Biomod/2011/HKBU/NBgamers:Project: Difference between revisions

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<tr> <td bgcolor="#232323" align="center"> <h3 align="center" style="color:white; font-size:20px;" >Project</h3> </td> </tr>

<h3>Contents</h3> <ol>

 <li><a href="#Project Title">Project Title</a></li>
 <li><a href="#Abstract">Abstract</a></li>
 <li><a href="#Design">Design</a></li>
 <ul>
   <li><a href="#Structure">Structure</a></li>
   <li><a href="#Highlights">Hightlights</a></li>
 </ul>
 <li><a href="#Animation">Animation</a></li>
 <li><a href="#Detection">Detection</a></li>
 <ul>
   <li><a href="#Visualization">Visualization</a></li>
   <li><a href="#Quantification">Quantification</a></li>
 </ul>
 <li><a href="#Project Scheme">Project Scheme</a></li>
 <li><a href="#3D Animation">3D Animation</a></li>
 <li><a href="#Instrument">Instrument</a></li>
 <li><a href="#Materials">Materials</a></li>
 <li><a href="#Experiment">Experiment</a></li>

</ol> <br />

<h3><a name="Project Title">Project Title</a></h3> <p> High Throughput and Ultrasensitive Beta-Amyloid based Nanosensor for the Detection of Biomolecules </p> <br />

<h3><a name="Abstract">Abstract</a></h3> <p> We develop a simple and efficient technique using self-assembling beta-amyloid (Aβ) nanofibrillar sensor for ultrasensitive and pretreatment-free detection of biomolecules. As a proof-of-concept, small DNA (ca. 20 nt) is used as target analyte model for demonstration. Herein, Aβ fibrils conjugated with complementary DNA probes are applied as the detection template to capture (by hybridization) and preconcentrate the target DNA in solution. With the aid of total internal reflection fluorescence microscopy (TIRFM), two types of fluorescence signals would be collected. YOYO-1 iodide, which is a DNA bis-intercalating dye, is used to label the DNA hybrids for quantification; while quantum dots of different emission wavelengths are tagged and utilized to differentiate various probes conjugated on the fibrillar sensors, and hence achieving simultaneous multiplexed detection of biomolecules. In our assay, each well-defined fibril serves as an individual sensor, and thus one may perform numerous detection assays in parallel. In summary, this design offers fast yet accurate detection of small DNA in high-throughput manner without the need of sample enrichment. It also brings insight in the development of novel biomaterial-based sensors in nanoscale. </p> <br />

<h3><a name="Design">Design</a></h3> <h5><a name="Structure">Structure</a></h5> <p>1. Preparation of biotinylated beta-amyloid fibril as biosensing template</p> <p align="center"> <a href="http://openwetware.org/images/3/3a/NBgamers_Structure_1.png"> <img src="http://openwetware.org/images/3/3a/NBgamers_Structure_1.png"> </a> </p> <p> <a href="">Beta-amyloid (Aβ) peptides</a> self assemble into Aβ fibrils once they misfold. Synthetic Aβ are commercially available and the fibrillation conditions of Aβ is also well-established by many research groups. In order to adapt it as one building parts of a sensor, the native Aβ monomers are co-incubated with monomeric biotinylated Aβ to form biotin-functionalized fibrils. The morphology of the fibril formed by native and biotinylated monomers is found to be the same as the native fibrils (~10 nm in diameter and few microns in length). This confirms that the self-assembled fibril is now ready to be manipulated as a biosensor template. </p> <br /> <p>2. Conjugation of streptavidin linker on beta-amyloid fibril</p> <p align="center"> <a href="http://openwetware.org/images/f/f2/NBgamers_Structure_2.png"> <img src="http://openwetware.org/images/f/f2/NBgamers_Structure_2.png"> </a> </p> <p> Quantum dot labeled streptavidin (commercially available) is attached onto the fibril by biotin-streptavidin conjugation. Quantum dot <a href="">(QD)</a> is a highly fluorescent nano-particle and different QDs have different emission colors of sharp peaks. Making use of this unique property of QDs, one can thus fluorescently label the fibrillar sensor with various colors respectively. [see <a href="">multiplex detection</a>] </p> <br /> <p>3. Attachment of biotinylated DNA probes on amyloid biosensor</p> <p align="center"> <a href="http://openwetware.org/images/1/15/NBgamers_Structure_3.png"> <img src="http://openwetware.org/images/1/15/NBgamers_Structure_3.png"> </a> </p> <p> As a proof-of-concept for short nucleic acid detection, short sequence single-stranded DNA (15 nt) probe is particularly chosen in this project. The commercially available biotinylated DNA probe is then attached to the fibrillar sensor by binding to previous QD-labeled streptavidin as illustrated in the schematics. After that, the detection of target DNA molecule is achieved by specific and complementary hybridization between the probes and targets. </p> <br />

