عنوان پایان‌نامه
ساخت چیپ میکروسیال برای اندازه گیری سطح سرمی مارکرهای استخوانی در بیمار
- رشته تحصیلی
- نانوبیوتکنولوژی
- مقطع تحصیلی
- دکتری تخصصی PhD
- محل دفاع
- کتابخانه دانشکده علوم و فنون نوین شماره ثبت: 298184;کتابخانه مرکزی -تالار اطلاع رسانی شماره ثبت: 75155;کتابخانه دانشکده علوم و فنون نوین شماره ثبت: 298184;کتابخانه مرکزی -تالار اطلاع رسانی شماره ثبت: 75155
- تاریخ دفاع
- ۳۰ تیر ۱۳۹۵
- دانشجو
- پاتریشیا خشایار
- استاد راهنما
- قاسم عموعابدینی
- چکیده
- چکیده تشخیص پوکی استخوان، که امروزه با استفاده از سنجش تراکم مواد معدنی استخوان (BMD) صورت می گیرد، از محدودیت های متعددی رنج می برد. به همین دلیل به نظر می رسد استفاده از مارکرهای استخوانی (BTMs) می توانند در بهبود تشخیص پوکی استخوان کمک کننده باشند. با وجود پیشرفت های اخیر، فن آوری های موجود برای اندازه گیری BTM، مانند سنجش ایمونوسوربنت متصل به آنزیم (ELISA) و الکتروکمی لومینسنس (ECLIA) ، نه تنها وقت گیر و پر زحمت بوده بلکه به برچسب های خاص و ابزار گران قیمت نیاز دارد. با توجه به این محدودیت های آشکار، تمرکز مطالعات مرتبط با سلامت در سالهای اخیر به سمت توسعه و ساخت حسگرهای زیستی نقطه مراقبت (POC) بدون برچسب، قابل حمل، قابل استفاده مجدد و در عین حال قابل استفاده برای اندازه گیری همزمان چند مارکر به ویژه برای تشخیص و نظارت بیماریهای مختلف می باشد. با توجه به بررسی های انجام شده، تاکنون، هیچ یک از حسگرهای زیستی استخوان موجود در این زمینه موفق نبوده اند. در این پروژه، گزارش ساخت یک میز پروتئومیک میکروسیالی که به راحتی قابل تبدیل به یک دستگاه تشخیص برای هر نشانگری (biomarker) می باشد ارائه شده است. این پلت فرم حاصل ادغام تکنولوژی میکروسیالی با سیستم سنجش الکتروشیمیایی بوده و از یک محفظه واکنش/تشخیص برای اندازه گیری سطح سرمی نشانگرهای زیستی متفاوت تشکیل شده است.
- Abstract
- Osteoporosis diagnosis, which is nowadays generally made based on bone mineral density (BMD) measurements, suffers from certain limitations. Thus it is believed that bone turnover markers (BTMs) can help improve osteoporosis detection. Despite recent advancements in proteomics and personalized medicine, available technologies for BTM measurement, enzyme-linked immunosorbent assays (ELISA) and electrochemiluminescence (ECLIA), are time consuming, laborious, require special labels and expensive instruments. With these apparent limitations, the focus of health-related studies has shifted towards the development of portable, reusable and at the same time multiplex, label-free Point-of-Care (POC) biosensors particularly for diagnosis and monitoring of different diseases. Also known as on-the-spot diagnostic devices, these POC biosensors should be simple, sensitive, rapid, specific, cheap, and easy to interpret. In this work, we report a microfluidic proteomic platform that can easily be translated into a biomarker diagnostic. This platform integrates microfluidic technology with electrochemical sensing and embodies a reaction/detection chamber to measure serum levels of different biomarkers. The unique design of the platform offers the potential for greater sensitivity as the microfluidic and electrochemical structures can be independently optimized. As the first step, through the inherent interaction between gold nanoparticles (AuNPs) and antibody biomolecules, a novel gold nanoprobe was developed to be used in the non-enzymatic electrochemical immunoassay. This one-pot method not only provides a simple method for loading high-content antibody on nanoparticles but also greatly improves the repeatability and controllability of the nanoprobe preparation. The next step was the immobilization of this probe on the electrode surface while saving the structure and functionalization of the antibodies. The deposition of gold nanoparticles (AuNPs) on the surface of gold electrode is believed to enhance the electrochemical characteristics of the surface. Our results showed that electrodeposition of AuNP using cyclic voltammetry improved measured current ratio (resulting in an increase in surface area by 2.3 times and in roughness by 3.49 times). These modified electrodes also showed several advantages with respect to stability and reproducibility (less than 5% variability). We developed a cheap, reliable and rapid method for the fabrication of microfluidic channels using double-sided tapes, enabling not only highly uniform cross-sectional dimensions along the channels but also proper adhesion in hybrid systems, composed of different layers. As the last step, the electrochemical sensor and the microfluidic chips were integrated to develop the Osteokit system. The sensing interface fabrication, sample incubation, and electrochemical detection in this antibody-antigen-based platform were all performed using microfluidic channels. Microfabricated Au electrodes encased in a microfluidic chamber were functionalized to immobilize the antibodies, which can selectively capture the corresponding antigen. Chronoamperometry technique at room temperature was used to measure the concentration of the antigen solutions. Chronoamperometry was used to measure the concentration of the antigens in room temperature. We demonstrated the implementation, feasibility and specificity of this platform (Osteokit) in assaying serum levels of Osteocalcin (Oc) and collagen type 1 cross-linked C-telopeptide (CTX). The detection limit of osteocalcin was 1.94 ng/mL, whereas that of CTX was 2.77 ?g/mL. The device was also used to measure serum levels of the both Oc and CTX and the results were compared to that of ECLIA. The results of which showed acceptable concordance. The developed sensor was sensitive and specific for serum Oc and could detect serum levels of the marker within the range of 2.5 - 100 ng/mL. This is while the normal reference of the marker is 9 - 42 ng/mL, suggesting that the sensor can acceptably detect Oc. Similarly, CTX levels were successfully measured from 1 - 1000 ?g/mL. This is while the normal reference of the marker is 50-450 ?g/mL, suggesting that the sensor can acceptably detect CTX. Our sensor showed no cross-reactivity for other biomarkers (b-CrossLaps and parathyroid hormone (PTH) for Oc sensor and osteocalcin and PTH for CTX sensor). The good correlation between the ECLIA and Osteokit showed that they can be used in the clinically relevant range and other macromolecules available in serum do not affect our results. It was concluded that compared to current state of art (ECLIA and ELISA), the proposed electrochemical sensor resulted in selective and sensitive measurement of the proposed BTM levels with reduced interference reactions, reduced measurement time, need for less amount of sample and better stability in a simplified detection system. Keywords: Osteoporosis, Fracture, Biomarker, Electrochemistry, Gold Nanoparticles, Biosensor, Microfluidic, Osteocalcin, CTX
