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.
