Nikita+Khlystov's+Proposal

Aptamer-based Sensing of Proteins using Optical Microresonators

// Nikita Khlystov // // April 9, 2009 //
 * Research Proposal**

//__Research Purpose__// //Develop a sensor array to detect protein concentration abnormalities in body fluid samples that can be used to indicate a broad range of diseases.//

//__Hypothesis/Engineering Goal__// Demonstrate the detection of thrombin as an example protein through aptamer-binding on the surface of an optical microresonator, with fluorescence as the control. Determine an optimal T-spacer length to use in future experiments on both SU-8 coated glass slides and optical microresonators.

//__Background__// Aptamers are single-stranded oligonucleotide sequences, which have been demonstrated, under certain conditions, to specifically form bonds with certain proteins, DNA, cells, and even small molecules. There is currently a significant research effort aimed at further investigating the potential of aptamers. In particular, aptamers’ high specificity and high association constants are desirable in the field of clinical diagnostics: the use of fluorescently tagged aptamers on gold-nanoparticles and DNA-tile arrays has been demonstrated to be highly efficient methods of detecting nanoscopic concentrations of biomolecules through observations of variance in fluorescence [3, 5]. In these experiments, thrombin, a protein found in blood as coagulation factor II and contributes to the process of blood clotting, was used because of its low price and the existence of a well-established aptamer sequence that is highly specific to thrombin. While there have been numerous novel studies conducted on thrombin-detection using fluorescently tagged aptamers, there is another, infrequently mentioned potential method of detection through optical microresonators. These are photonic-based sensors made of silicon that are coated in SU-8 DNA-binding-facilitating polymer and that have micron-wide waveguide patterns cut into them, including a ring in which light can resonate. They utilize refraction index changes due to the presence of additional substance on the surface of the ring, which induces a different resonant wavelength in the output light. This change in wavelength can be detected by using a tunable scanning laser and measuring amplitude versus wavelength. When this is performed over the course of an hour, the amplitude peaks should undergo a wavelength shift, indicating successful binding. After the procedure utilizing fluorescence has been demonstrated to be successful, this method will be used to detect the presence of thrombin bound to exosite I aptamers bound to the SU-8 surface. The surface chemistry involving the immobilization of aptamers onto SU-8 polymer surfaces has not been fully understood. Specifically, the efficiency of thrombin-aptamer binding may be highly contingent on the distance between the binding site of the aptamer and the immobilization surface. Therefore, in addition to demonstrating that thrombin can successfully bind to aptamers on SU-8 coated glass slides and microresonators, the goal of this experiment is to investigate whether this distance has an effect on thrombin-aptamer binding efficiency and, if so, to determine an optimal distance to use in conducting future aptamer-thrombin experiments. This will be done using varying lengths of dT-oligonucleotide spacers attached to the surface-binding end of the thrombin-specific aptamer oligonucleotide sequence. If the detection of thrombin is demonstrated to be possible using microresonators, then further development is possible, namely in designing and manufacturing a sensor array using multiple microresonators to detect various proteins, red and white blood cells, and potentially other biomolecules present in a single drop of blood. This has indispensible applications in the field of clinical diagnostics and can be used to identify numerous human diseases such as cancer, leukemia, Alzheimer’s disease, Addison’s disease, sickle-cell anemia, malaria, and the West Nile Virus in patients by detecting abnormalities in concentrations.

