SIMPLIFIED NUCLEIC ACID ISOLATION AND DETECTION TECHNIQUES FOR POINT-OF-CARE APPLICATIONS

The first step towards the treatment of any disease is diagnosis; however, a major hindrance towards monitoring diseases remains the availability of rapid diagnostic tests to detect DNA and RNA biomarkers.Unfortunately, traditional assays to test for these analytes are limited to centralized laboratories. For example, the gold-standard sample preparation methods to isolate and purify nucleic acid analytes require multiple hands-on steps, numerous chemicals, and specialized equipment. Recent research endeavors have made incremental progress to simplify these procedures by miniaturizing these steps, but still require the numerous chemicals and bulky peripheral instrumentation for operation. Similarly, gold-standard detection methods, namely quantitative PCR (qPCR), rapidly cycle between near-boiling temperatures, thus mandating the use of sophisticated instrumentation with high power demands. These methods for sample preparation and target detection are overall laborious, time-consuming, and require technical expertise thereby imposing large time, and financial costs for diagnoses. To address these challenges, this dissertation aims to shift the paradigm of how we approach sample preparation for nucleic acid isolation, and to design novel assays for rapid, specific, and multiplexed detection in a single reaction. First, a new sample preparation method is presented that simultaneously accomplishes three time-consuming sample preparation steps (cell lysis, DNA capture, and purification) in less than ten minutes. This platform enables DNA isolation from whole blood droplets, with subsequent amplification and detection of both genomic and pathogenic DNA bound to the microparticle surface. Second, we explore optimizations of a popular isothermal DNA amplification technique that previously demonstrated multiplex detection of bacterial genomes. By developing a triplex assay towards the identification of drug resistant bacteria, we address inhibition that is normally observed with this technique, and present strategies towards achieving amplification within 30 minutes. Lastly, we outline design consideration towards the development of a novel amplification scheme specifically for microRNA targets. This system leverages both DNA and RNA polymerases to achieve positive feedback and thus, requires evaluation of enzymes, sequence design, and buffers to inform assay design. Together, this work advances the development of NAATs towards a simplified, specific and multiplexed system

Improving Assay Performance using Microfluidic Cavity Acoustic Transducers

The development of an integrated immunoassay platform is crucial for providing diagnostic tools for global health applications. With the emergence of microfluidics, there is an increased focus in addressing this need. While many platforms have been created to successfully complete an immunoassay, they still require bulky external equipment to manipulate fluid within the device. Using technology developed in the BioMiNT lab, we've created an immunoassay platform that is capable of on-chip pumping and mixing with lateral and vertical-cavity acoustic transducer (LCAT & VCAT respectively) coupled to a piezoelectric transducer (PZT). PDMS devices were fabricated, molded and bonded to glass slides adhered with antigen-spotted nitrocellulose pads. Once they are primed with blocking buffer to introduce air bubbles, the devices are actuated with a PZT to drive fluid pumping and mixing. The reagents are pumped into the device serially and after completion, the spotted antigens on the pad become a dark purple hue, indicating an antibody-antigen binding event. For quantitative results, the intensities are evaluated using computer software. Results show that the resulting spot intensities are comparable to an established, optimized microarray platform in one-fifth of the time. We also show that these results cannot be reproduced in a passive or flow-through microfluidic device, indicating that convective mixing is necessary for improved signal intensity. Using the LCAT/VCAT platform, an immunoassay can be performed in 20 minutes and with increased sensitivity. Future work will involve expanding the device to allow for an automated point-of-care platform

Improving Assay Performance using Microfluidic Cavity Acoustic Transducers

The development of an integrated immunoassay platform is crucial for providing diagnostic tools for global health applications. With the emergence of microfluidics, there is an increased focus in addressing this need. While many platforms have been created to successfully complete an immunoassay, they still require bulky external equipment to manipulate fluid within the device. Using technology developed in the BioMiNT lab, we've created an immunoassay platform that is capable of on-chip pumping and mixing with lateral and vertical-cavity acoustic transducer (LCAT & VCAT respectively) coupled to a piezoelectric transducer (PZT). PDMS devices were fabricated, molded and bonded to glass slides adhered with antigen-spotted nitrocellulose pads. Once they are primed with blocking buffer to introduce air bubbles, the devices are actuated with a PZT to drive fluid pumping and mixing. The reagents are pumped into the device serially and after completion, the spotted antigens on the pad become a dark purple hue, indicating an antibody-antigen binding event. For quantitative results, the intensities are evaluated using computer software. Results show that the resulting spot intensities are comparable to an established, optimized microarray platform in one-fifth of the time. We also show that these results cannot be reproduced in a passive or flow-through microfluidic device, indicating that convective mixing is necessary for improved signal intensity. Using the LCAT/VCAT platform, an immunoassay can be performed in 20 minutes and with increased sensitivity. Future work will involve expanding the device to allow for an automated point-of-care platform

Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles

Tremendous advances have been made in the development of portable nucleic acid amplification devices for near-patient use. However, the true limitation in the realization of nucleic acid amplification tests (NAATs) for near-patient applications is not the amplification reaction, it is the complexity of the sample preparation. Conventional approaches require several precise intervention steps during the protocol. There are numerous reports in the literature that mimic the sample preparation procedure within a lab-on-a-chip device or cartridge, but these systems require a high number of integrated steps, making the devices and/or their supporting equipment too complex to meet the necessary cost targets and regulatory requirements for near-patient applications. Here we report a simplified method to purify and amplify DNA from complex samples in a minimal number of steps. We show that chitosan-coated microparticles can lyse human cells and capture the released DNA in a single mechanical agitation step, and we show that bound DNA can be amplified directly from the microparticle surface when the magnetic microparticles are transferred to a polymerase chain reaction (PCR). This procedure eliminates (i) the use of PCR-inhibiting reagents (e.g., chaotropic salts and alcohol) and (ii) the washing and elution steps that are required to remove these reagents and release DNA in typical NAAT sample preparation methods. To illustrate the use of this direct PCR method in diagnostics, we amplify human genomic DNA sequences from a ∼1 μL droplet of whole blood, and we amplify plasmid DNA spiked into whole blood droplets to represent circulating viral DNA or cell-free DNA. The qPCR threshold cycle for direct PCR from whole blood is comparable to that of direct PCR with purified DNA, demonstrating that the lysis and capture steps effectively bind DNA and sufficiently enable its amplification. Furthermore, the efficient amplification of plasmid DNA spiked into whole blood proves that the large mass of human genomic DNA captured from the lysed cells does not inhibit the capture and amplification of other circulating DNA. We anticipate that this new streamlined method for preparing DNA for amplification will expand the diagnostic applications of nucleic acid amplification tests, in particular for near-patient applications