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INNOVATIVE NUCLEIC ACID BASED STRATEGIES FOR FIBER OPTIC SURFACE PLASMON RESONANCE BIOSENSOR DESIGN

Boek - Dissertatie

The diverse information embedded in the specific sequence of DNA molecules has been feeding our understanding and insights in disease heritage, spreading and evolution. Ultrasensitive detection of the DNA molecules themselves is crucial for the early-stage detection of several diseases, as it facilitates the minimization of actual disease burden. Gold standard technologies, such as the polymerase chain reaction (PCR), are mainly restricted to sophisticated laboratory settings, relying on trained personal, bulky equipment and large batches of samples. In order to improve the accessibility of nucleic acid (NA) analysis and provide its valuable information outside these sophisticated settings, alternative biosensors are being developed. These biosensors can offer more user-friendly, robust, fast and cost-effective devices, thereby focusing on the needs of the end-user. In addition, biosensors do not limit themselves to the use of DNA as target molecule. Instead, the unique structure of DNA is also exploited as a building block for smart biosensor design. Fiber optic surface plasmon resonance (FO-SPR) is a mass-sensitive biosensing technique with some advantageous characteristics, such as its real-time aspect, user-friendliness and fast analysis. While these FO-SPR characteristics match several of the features mentioned above, DNA-based FO-SPR assays are mainly restricted to classical hybridization strategies, limiting the sensitivity to the lower nM range. Ultrasensitive detection of NAs continues to be challenging and alternative FO-SPR designs to generate and amplify this signal have not been fully explored. Upcoming fields like DNA nanotechnology drive these innovative designs by discovering new functional molecules, such as DNAzymes. These enzyme-like molecules are of great interest in biosensor development due to their easy coupling to existing signal generation systems and their inherent amplification. Because of their NA-based structure, they are more stable, cost-effective, and show superior flexibility compared to their protein counterparts in their design towards different target molecules. However, DNA nanotechnology is not yet mature and surface-based DNAzyme strategies have been concentrating mainly on metal ion detection. NA and protein applications are rare and demand more advanced design and implementation of current DNAzyme-based strategies. In this framework, the main objective of this dissertation was to design innovative NA-based strategies using a FO-SPR platform for the sensitive and specific detection of NAs. To do so, three different NA-based strategies were presented: (i) a FO-PCR melting assay, (ii) a FO-SPR sensor design sensitive to catalytic DNAzyme activity and (iii) a DNAzyme-based FO-PCR assay. The first design was based on PCR amplification of the target sequences, which subsequently hybridized to the FO sensor surface and DNA-functionalized gold nanoparticles (AuNPs) for amplification. As a proof-of-concept, the assay was explored for the detection of celery DNA, one of the most important provokers of food allergic reactions. Different concentrations of celery DNA (1 pM - 0.1 fM) were detected and inclusion of a following melting step enabled distinction from closely related sequences, such as carrot DNA. The concept was validated in cleaning water samples and benchmarked against a reference qPCR followed by high resolution melting, which showed excellent agreement (R² = 0.96). This way the FO-PCR melting assay provided a rapid and simple detection method, suitable for one-step quantification and discrimination of single stranded DNA (ssDNA) with great potential in fields such as food quality and safety assurance. For the second design, first a robust ligation strategy was developed to functionalize the FO-SPR sensing surface with ssDNA, which in turn was labelled with AuNPs, to serve as the DNAzyme substrate. Next, we studied the relation between the change in FO-SPR shift and the DNAzyme cleavage activity, which showed faster cleavage kinetics for higher DNAzyme concentrations. Finally, we translated this real-time cleavage activity into a generic biosensing concept for the detection of ssDNA targets. A DNAzyme-inhibitor complex was designed to release active DNAzymes in a controlled way as function of the target concentration. Reproducible target detection was demonstrated with a theoretical LOD of 1.4 nM. In summary, an innovative DNAzyme-based FO-SPR biosensor was established with great potential to act as universal sensor for promising applications in the medical and agrofood sector. The final design in this dissertation combined insights from both the first and the second design by boosting the catalytic cleavage activity through PCR-amplification of the DNAzymes. Based on the readout system of the second design, the surface cleavage efficiency of the DNAzyme-extended amplicons (DNAzyme-amps) was first evaluated and confirmed. Next, the PCR and cleavage reaction conditions were fine-tuned to assure compatibility with the FO-SPR system and establish a one-step assay. As a proof-of-concept, the primer pair with the built-in DNAzyme complement was tested for the detection of the antimicrobial resistance gene MCR-2. PCR-amplified DNAzyme-amps generated in the presence of the MCR-2 gene were monitored in real-time, resulting in an experimental LOD of 4 × 10^5 copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, further proving the specificity of this assay. In short, this DNAzyme-based FO-PCR assay provides a universally applicable, real-time system for the detection of virtually any NA target, in a specific and sensitive manner.
Jaar van publicatie:2020
Toegankelijkheid:Open