//__Procedure__// The first step in the experimental process for this project will involve the review and refinement of my original procedure for binding fluorescently labeled thrombin to thrombin-specific aptamers. If approved by supervising Duke University professors Dr. Nan Jokerst and Dr. Thomas LaBean, the materials for the experiment will be ordered (see materials below). As the procedure stands now, this initial experiment will be performed on an SU-8 coated glass slide under incubation conditions of 37 degrees Celsius. Aptamers specific to thrombin’s fibrinogen-binding site (exosite I) in buffer solution will be first immobilized to the SU-8 surface through prolonged surface-to-surface exposure and 37 degree Celsius humidified incubation conditions. To determine the optimum aptamer length and distance from surface, three different spacers of 8, 16, and 24 dT-oligonucleotide lengths will be attached to the surface-binding end of the aptamer. Thrombin and buffer solution will then be applied by covering the immobilized aptamers with an aluminum-sealed chamber containing thrombin solution. After two hours of incubation, the first chambers will be removed and the glass slide will be washed with buffer solution and dried under nitrogen stream. New chambers that will hold the fluorescently tagged aptamers that are specific to thrombin’s heparin-binding site (exosite II) will then be placed on the glass slide. Fluorescent thrombin is not commercially available, so these exosite II aptamers, labeled with a Cy3-fluorescent marker, will be bound to the immobilized thrombin and will allow for fluorescence observations as a function of degree of thrombin binding. The exosite II aptamer solution in the chamber attached to the glass slide will be incubated for an additional hour. The chamber will then be removed and the slide washed again in buffer solution and dried under a nitrogen stream. Fluorescence will be observed using a fluorescence microscope and as data collection, images will be taken with a high-resolution fluorescence camera. The control for data analysis will be fluorescently tagged, thiolated thrombin exosite I aptamer immobilized to the SU-8 surface without exposure to thrombin. Fluorescence is the response variable, and the data collection size is three samples of five 1 microliter spots of immobilized exosite I aptamer that has spacers of different lengths attached and that has been exposed to thrombin and fluorescent exosite II aptamers. The strength of fluorescence of the three samples will determine the optimum spacer length. The time period for this initial experiment has been set to four weeks after the completion of safety training and ordering of materials; in these four weeks, thrombin-binding on SU-8 coated glass slides should be successfully demonstrated and a optimum spacer length determined. Provided that Matt Royal, graduate student at Duke University working under supervision of Dr. Jokerst in developing original optical microresonators, develops a successful protocol for immobilization of DNA on optical microresonators, thrombin binding to unlabeled aptamers should be demonstrated to be successful and a new optimum spacer length for immobilization on microresonators should be determined in six weeks after the completion of safety training and ordering of materials. If this protocol is not developed after four weeks after the completion of safety training and ordering of materials, the proposed experimentation on microresonators cannot proceed; therefore, all work will be aimed towards helping Matt develop this protocol.

//__Data Measurement__// The response variable of the experiment on a microresonator ring is the output amplitude as a function of wavelength; a peak wavelength shift should be observed if thrombin successfully binds. Optimum spacer length will be determined by the maximum overall amplitude of the signal response from each of the three samples. The control for this experiment will be the results of the fluorescence experiment on SU-8 coated glass slides. Sample size in this experiment will be billions of data points of amplitude over a range of wavelengths scanned over approximately an hour; these data points will be efficiently analyzed and presented in a simple graphical manner by using Matt Royal’s original computer program written in Matlab.

//__Materials__// Buffer solution will be mixed using solid sodium dodecyl sulfate (also known as sodium lauryl sulfate) and saline sodium citrate (both to be ordered from Sigma-Aldrich USA) and de-ionized water using Matt Royal’s procedure, provided it will be demonstrated to be functional in the near future. However, based on literature, another buffer solution may be tried, namely diluted Tris-HCl buffer, to be ordered from Sigma-Aldrich USA. Custom thiolated and fluorescently tagged aptamer sequences for thrombin’s exosite I and exosite II based on literary sources will be ordered from Integrated-DNA Technologies; the exosite I sequence was taken from [8] and the exosite II sequence was taken from [7]. Powdered human plasma alpha thrombin will be ordered from Sigma-Aldrich USA. Solid potassium-monobasic and potassium-dibasic phosphate, solid sodium hydroxide, and concentrated hydrochloric acid will be ordered from Sigma-Aldrich USA to establish an additional buffer solution of pH 7.4; a digital pH meter will be needed to ensure correct pH of this buffer. SU-8 coated glass slides and microresonators will be used as premade by Matt Royal. The microresonators are part of his novel research work and made according to his original designs. To observe fluorescence, a fluorescence microscope with high-resolution imaging capabilities located at Duke University will be used. Multiple 50-mL and 50-microliter centrifuge tubes will be used to mix solutions; a Vortex machine will be used to ensure proper mixing of buffer solutions. A sonicator will be used to properly mix and heat refrigerated solutions. A large 500-mL beaker and a small 100-mL beaker will be used to create humidified conditions. 3-mL and 50-mL syringes and syringe filters will be needed to transfer and purify large quantities of solutions (i.e. greater than 1 mL); adjustable Eppendorf pipettes capable of transferring 1 to 1000 microliters of solution and the corresponding disposable pipette tips will be also needed. Scapulas will be used to handle solid chemicals. A hot plate capable of holding a constant temperature of 37 degrees Celsius will be needed. All of these materials are provided at the lab at Duke University; however, an additional supply of all materials except aptamers, thrombin, SU-8 covered glass slides, microresonators, sodium dodecyl sulfate, and saline sodium citrate will be needed if some experimentation is to be performed at NCSSM if occasional transportation or supervision issues arise.

