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>Content</h3> <ol>

 <li><a href="#Project Title">Project Title</a></li>
 <li><a href="#Abstract">Abstract</a></li>
 <li><a href="#Background">Background</a></li>
 <li><a href="#Idea">Idea</a></li>
 <li><a href="#Aim">Aim</a></li>
 <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="Background">Background</a></h3> <h5>What are biomolecules?</h5> <p> Let’s begin from the word “biomolecule”, it consists two parts: “bio” and “molecule”. So it simply means the molecule that is produced by a living organism. These biomolecules are nucleic acids (DNA and RNA), glycans (i.e. glucolipids, glycoproteins), proteins, and lipids. (Reference: <a href="http://en.wikipedia.org/wiki/Biomolecule">Wikipedia: Biomolecule</a>) They are the building blocks of our life; each of them serves different functions as well as associated with different biological events. </p> <h5>Why we need to detect biomolecules?</h5> <p> The reason is obvious: living organisms are made of biomolecules, and the capability of detectin g biomolecules translates directly in the capability of monitoring life. Let us give an example of how biomolecule detection can improve our lives. </p> <p> Cancer is a good example of a worrying problem. Cancer is a genetic disease that begins with one or several point variations in a biomolecule called DNA. These mutations in DNA translate into the production of mutant proteins which are the effectors of the cells, and mutations can cause the appearance of proteins with truncated functions with respect to the standard ones. As a consequence, the cell cycle may undergo unregulated and, sooner or later, the cell becomes immortal and a tumor appears. </p> <p> Now, what can be done to fight cancer? The better way is to detect it before it happens, i.e., prognosis. For instance, people with previous cases of this disease in their families could be genetically mapped in order to check whether they carry inherited mutations. If the prognosis fails, an early diagnostic would be a powerful tool for the treatment of malignant disease. Early detection is particularly important because the early stages of disease are typically treated with the greatest probability of success. </p> <p> <table border="1" align="center"> <tr>

 <td><h5>Gene</h5></td>
 <td><h5>Cancer</h5></td>

</tr> <tr>

 <td>HSPA</td>
 <td>Breast caner</td>

</tr> <tr>

 <td>MTHFR</td>
 <td>ColoRectal cancers</td>

</tr> <tr>

 <td>TCTA</td>
 <td>Leukaemia</td>

</tr> <tr>

 <td>PAP</td>
 <td>Liver caner</td>

</tr> </table> (Reference: <a href="http://www.cancerindex.org/">CanerIndex</a>) </p> <h5>What is a nano-biosensor?</h5> <p> A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component. Nano means the biosensor is in the diameter of nano scale (10-9 m). </p> <p> Usually, the biosensor consists of 3 parts: <ol>

 <li>Sensitive biological element which the analyte under study can “react” with;</li>
 <li>Detector element that transforms the signal resulting from the interaction of the analyte with the biological element into another signal that can be more easy measured and quantified such as optical signal;</li>
 <li>Biosensor reader device.</li>

</ol> </p> <p> To be a good nano-biosensor, it must have ultrasensitive, reliable and low resources consumption. The more sensitive the sensor, the less sample is needed. Also we need to consider the time and cost of a particular detection method. </p> <p> In the detection of real life samples, many treatments and modifications are performed before the detection of specific target. There are drawbacks for pretreatments: you have to bear the risk of sample lost at the same time it is time consuming. </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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