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

Revision as of 10:31, 2 November 2011

<html>

<body>

 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Home
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Mission" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Mission
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Project" style="color:white; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='white';">
 Project
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Results" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Results
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Video" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Video
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Team" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Team
 </a>
 </td>
 <td align="center" height="50">
 <a href="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Resources" style="color:black; font-size:20px;" onmouseover="this.style.color='white';" onmouseout="this.style.color='black';">
 Resources
 </a>
 </td>

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

<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="#Material and Method">Material and Method</a></li>
 <ul>
   <li><a href="Experimental Procedures">Experimental Procedures</a></li>
   <li><a href="Instrumental Configurations">Instrumental Configurations</a></li>
 </ul>

</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="http://openwetware.org/wiki/Biomod/2011/HKBU/NBgamers:Beta-amyloid (Aβ) peptides">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. <a href="http://en.wikipedia.org/wiki/Quantum_dot">Quantum dot (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="Material and Method">Material and Method</a></h3> <h5><a name="Experimental Procedures">Experimental Procedures</a></h5> <h5>Preparation of cover glasses</h5> <p>All coverslips were pre-washed prior to experiments.</p> <ol>

 <li>No.1 22 × 22 square mm cover glasses (Corning, NY) were successively sonicated in absolute ethanol, sodium hydroxide (NaOH) solution, glacial acetic acid and distilled water successively.</li>
 <li>The cleaned coverslips were dried completely at 140 °C oven for approximately 15 minutes and stored for future usage.</li>

</ol> <br /> <h5>Preparation of Aβ<sub>1–40</sub> fibrils for seeding</h5> <ol>

 <li>Monomeric Aβ<sub>1–40</sub> (Invitrogen) Aβ<sub>1–40</sub> was prepared by dissolving 1 mg of Aβ1–40 monomers in 400 μL ice-cold 0.02 % ammonia solution and stored at −80 °C until use.</li>
 <li>Stock solution of Aβ<sub>1–40</sub> was diluted to 50 μM with phosphate buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.4). The mixture was incubated in water bath at 37 °C with gentle shaking for 20 hours.</li>
 <li>The resultant fibrils were sonicated for 5 seconds thrice and used as seedings in the later experiments for the seed-mediated fibrillation.</li>

</ol> <br /> <h5>Preparation of biotinylated Aβ<sub>1–40</sub> fibrils</h5> <ol>

 <li>Stock biotin-Aβ<sub>1–40</sub> monomers (Anaspec, CA) were diluted with 0.02 % ammonia solution to 100 μM. </li>
 <li>Biotinylated-Aβ1<sub>1–40</sub> fibrils were prepared by mixing monomeric biotin-Aβ<sub>1–40</sub> and Aβ<sub>1–40</sub> in a molar ratio 1:4, 1:9, 1:19, 1:49 respectively, with a total Aβ concentration of 50 μM.</li>
 <li>0.87 μg/mL of prepared Aβ<sub>1–40</sub> seeding was added to the solution. The peptide mixture was then incubated at 37 °C for 60 minutes. </li>

</ol> <br /> <h5>Immobilization of amyloid fibrils on the surface of flow cell</h5> <ol>

 <li>Sealed flow cell was prepared by combining two pre-cleaned cover glasses with double-sided adhesive tapes with a channel width of approximately 3 mm each.</li>
 <li>10 μL of diluted amyloid fibrils and buffer solution was flowed into the flow cell.</li>
 <li>Solutions in excess were withdrawn at the outlet with Kimwipes based on capillary force such that fibrils were stretched under the capillary force. </li>
 <li>4.	The flow cell was placed under the home-built prism-type total internal reflection fluorescence (TIRFM) system for imaging.</li>

</ol> <br /> <h5>Optimization of fibril density and fibril co mposition</h5> <ol>

 <li>Fibrils were immobilied on the surface of flow cell as described in the previous section.</li>
 <li>10 μL of Qdot 625 streptavidin conjugate (QD625-Stv, Invitrogen) solutions was flowed into the channel after the flow of phosphate buffer and PBS buffer with 1 % of bovine serum albumin (PBS-1% BSA).</li>
 <li>The sample was incubated at room temperature for 30 minutes before imaging.</li>
 <li>The flow cell was placed under the home-built TIRFM system for imaging.</li>

