New Bioassays Based On Functional Nucleic Acids And Microfluidic Paper-based Analytical Devices

Functional nucleic acid refers to a class of nucleic acid molecues that can specifically bind analytes of interest or have a unique catalytic ability. Due their avantages of excellent biocompatibility, flexible designs of signal transduction mechanisms and ease of synthesis,etc., in recent years the functional nucleic acid-based molecular probe has attracted increasing attention. A great number of new bioasssays and biosensors based on biofunctional nucleic acids have been developed. Nevertheless, most of them can not allow for quantitative analysis or have to use expensive equipments to provide quantitative results. Inorder to addree these issues, herein low-cost microfluidic paper-based analytical device(μPAD) have been introduced to develop new equipment-free quantitative bioassays based on functional nucleic acids. The details are summarized as follows:The Chapter 2 describes a portable peroxidase-mimicking hemin/G-quadruplex DNAzyme bioassay that integrates a μPAD with a timing quantitative detection motif based on the DNAzyme-mediated wettability change of paper from hydrophilic to hydrophobic.Using K+ as an example, we demonstrate that this method only requires an quite inexpensive timer or a cell phone with a timing function and the ability to see color to allow sensitive,selective, portable quantification of K+ in artificial samples as well as in complex matrices such as human serum. The K+ assay method involves three core operations: formation of the peroxidase-like K+-stabilized hemin/G-quadruplex DNAzyme in solution; DNAzymecatalyzed reactions between H2O2 and TMB that is pre-loaded in the paper device; and measurement of time required for a colored solution(i.e., hydrophilic red ink used herein) to flow through a paper zone of a specified length. The poly-TMB products can change the wetting property of the TMB-loaded zone from hydrophilic to hydrophobic and in turn prolong the flow-through time of the red ink solution. The change level of paper wettability and the ink’s flow-through time depend on the K+concentration in the sample. The obtained calibration curve has two linear sections, i.e., 500 nM ~ 25 μM and 25 ~ 200 μM. The limit of detection for K+ was estimated to be ~490 nM using the first equation according to the 3σ rule.Moreover, it showed a good detection reproducibility. The results obtained from the recover tests of diluted human serum samples further validated its acceptable reliability and practicability.In Chapter 3, a new quantitative, point-of-care(POC) aptamer-based assay without using external electronic readers was proposed by using the μPAD. Visible identification and quantification of a model analyte, adenosine, is realized by simply counting the number ofμPAD’s microzones stained with detection reagent that become colorless. The design integrates two signal amplification processes, namely hybridization chain reaction and enzyme-catalyzed reaction. Magnetic separation/enrichment technique is additionally adopted to enhance detection specificity. This POC assay only requires the ability to see color and the ability to count in order to measure the level of adenosine in sample. It is quantitatively sensitive to adenosine concentrations ranging from 1.1 μM to 17.5 mM, with a detection limit of 1.1 μM. Multiple linear concentration ranges to meet different customized requirements could be further expected via adjustment of the colored detection reagent concentrations deposited in the μPAD’s microzones. This equipment-free method described herein is simple, low-cost, quantitative, portable, and thus offer new opportunities for the development of POC aptamer-based assays for use especially in resource-poor environments including less-industrialized countries and remote regions.