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 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&#160;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&#160;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&#160;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&#160;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&#160;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 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 work17. Briefly, this system uses re-usable 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&#160;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&#160;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&#160;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&#160;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&#160;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&#160;2.3.2).</li>
		<li>Dilute the BlaP-cAb-Lys3 solution to 20 &#181;g/mL in binding solution (see step&#160;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 antigen-nanobody reaction, wash three times as described in the previous step using the wash solution (see step&#160;2.1.4). Then rinse the electrode once with PBS (see step&#160;2.1.1).</li>
		<li>For detection, plug the sensor chip via the copper-circuitry part to a digital multimeter. Then, initiate the sensor response by applying a 50 &#181;L drop of detection solution (see step&#160;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&#160;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 colleagues17 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>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldricht</td><td>V000225&amp;nbsp;</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldricht</td><td>1551128</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldricht</td><td>S7653</td><td></td></tr><tr><td>Tris&amp;ndash;HCl</td><td>Roche</td><td>10812846001</td><td></td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma-Aldricht</td><td>E9884</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldricht</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldricht</td><td>NIST2186II</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldricht</td><td>M6250</td><td></td></tr><tr><td>alanine</td><td>Sigma-Aldricht</td><td>A7627</td><td></td></tr><tr><td>HClO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fluka</td><td>34288</td><td>1M HClO4 solution, distributor : Sigma-Aldricht</td></tr><tr><td>casein hydrolysate</td><td>Sigma-Aldricht</td><td>22090</td><td></td></tr><tr><td>benzylpenicillin sodium</td><td>Sigma-Aldricht</td><td>B0900000</td><td></td></tr><tr><td>hen egg white lysozyme</td><td>Roche</td><td>10837059001</td><td></td></tr><tr><td>heptane</td><td>Sigma-Aldricht</td><td>246654</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldricht</td><td>322415</td><td></td></tr><tr><td>ammonium hydroxide solution</td><td>Sigma-Aldricht</td><td>380539</td><td>28% NH3&amp;nbsp;in H2O, purified by double-distillation (concentrated?)</td></tr><tr><td>&lt;strong&gt;Laboratory consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6-well plate&amp;nbsp;</td><td>Greiner Bio-One</td><td>657165</td><td>CELLSTAR 6-Well Plate</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pH meter</td><td>WTW</td><td>1AA110</td><td>Lab pH meter inoLab pH 7110</td></tr><tr><td>vacuum and filtration system</td><td>Nalgene</td><td>NALG300-4100</td><td>Filter holders with receiver, distributor : VWR</td></tr><tr><td>potentiometric sensor chips</td><td></td><td></td><td>manufactured by Yunus and colleagues (ref 16)</td></tr><tr><td>PGSTAT30 Autolab</td><td>Metrohm Autolab</td><td></td><td>discontinued, succesor Autolab PGSTAT302N</td></tr><tr><td>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 &#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.

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

the-use-lactamase-based-conductimetric-biosensor-assay-to-detect

Research

JoVE Journal

Bioengineering

8.9K Views.  University of Liège. In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid β-lactamase technology. This method relies on release of protons upon hydrolysis of β-lactams.

Video: The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Chemistry

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Biochemistry

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Immunology and Infection

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte

Source: Desai, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59687">Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription.</a> J. Vis. Exp. (2019)
In this video, we demonstrate biolayer interferometry (BLI) technology to detect the intermolecular interactions between ligands and target analytes in real-time. BLI measures changes in the interference pattern of white light reflected off a layer of immobilized ligands on a biosensor surface as they interact with analytes in solution.

Source: Desai, M., et al. Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription. J. Vis. Exp. (2019)
In ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

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

Source:&#160; Vandevenne, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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

The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

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