//__Hazards and Safety Procedures__// No research will be conducted on human or vertebrate animal subjects. Some of the chemicals used may be hazardous if ingested, inhaled, or in contact with skin or eyes. None of these chemicals, however, is identified on Duke University’s official list of Potentially Hazardous Substances; similarly, none of them is considered hazardous chemicals by page 25 of the 2008 International ISEF Rules and Regulation. Nevertheless, to ensure safety, Material Safety Data Sheets have been downloaded and attached for all chemicals that are to be potentially used in experimentation. While the use of DNA and thrombin are included in this experimentation, they are not considered potentially hazardous biological agents by page 21 of the 2008 International ISEF Rules and Regulations. Tunable near-infrared lasers will be used in experimentation with microresonator rings; the risks involved include eye damage. As safety precautions, chemical safety goggles and gloves will be worn at all times in the lab when the use of above chemicals is involved; laser safety goggles will be worn when the laser is in use. Proper established chemical disposal procedures will be followed at all times. Additionally, Duke University mandates that all minors successfully complete an online chemistry, laser, and laboratory safety training course for this kind of experimentation; during experimentation in the laboratory, minors are also to be accompanied by designated supervisors at all times. See the attached Risk Assessment Form (ISEF form 3) for more details.

//__Contact Information of Supervising Faculty__// **Nan M Jokerst, Ph.D.** Professor at Duke University Electrical Engineering nan.jokerst@duke.edu +1 919 660 5503 (tel) Associate Research Professor at Duke University Computer Science, Chemistry, and Biomedical Engineering thl@cs.duke.edu +1 919 660 1565 (tel) +1 919 660 6519 (fax)
 * Physical Address:** FCIEMAS 3589, Durham, NC 27708
 * Postal Address:** Box 90291, Durham, NC 27708-0291
 * Thomas LaBean, Ph.D.**
 * Physical Address:** D212 French Sciences Building, Durham, NC 27708
 * Postal Address:** Box 90129, Durham, NC 27708-0129

Research in Chemistry Teacher at the North Carolina School of Science and Mathematics halpin@ncssm.edu +1 919 416 2764 (tel) Physical Address: 2nd Floor Bryan Building, North Carolina School of Science and Mathematics, Durham, NC, 27708 Graduate Student at Duke University mwr.134@duke.edu +1 215 801 4900 (tel)
 * Myra Halpin, Ph.D.**
 * Matt Royal**

//__Location of Workplace__// Center for Interdisciplinary Engineering, Medicine, and Applied Science (CIEMAS) Building, Lab 3584, Duke University, Durham, North Carolina. Bryan Building, First Floor Research Lab, North Carolina School of Science and Mathematics, Durham, North Carolina.

//__Literary References__// 1. Hamaguchi, Nobuko, Andrew Ellington, and Martin Stanton. “Aptamer Beacons for the Direct Detection of Proteins.” 15 Jun 2001. 8 Apr 2009 . 2. Tasset, Diane M., Mark F. Kubik, and Walter Steiner. “Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.” //Journal of Molecular Biology// 272.5 (1997): 688-698. 3. Wang, Wenjuan et al. “Aptamer biosensor for protein detection using gold nanoparticles.” //Analytical Biochemistry// 373.2 (2008): 213-219. 4. Yang, Hui et al. “An aptamer-based biosensor for sensitive thrombin detection.” //Electrochemistry Communications// 11.1 (2009): 38-40. 5. Lin, Chenxiang, Evaldas Katilius, Yan Liu, Junping Zhang, and Hao Yan. "Self-Assembled Signaling Aptamer DNA Arrays for Protein Detection." __Angewandte Chemie International Edition__ (2006): 5296-301. 6. Shangguan, Dihua, Zehui Zehui Cao, Ling Meng, Prabodhika Mallikaratchy, Kwame Sefah, Hui Wang, Ying Li, and Weihong Tan. “Cell-Specific Aptamer Probes for Membrane Protein Elucidation in Cancer Cells.” __Journal of Proteome Research__ (2008): 2133-2139. 7. Liu, Xuemei, Dajin Zhang, Guojun Cao, Guang Yang, Hongmei Ding, Gang Liu, Ming Fan, Beifen Shen, and Ningsheng Shao. “RNA aptamers specific for bovine thrombin.” __Journal of Molecular Recognition__ (2003): 23-27. 8. Ostatna, Veronika, Hana Vaisocherova, Jiri Homola, and Tibor Hianik. “Effect of the immoblisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance.” //Journal of Analytical and Bioanalytical Chemistry// (2008) 39: 1861-1869.