Development of Diagnostic Biosensors Using Aptamers and Nucleic Acid Amplification Strategies

Abstract

Biomarkers are measurable indicators of certain biological states or conditions, such as antigens, DNA, mRNA, enzymes, etc. Measuring the concentration of biomarkers in a biological environment provides information to predict early the risk or progress of a disease. Also, it provides the degree of effectiveness of a treatment process so that proper treatment can be chosen. Accordingly, a great deal of effort in improving human health is focused on developing reliable, cost-effective, and powerful analytical methods to detect biomarkers. It is important to select an appropriate probe and amplification strategy to establish such detection methods because of the low abundance of biomarkers, and complex physiological environment. In this Doctoral Thesis, aptamer was selected as a recognition probe for biomarker detection and its intrinsic characteristics were utilized to amplify signals. Prior to presenting the development progress and results, Chapter Ⅰ introduces aptamer as a newly emerging recognition material, and explains its advantages and diagnostic applications in detail. In addition, various signal amplification strategies using aptamers and nucleic acid amplification techniques will be covered. Chapter Ⅱ and Ⅲ present each research on the development of a detection method based on the above considerations in earnest. The leverage of aptamers and signal amplification strategies enabled highly sensitive and efficient detections of protein biomarkers in a complex media (serum or urine), overcoming the current diagnosis methods for various diseases.

Chapter Ⅱ. Safe and sensitive detection of ESAT6 in non-infectious clinical samples via an aptamer-based qPCR strategy for tuberculosis diagnosis Tuberculosis (TB) is one of the leading causes of infection-related deaths worldwide. Conventional diagnostic methods for TB require infectious samples and are time-consuming. In Chapter Ⅱ, I aimed to develop an aptamer-based qPCR (Apta-qPCR) assay to diagnose TB safely and rapidly. The assay uses a single-stranded DNA aptamer with an affinity for a TB biomarker, ESAT6, and molecular beacon capable of hybridizing to the aptamer. Apta-qPCR enables aptamer-mediated detection of ESAT6 via qPCR, wherein the aptamer serves as both a target recognition agent and template for quantification. This diagnostic method achieved a desirable detection limit of 2.56 nM in serum-spiked conditions with high reproducibility and reliability. In addition, the diagnostic performance was verified in a clinical application using human serum samples from normal controls and patients with active TB. The assay showed 50.0% sensitivity, 91.7% specificity, and 78.1% accuracy, similar to sputum smear microscopy, which is currently the most used method for TB diagnosis. Importantly, this assay has an advantage over smear microscopy in that it reduces the risk of infection by avoiding the use of sputum-containing infectious pathogens.

Chapter Ⅲ. Aptamer-antibody hybrid ELONA that uses a hybridization chain reaction to detect a urinary biomarker EN2 for bladder and prostate cancer In Chapter Ⅲ, I suggest an EN2-specific (Kd = 8.26 nM) aptamer, and a sensitive and specific enzyme-linked oligonucleotide assay (ELONA) for rapid and sensitive colorimetric detection of bladder and prostate cancer biomarker EN2 in urine. The assay relies on an aptamer-mediated hybridization chain reaction (HCR) to generate DNA nanostructures that bind to EN2 and simultaneously amplify signals. The assay can be performed within 2.5 h and has a limit of detection of 0.34 nM in buffer and 2.69 nM in artificial urine. Moreover, this assay showed high specificity as it did not detect other urinary proteins, including biomarkers of other cancers. The proposed ELONA is inexpensive, highly reproducible, and has great chemical stability, so it may enable the development of a simple, sensitive, and accurate diagnostic tool to detect bladder and prostate cancers early.