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

Protocolo

1. Protein Sample Preparation

1. 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  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.
2. 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 μm cutoff.

2. Biosensor Assays

1. Solution and buffer preparation
   1. 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 °C.
   2. 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 °C.
   3. 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 °C.
   4. Prepare a washing solution (0.1% Tween - PBS) by adding 100 μL of Tween 20 (100%) in 100 mL of PBS prepared as described above (see step 2.1.1). Store at 4 °C.
   5. 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 °C.
   6. 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 °C.
   7. 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 °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 °C.
2. 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’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.
   1. Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/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.
   2. Rinse the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of distilled water for 2 min with gentle mixing at room temperature.
   3. Regenerate the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of regeneration solution (3.5 M KCl, see step 2.1.6) overnight at 4 °C or 1 h at room temperature.
   4. Perform 3 washes of the electrodes by dipping the tips into wells of a 96-well plate containing 300 µL/well of phosphate buffer saline (see step 2.1.1.). Perform each wash for 2 minutes with gentle mixing at room temperature.
3. Binding assay performed on the sensor
   1. Coat HEWL onto the PANI (polyaniline) surface of the electrode by depositing a 15 µL drop of 40 µg/mL HEWL prepared in PBS onto the electrode surface. Incubate overnight at 4 °C or 1 hour at room temperature.
   2. 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 µL/well of phosphate buffer saline. Perform each wash for 2 min with gentle mixing at room temperature.
   3. Saturate the electrodes by adding a 50 µL 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).
   4. Dilute the BlaP-cAb-Lys3 solution to 20 µg/mL in binding solution (see step 2.1.3) and apply a 15 µL 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).
   5. For detection, plug the sensor chip via the copper-circuitry part into a digital multimeter. Then, initiate the sensor response by applying a 50 µL drop of detection solution (see step 2.1.7) onto the positive electrodes and applying a 50 µL 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’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.

Materiais

Name	Company	Catalog Number	Comments
Reagents
KH2PO4	Sigma-Aldrich	V000225
K2HPO4	Sigma-Aldrich	1551128
NaCl	Sigma-Aldrich	S7653
Tris–HCl	Roche	10812846001
EDTA	Sigma-Aldrich	E9884
KCl	Sigma-Aldrich	P9541
Na2HPO4	Sigma-Aldrich	NIST2186II
2-mercaptoethanol	Sigma-Aldrich	M6250
Alanine	Sigma-Aldrich	A7627
HClO4	Fluka	34288	1M HClO4 solution, distributor : Sigma-Aldrich
Casein hydrolysate	Sigma-Aldrich	22090
Benzylpenicillin sodium	Sigma-Aldrich	B0900000
Hen egg white lysozyme	Roche	10837059001
Heptane	Sigma-Aldrich	246654
Methanol	Sigma-Aldrich	322415
Ammonium hydroxide solution	Sigma-Aldrich	380539	28% NH3 in H2O, purified by double-distillation (concentrated?)
Laboratory consumables
6-well plate	Greiner Bio-One	657165	CELLSTAR 6-Well Plate
Equipment
pH meter	WTW	1AA110	Lab pH meter inoLab pH 7110
Vacuum and filtration system	Nalgene	NALG300-4100	Filter holders with receiver, distributor : VWR
Potentiometric sensor chips			manufactured by Yunus and colleagues (ref 16)
PGSTAT30 Autolab	Metrohm Autolab		discontinued, successor Autolab PGSTAT302N
Digital multimeter, METRAHit 22M	Gossen Metrawatt		discontinued, successor Metrahit Base