تك زيست‌حسگر DNA براي تشخيص اهداف نوكلئيك و غيرنوكلئيك اسيدي براساس واكنش گسترش آغازگر

A Primer Extension reaction driven DNA Biosensor for the detection of nucleic an‎d non-nucleic acid Targets

شيمي تجزيه

Among various types of biosensors, electrochemical DNA-based biosensors hold a prominent position due to their possession of the highest levels of sensitivity an‎d selec‎tivity. Accordingly, the primary objective of this thesis is to design a DNA biosensor capable of detecting an‎d responding to a wide range of targets, including both nucleic acids an‎d non-nucleic acid targets.The key innovation of this method is the employment of the Primer Exchange Reaction (PER) to convert any target input into a specific DNA stran‎d with a predetermined sequence. This mechanism completely eliminates the need for the costly an‎d time-consuming process of re-optimizing the sensor for each new target. In fact, by using this programmable an‎d automated approach, which utilizes catalytic DNA hairpin structures, the simultaneous detection of non-nucleic acid markers an‎d nucleic acids within a single system is made possible. In this reaction, the primer binds to these structures an‎d, with the help of polymerase, the DNA stran‎d is extended in a pre-defined sequence before automatically dissociating to allow the reaction to repeat. Subsequently, in each biosensor, after synthesizing complementary sequences via the PER method, these sequences were hybridized with thiolated stran‎ds on a disposable gold electrode fabricated from an archival CD. Finally, detection was carried out using an integrated bipolar electrochemical method combined with electrochemiluminescence in a single system (BPE-ECL). This method holds significant potential for versatile an‎d rapid molecular diagnostics. For nucleic acid detection (such as specific genomic sequences of SARS-CoV-2), the following process was performed: Genomic RNA was first reverse transcribed into cDNA (A single-stran‎ded DNA copy is synthesized from RNA through reverse transcription). During the Primer Extension Reaction (PER), the target sequence was tagged with a universal sequence (e.g., AGGGTT). The extended product was then hybridized with a thiolated probe immobilized on the bipolar electrode.The electrochemical response was manifested through light emission, which was quantitatively detected using a digital camera. The developed system demonstrated a wide linear detection range from 10⁻⁷ to 10⁻¹⁷M with a low detection limit of 2.31 × 10⁻¹⁷ M. The capability to distinguish between clinical samples with different threshold cycle (Ct) values without requiring complex instrumentation. All measurements were performed on an open bipolar electrochemiluminescence (BPE-ECL) system, highlighting the platformʹs simplicity an‎d effectiveness for clinical diagnostics. For non-nucleic acid targets (such as breast cancer cells), we employed an integrated PER-aptamer strategy utilizing an "apta-primer" system. The apta-primer (e.g., AS1411) specifically binds to nucleolin expressed on cancer cell surfaces. Following target recognition, it initiates a Primer Extension Reaction (PER) to attach the same universal DNA tag (AGGTTT) previously implemented for COVID-19 detection, thereby establishing a stan‎dardized detection framework. The resulting electrochemiluminescence (ECL) signal demonstrates a direct linear correlation with target concentration. Measurements were acquired using a photomultiplier tube (PMT). Implemented on a fully integrated, closed bipolar electrochemistry system featuring: Elimination of external driving electrodes. Minimal electrolyte consumption (10 µL). This system shows a detection limit of 4 cells/µL in a linear range of 12 to 60 cells/µL. This biosensor is capable of simultaneously distinguishing cancer cells from normal cells in mixed samples with different cell percentage ratios. The core feature of this platform is its consistent bipolar electrode design, which remains unchanged across different targets. A broad spectrum of analytes can be detected simply by modifying the primer extension reaction (PER) input.

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