Development of DNA aptamers in antibody-antigen complex, and their application in biosensor for detecting various biomarkers

Abstract

Biosensors are analytical devices that combine biological components with physicochemical methods to detect various chemical signals in complex environments. All biosensors include a bioreceptor which is designed to interact with and detect the target signal. Bioreceptors are a fundamental component of biosensors and are responsible for both the selectivity and sensitivity of the respective biosensors. However, conventional bioreceptors, including antibodies, have limitations in their detection of various biomarkers at low concentrations in bodily fluids. In order to overcome this limitation, this study focused on the development of DNA-based bioreceptors (i.e. aptamers) selected by newly developed method and applied in various sensitive detection assays. Chapter 2 describes the selection method applied in the development of this study’s heterogeneous sandwich-type biosensors. Enzyme-linked immunosorbent assay (ELISA) is widely applied to detect various biomarkers in body fluids. Here, enzyme-linked secondary antibodies or aptamers serve as detection reagents for signal amplification or colorimetric analysis. Aptamers have similar target affinities and specificities as antibodies, and are relatively easy to label making them applicable in a number of methods other than ELISA. For this reason, many quantitative analysis methods have recently focused on the development of heterogeneous sandwich format assays that use a capture antibody with high specificity for a target, and aptamers as the detection probe for signal amplification. However, the application of aptamers selected using the conventional SELEX method in heterogeneous sandwich-type assays requires additional screening procedures to increase signal to noise ratios between the aptamer and the antibody. To avoid this complex procedure, there is a need for specially designed aptamer selection systems. Here we developed one such method and applied it to the identification of various target-specific aptamers which were shown to have high binding affinity for their targets. Using this method, we developed four aptamer candidates with Kd values ranging from 77.6 to 125.7 nM against NP of influenza A virus. Novel DNA aptamers specific for the nucleocapsid protein (NP) of severe fever with thrombocytopenia syndrome (SFTS) virus were also developed using this selection method. In addition, a total of four more aptamers specific to the influenza B virus nucleoprotein (NP) were designed and optimized using this selection strategy. Finally, aptamers specific for the p24 protein of HIV-1 and the NP of Ebola virus were also developed using this method. These aptamers demonstrated that our advanced SELEX method can be used to successfully screen heterogeneous sandwich-type bioreceptors while reducing costs and development time. Chapter 3 describes the development of biosensors on various platforms using the DNA properties of the selected aptamers and applying protein engineering methodologies to complete the sensors. A key requirement of a biosensor is high selectivity and sensitivity for the target analyte in a matrix of other chemicals or biological components. In an attempt to increase sensitivity and specificity, biosensor technology has evolved considerably from conventional ELISA. Advanced point-of-care testing (POCT) immunosensor technologies have been developed for both in situ diagnostics as well as laboratory-scale testing. These use two or more different bioreceptors, and various novel labels conjugated to their detection elements. To amplify the signal generated in the immunocomplex, the bioreceptor may be altered to increase target affinity. Here we developed two novel sensitive sensing platforms using the characteristics of our aptamers for application in laboratory-scale and POCT, respectively. In each case, the sensitivity was improved more than 10 times when compared to the conventional methods, and the efficacy of the novel methods was confirmed in clinical samples. Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used POCT technologies. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet the sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in bodily fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we developed a colorimetric sensing platform using gold nanoparticles and aptamers. In this method, capture antibodies were immobilized on the LFIA and an aptamer was used as the detection probe in heterogeneous sandwich immunocomplex. Then, single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs were used to detect the aptamers, thus, enabling signal amplification through multiple AuNP interactions per aptamer. This technique showed significantly higher sensitivity than conventional methods widely used in LFIAs. In addition, we developed a high sensitivity sensing platform for laboratory-scale applications, this method relies on the ultrasensitive detection of target molecules through heterogeneous sandwich-type recombinase polymerase amplification using target-specific aptamers. The detection of protein biomarkers present at very low concentrations in the body is very important in diagnostics. Here we developed a sensing platform that selectively amplifies only the DNA bound to the target in the form of a heterogeneous sandwich and detects the fluorescent signal emitted from these complexes following the addition of an intercalating dye enabling high specificity, ultra-sensitive target detection. This method was applied in the detection of various infectious disease protein biomarkers including influenza A, influenza B, HIV, and Ebola. This method was shown to facilitate attomolar-level detection of biomarkers as well as low cross-reactivity between different types of viruses. In addition, this platform was evaluated for the clinical application in influenza A and B detection. In this dissertation, we discuss the target-specific aptamer selection methodologies and detection strategies developed and evaluated in this study. We were able to develop a novel SELEX method, which could be used to create aptamers that were successfully substituted for other bioreceptors in heterogeneous sandwich-type biosensor assays. These alternative assays were then validated using clinical samples and were shown to enhance the detection of various infectious diseases even in ultrasensitive applications. The work described here will help to develop novel ultrasensitive sandwich based assays that can be used for more reliable diagnosis of various diseases.