Name: the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions
Uploaded: 2018-02-01T13:00:00
Description: Watch this Scientific Journal Video about The Use of a Î²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

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>
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			<td height="25" style="height:25px;">KH2PO4</td>
			<td>Sigma-Aldricht</td>
			<td>V000225&#160;</td>
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			<td height="25" style="height:25px;">K2HPO4</td>
			<td>Sigma-Aldricht</td>
			<td>1551128</td>
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			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldricht</td>
			<td>S7653</td>
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			<td height="21" style="height:21px;">Tris&#8211;HCl</td>
			<td>Roche</td>
			<td>10812846001</td>
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			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>E9884</td>
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			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldricht</td>
			<td>P9541</td>
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			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td>Sigma-Aldricht</td>
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			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Sigma-Aldricht</td>
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			<td height="21" style="height:21px;">alanine</td>
			<td>Sigma-Aldricht</td>
			<td>A7627</td>
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			<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.

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

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...