Instrument-Free Quantitative Bioassays Based On Microfluidic Paper-Based Analytical Devices

Instrument-free quantitative bioassays have attracted increasing attention in recent years due to their many useful features.Key among these features includes low cost and portability,which make them ideally suitable for point-of-care use in less-industrialized countries.In this thesis,several new instrument-free quantitative bioassays based on distance-measuring readout in microfluidic paper-based analytical device??PAD?platform have been developed for detection of three initial model analytes:Cu²⁺,alkaline phosphatase and denosine,and described as follows:?1?In Chapater 2,a distance-measuring method for equipment-free quantitative detection of Cu²⁺ion has been proposed for the first time.In the presence of Cu²⁺,the activity of glucoamylase for hydrolyzing soluble starch is inhibited,and the starch remains its poor water solubility and thus changes hydrophilicity of the addition zone in a strip-like?PAD into hydrophobicity.The flowing of the red ink solution through the addition zone is limited and a short flowing length would be obtained in the detection zone of the paper device.The ink's flowing length in the detection zone negatively relies on the Cu²⁺level in the sample.The results demonstrate that this proposed instrument-free method requires only a ubiquitous cheap ruler?rather than any expensive,complex instruments?to quantify the concentration of Cu²⁺ion.Under optimal conditions,this new method is quantitatively sensitive to Cu²⁺concentrations ranging from 3.2×10^-9 to 5.0×10^-55 M,with a visual detection limit of 3.2×10^-99 M.?2?Encouraged by the good results of Chapter 2,a distance-measuring method for equipment-free quantitative detection of alkaline phosphatase?ALP?activity on a strip-like?PAD has been proposed in Chapter 3,by combining the special and strong pyrophosphate acid?PPA?-Cu²⁺complexation reaction with the ALP-catalyzed hydrolysis of PPA.In the absence of ALP,after the complexation of Cu²⁺ion by the PPA,the glucoamylase could effectively hydrolyze the soluble starch to produce glucose.Since the glucose is highly water-soluble and could not enhance the hydrophobicity of the addition zone in the paper device,the red ink solution is able to flow rapidly through this zone into the detection zone to produce a long flowing length.The ink's flowing length in the detection zone positively depends on the ALP activity in the sample.Under optimized conditions,this new method is quantitatively sensitive to ALP levels in buffer samples ranging from⁰.037 to 5 U/mL with a visual detection limit of 0.037 U/mL.Moreover,this new approach shows high specificity to the ALP.In addition,satisfactory recoveries are obtained in the assays of human serum samples.To the best of our knowledge,this is the first report of quantifying ALP activity without using any external electronic instruments.?3?In Chapter 4,an equipment-free quantitative aptamer-based assay has been developed initially by combining the glucoamylase-starch catalysis reaction-mediated wettability change of paper and the strip-like?PAD-based distance-measuring strategy developed in Chapters 2 and 3.It additionally uses superparamagnetic bead platform for minimizing non-specific effect.This new method is conducted for the analysis of adenosine as a model analyte and red ink as the colorful detection reagent.The ink's flowing length in the detection zone of paper devices positively depends on the adenosine level in the sample.The results show that the developed approach for adenosine detection has a liner concentration range of 1.7⁶2.5?M,with a detection limit of ca.1.6?M.Moreover,it shows excellent assay specificity.The recovery results of assaying human urine samples are also satisfactory.