic substrate by the &#946;-lactamase enzyme generates an electrical signal &#8212; confirming the antigen-antibody interaction.

&beta;-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions

Source:&#160; Vandevenne, M., et al. The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions. J. Vis. Exp. ...

To begin the &#946;-lactamase-based conductimetric biosensor assay, take a transducer chip with working, counter, and reference electrodes. The working electrode has a conductive polymer coating, facilitating target antigen immobilization. The reference and counter electrodes lack this polymer coating.
Add a solution containing the target antigen onto the chip. Incubate. The antigen gets immobilized on the working electrode surface via non-covalent interactions. Wash to remove unbound antigens.
Add a blocking solution. Proteins in the solution attach to unbound sites on the working electrode and block them.
Add a bifunctional chimeric protein to the chip to detect the immobilized antigen. The chimeric protein comprises a &#946;-lactamase enzyme &#8212; a bacterial protein responsible for antibiotic resistance. The enzyme is fused to a nanobody &#8212; the N-terminal variable region of single-domain antibodies.
Upon incubation, the nanobody of the protein binds to the immobilized target antigen. Wash to remove unbound chimeric proteins.
Plug the chip into a computer-controlled digital multimeter. Add a detection solution containing the antibiotic benzylpenicillin &#8212; a substrate for &#946;-lactamase.
Benzylpenicillin binds to the &#946;-lactamase of the antigen-bound chimeric protein and gets hydrolyzed &#8212; causing the release of protons. The protons induce a change in the electrical conductance of the polymer.
Plot the real-time difference in conductance between the reference electrode and the working electrode with the immobilized target antigen, confirming its interaction with the chimeric protein.

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84 Views. Source:  Vandevenne, M., et al. The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions. J. Vis. Exp. (2018) This video demonstrates a conductimetric biosensor assay to study the interaction of an antibody with a target antigen immobilized on a transducer chip. The biosensor is engineered to have a bifunctional chimeric protein — a β-lactamase enzyme with a functional catalytic site fused to a nanobody for binding to the immobilized...

β-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions

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