Development of Flow Controllable Paper Sensor and Film-Formed Sensor for Enhancement of Color Uniformity and Reagent Stability

Abstract

The prevalence of chronic diseases such as diabetes mellitus (DM) and gout which are necessary for sustainable care and monitoring the patient condition, is increasing sharply. As a monitoring tool of chronic diseases, pointcare-of testing (POCT) which has advantages including simple, cost-effective, and rapid was employed. However, most of POCTs have been required high-performed biochemical analyzer, technician, and labile materials (chemicals, enzymes, and antibodies). Therefore, instrument-free, easy-to-use, and the stabilization method of labile components in the sensor were essentially required.

To develop a more adaptable platform to POCT, microfluidic paper-based analytical devices (μPADs) were employed. μPADs offering low-cost, portability, simplicity, and rapid, induced the flow of fluid using mesoporous materials like paper without external power. Depending on the method of detection, platforms were classified as optical (including color, luminescence, fluorescence), electric, and electrochemical. Among these methods, colorimetric detection is one of the most popular because it is a rapid and simple method.

However, the colorimetric detection using water-soluble color-forming reagents in μPADs was suffered by washing the colorimetric signal to the edge of the sensor. Because the washout of colorimetric signal affected the reproducibility and sensitivity of the sensor, many researchers have been reported the advanced platforms for preventing the signal washout. Although these reports significantly increased the uniformity of signal, the additives for color uniformity reduced the stability of the enzyme, and washout of the signal was not perfectly solved.

This dissertation reports and demonstrates the development of flow-controllable and stable bio-sensing platforms using chitosan oligosaccharide lactate (COL) and polyvinyl alcohol (PVA). Each chapter included (i) colorimetric signal focusing effect using induced asymmetric flow on the paper membrane by COL, (ii) quantification of 1,5-anhydroglucitol using glucose elimination of enzyme bridge, and (iii) stabilization of labile reagents by PVA film fabrication.

In Chapter 2, the mechanism of signal focusing without colorimetric signal loss, the introduction of glucose and uric acid, and quantification of targets were shown. To prevent the washout of color signal, the chitosan oligosaccharide lactate (COL) was employed, and mixed with enzymes and color-forming reagents. After spotting the reaction mixture with COL, the mesoporous structure of the membrane was blocked and reduced by COL. The flow rate of the COL-treated area dynamically decreased, and this phenomenon induced the asymmetric flow between the COL-treated area and the non-treated area. Thus, the colorimetric signal was well focused without signal loss by the asymmetric flow on the paper membrane. Besides, COL showed the highest decreasing rate of flow rate than the other chitosan derivatives. Using the induced color-focusing effect by COL, glucose, and uric acid which are biomarkers for DM and gout were quantified in water and human urine.

In Chapter 3, a glucose elimination bridge was used to quantify the 1,5-anhydroglucitol (1,5-AG). One biomarker for DM, 1,5-AG is not mostly metabolized in the human body, and its blood levels are maintained by oral intake and urinary excretion. The re-absorption of 1,5-AG competes for the glucose, thus, it was gradually decreased by the increment of glucose level in blood. Therefore, the level of 1,5-AG reflects the status of hyperglycemia and was used to monitor the DM. However, pyranose oxidase (PROD) as the essential enzyme for quantifying the glucose could not distinguish between 1,5-AG and glucose. To eliminate the interference from glucose, conventional methods have multiple steps for the elimination of glucose and colorimetric signal generation from 1,5-AG. In this chapter, one step 1,5-AG quantification was demonstrated by using enzyme-based glucose elimination to fabricate a simple and rapid 1,5-AG sensor.

In Chapter 4, the film-formed biosensor for stabilizing the labile reagents (chemical, antibody, and enzymes) was fabricated using polyvinyl alcohol (PVA). As a biocompatible and water-soluble polymer, PVA was mixed and cast to fabricate the film with the labile reagents including glucose oxidase, horseradish peroxidase, 4-AAP, and MADB. After drying in a vacuum, the reagents film was peeled and resized. To prevent the effect of oxygen and humidity, the film was packaged using vacuum conditions. As a result, the stability of reagents is perfectly retained at 4℃ and 25℃ for 5 months, while the stability without PVA sharply decreased by exposing at 25℃. In addition, the fabricated film for glucose detection was rehydrated using the sample solution. Following the concentration of glucose in the sample, the intensity of the colorimetric signal increased.