</ol> <br /> <h5>Detection of DNA sequences with biotinylated Aβ<sub>1–40</sub> fibrils</h5> <ol>

 <li>Fibrils were immobilied on the surface of flow cell as described in the previous section.</li>
 <li>Excess QD625-Stv were then added into the channel, incubated for 15 minutes and successively washed with buffer.Herein QD625-Stv was added for localization of fibrils in the images. </li>
 <li>Excess biotinylated-poly(T)15 probe (5’-Biotin-TTT TTT TTT TTT TTT-3’, Invitrogen, HPLC-purified) were added into the channel and incubated for 15 minutes to saturate the available Stv sites.</li>
 <li>The channel was then washed with phosphate buffer. Target poly(A)15 (5’-AAA AAA AAA AAA AAA-3’) of different concentrations were added into the channel, hybridized for 30 minutes at r.t. for detection and calibration respectively.</li>
 <li>YOYO-1 iodide (YOYO, Invitrogen) was finally added to label and produce fluorescence signals from the hybridized DNA duplex.</li>
 <li>The flow cell was placed under the home-built TIRFM system for imaging with the excitation of 488 nm laser.</li>

</ol> <br /> <h5>Multiplexed Detection of DNA sequences with biotinylated Aβ<sub>1–40</sub> fibrils</h5> <ol>

 <li>Fibrils were immobilied on the surface of flow cell as described in the previous section.</li>
 <li>Excess QD625-Stv were then added into the channel, incubated for 15 minutes and successively washed with buffer.Herein QD625-Stv was added for localization of fibrils in the images.</li>
 <li>Excess biotinylated-probe A were added into the channel and incubated for 15 minutes to saturate the available Stv sites.</li>
 <li>Additional fibrils were immobilized on the surface of flow cell.</li>
 <li>QD565-Stv and biotinylated–probe B were added successively to the channel with a similar procedure as described in (2) and (3).</li>
 <li>YOYO-1 iodide (YOYO, Invitrogen) was finally added to label and produce fluorescence signals from the hybridized DNA duplex.</li>
 <li>The flow cell was placed under the home-built TIRFM system for imaging with the excitation of 488 nm laser using 3 different filter cubes.</li>
 <li>Herein, fluorescence signals from QD625-Stv (by using HQ625/20 filter) and QD565-Stv (by using HQ565/18 filter) were monitored to distinguish the location of fibrils immobilized with probe A and probe B respectively; while fluorescence signals from YOYO (by using HQ535/50) were measured for quantification purpose.</li>

</ol> <br /> <h5><a name="Instrumental Configurations">Instrumental Configurations</a></h5> <h5>Total Internal Reflection Fluorescence Microscopy (TIRFM) imaging system</h5>

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

</td> </table>

</body>

</html>

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

Revision as of 10:31, 2 November 2011

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

research

Tools

@@ Line 90: / Line 90: @@
    <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="#Material and Method">Material and Method</a></li>
-   <li><a href="#Materials">Materials</a></li>
+   <ul>
-  <li><a href="#Experimental Procedures">Experimental Procedures</a></li>
+    <li><a href="Experimental Procedures">Experimental Procedures</a></li>
+    <li><a href="Instrumental Configurations">Instrumental Configurations</a></li>
+  </ul>
 </ol>
 <br />
@@ Line 216: / Line 218: @@
 <br />
-<h3><a name="Experimental Procedures">Experimental Procedures</a></h3>
+<h3><a name="Material and Method">Material and Method</a></h3>
+<h5><a name="Experimental Procedures">Experimental Procedures</a></h5>
 <h5>Preparation of cover glasses</h5>
 <p>All coverslips were pre-washed prior to experiments.</p>
@@ Line 276: / Line 279: @@
 </ol>
 <br />
+<h5><a name="Instrumental Configurations">Instrumental Configurations</a></h5>
+<h5>Total Internal Reflection Fluorescence Microscopy (TIRFM) imaging system</h5>
 </td>