<h3><a name="Idea">Idea</a></h3> <p> We want to develop a simple and efficient sensor for biomolecular detection. This sensor is using self assembling beta-amyloid (Aβ) as a backbone, then we functionalized the Aβ fibril by conjugation with biotin. The probe can then be attached on the fibril by biotin-strepavidin conjugation. In addition to monoplex quantitive detection we also want to extend it to multiplexed detection. </p> <br />

 <li>To functionalize the beta-amyloid fibril with biotin but not influence self-assembly.</li>
 <li>To form the sensor by adding biotinlyated DNA probe on the fibril using biotin-streptavidin conjugation.</li>
 <li>To test the viability of our sensor for DNA detection.</li>
 <li>To use QDs to label the fibril so that multiplexed detection can be achieved.</li>

</ol> </p> <br />

<h3><a name="Project Scheme">Project Scheme</a></h3> <p align="center"> <a href="http://openwetware.org/images/7/74/NBgamers_project_scheme.png"> <img src="http://openwetware.org/images/7/74/NBgamers_project_scheme.png" width="750" height="200" alt="Schematic of β-Amyloid based nanosensor (Click to enlarge.)" title="Schematic of β-Amyloid based nanosensor (Click to enlarge.)"> </a> </p> <br />

<h3><a name="3D Animation">3D Animation</a></h3> <p align="center"> <iframe width="420" height="315" src="http://www.youtube.com/embed/MK6sycHd2wM?hl=zh&fs=1&rel=0" frameborder="0" allowfullscreen></iframe> </p> <br />

<h3><a name="Instrument">Instrument</a></h3> <p> Total internal reflection fluorescence (TIRF) is a technique where only a very small region close to the coverslip is being exceted. This diagram below from the Leica brochure nicely explains TIRF. <p align="center"> <a href="http://openwetware.org/images/f/fe/TIRFM.png" alt="TIRF Microscopy (Click to enlarge.)" title="TIRF Microscopy (Click to enlarge.)"> <img src="http://openwetware.org/images/f/fe/TIRFM.png" width="300" height="300"> </a> </p> <p> The excitation from a laser is sent off-center up a high-NA objective. The light hits the coverslip at an angle such that total internal reflection occurs and light passes through the coverslip and generates an evanescent wave. This layer of excitation is approximately 100 nm thick, such that only fluorophores within the layer would be excited, while others beyond remains silent. Therefore, TIRF is commonly used in single molecule detection. As in our demonstration, the beta-amyloid is attached on the upper slide of the flow cell; we used TIRF microscopy to achieve a high resolution.<br /> (Reference: <a href="http://microscopy.duke.edu/introtomicroscopy/tirf.html">Duke University Light Microscopy Core Facility</a>) </p> <br />

<h3><a name="Materials">Materials</a></h3> <p> All the materials we used are commercially available which is easy to access. Below is the list of all the materials. You can find more information by click the link.<br /> <br /> Beta amyloid (1-40) monomer<br /> Brand: invitrogen<br /> Species: Human<br /> Product size: 1 mg<br /> <a href="http://products.invitrogen.com/ivgn/product/03136?ICID=search-product">http://products.invitrogen.com/ivgn/product/03136?ICID=search-product</a><br /> <br /> Biotin-beta-amyloid<br /> Brand: AnaSpec<br /> Size: 0.1 mg<br /> Molecular weight: 4556.2<br /> <a href="http://www.anaspec.com/products/product.asp?id=30266&productid=14474">http://www.anaspec.com/products/product.asp?id=30266&productid=14474</a><br /> <br /> Qdot 625 streptavidin conjugate<br /> Brand: Qdot®<br /> Product size: 1 μM, 200 μL<br /> Emission maxima: 625 nm<br /> <a href="http://products.invitrogen.com/ivgn/product/A10196?ICID=search-product">http://products.invitrogen.com/ivgn/product/A10196?ICID=search-product</a><br /> <br /> Qdot 565 streptavidin conjugate<br /> Brand: Qdot®<br /> Product size: 1 μM, 200 μL<br /> Emission maxima: 565 nm<br /> <a href="http://products.invitrogen.com/ivgn/product/Q10131MP?ICID=search-product">http://products.invitrogen.com/ivgn/product/Q10131MP?ICID=search-product</a><br /> </p> <br />

<h3><a name="Experiment">Experiment</a></h3> <h5>Monoplex</h5> <p> Fibril incubation → loading fibril into flow cell channel → Loading PBS buffer with 10% BSA → Loading PBS buffer → Loading Qdot labeled streptavidin → Loading probe → Loading target → Adding YOYO into the flow cell channel </p> <h5>Remarks</h5> <ol>

 <li>Fibril growth</li>
 <p>For efficient fibril formation, adding seeds.<br /> Added biotinlyted and native beta-amyloid monomer first and seed last.<br /> Sonitation the mixture for 3 seconds before incubation.<br /> Incubate 1 hour in 37℃ water bath.<br /> Use capillary force for loading sample.</p>
 <li>PBS buffer with 10% BSA</li>
 <p>Bovine serum albumin has non-specific absorption so that block the room on the slide without fibril attached. Thus Qdot added after could only occupy the position on the fibril.<br /> As Qdot all conjugated with the biotin on the fibril there will be less unconjugated streptavidin.</p>
 <li>Loading probe</li>
 <p>Calculate the amount of probe needed; make each fibril saturated with probes.<br /> Give time for probe conjugated on the fibril.</p>
 <li>Loading target</li>
 <p>Give time for target and probe hybridization before adding YOYO.</p>

</ol> <h5>Multiplex</h5> <p> Fibril incubation → Loading fibril into flow cell channel → Loading PBS buffer with 10% BSA → Loading PBS buffer → Loading Qdot 1 labeled streptavidin → Loading probe 1 → Loading fibril into flow cell channel → Loading Qdot 2 labeled streptavidin → Loading probe 2→Loading sample → Adding YOYO into the flow cell channel </p> <h5>Remarks</h5> <ol>

 <li>Well labeled samples, don’t mix up.</li>
 <li>Loading two times of fibrils but only one time of BSA.</li>

</ol> <br />

</td> </table> </td> </tr>

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Biomod/2011/HKBU/NBgamers:Project: Difference between revisions

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@@ Line 119: / Line 119: @@
 <a href="">Beta-amyloid (Aβ) peptides</a> self assemble into Aβ fibrils once they misfold. Synthetic Aβ are commercially available and the fibrillation conditions of Aβ is also well-established by many research groups. In order to adapt it as one building parts of a sensor, the native Aβ monomers are co-incubated with monomeric biotinylated Aβ to form biotin-functionalized fibrils. The morphology of the fibril formed by native and biotinylated monomers is found to be the same as the native fibrils (~10 nm in diameter and few microns in length). This confirms that the self-assembled fibril is now ready to be manipulated as a biosensor template.
 </p>
+<br />
 <p>2. Conjugation of streptavidin linker on beta-amyloid fibril</p>
 <p align="center">
@@ Line 128: / Line 129: @@
 Quantum dot labeled streptavidin (commercially available) is attached onto the fibril by biotin-streptavidin conjugation. Quantum dot <a href="">(QD)</a> is a highly fluorescent nano-particle and different QDs have different emission colors of sharp peaks. Making use of this unique property of QDs, one can thus fluorescently label the fibrillar sensor with various colors respectively. [see <a href="">multiplex detection</a>]
 </p>
+<br />
 <p>3. Attachment of biotinylated DNA probes on amyloid biosensor</p>
 <p align="center">