The Development of Portable Microfluidic Platform with Distance-based Display for Visual and Quantitative Detection of Analytes

開發基於距離顯示的便攜式微流體平台用於分析物的視覺和定量化檢測

Student thesis: Doctoral Thesis

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Award date29 Nov 2022

Abstract

Microfluidic systems with the advantages of miniaturization, less sample consumption, and high throughput have earned considerable attention over the past decades. It is designed to integrate all the processes required for biochemical detection, such as sampling and pretreatment, reaction and signal output, and realize its automation and integration on a miniaturized chip. As a research field covering interdisciplinary backgrounds, microfluidic technology has advanced the performance of detection and analysis in areas of environmental monitoring and early diagnosis of cancers for personalized medicines. Moreover, with the urgent demand for real-time, on-site detection, efforts dedicated to improving its portability and affordability for unskilled end users have been made with the ease of handling and providing a user-friendly interface as signaling. However, most developed platforms enabling quantitative measurement are based on the reporting of fluorescence or electrochemical signals, which requires interpretation by professional trainees and lacks portability. On the other hand, portable devices with high portability are limited to providing qualitative or semi-quantitative results which cannot meet the demand for biosensing. Therefore, a portable device with a user-friendly interface and the capacity of providing quantitative measurement is urgently needed. In this thesis, a portable microfluidic device with a distance-based display was developed based on the accumulation of polystyrene microparticles (PMPs) by forming a trapping bar that can be visualized by the naked eye. First, the proposed device was applied for lead (Pb2+) and cadmium (Cd2+) detection. Moreover, an integrated automated microfluidic platform was proposed to make it in a ready-to-use state and more accessible to the end-users, which was applied for the simultaneous detection of multiple heavy metal ions in the water sample. Finally, to explore the application of the microfluidic platform with distance-based display for cancer diagnosis, the diagnosis of malignant melanoma has been investigated by combining the microneedle patch for interstitial skin fluid (ISF) sampling with the microfluidic device for visual and quantitative detection of S100A1 protein, a biomarker for the malignant melanoma. The detailed experimental design and primary results and conclusions can be as follows.

First, the portable microfluidic device with a distance-based readout was applied for lead ion detection. By providing a simple, rapid, label-free, and power-free microchip, this platform enables the visual quantification of lead ions (Pb2+) by the naked eye. GR-5 DNAzyme (Deoxyribozyme) was utilized to connect magnetic microparticles (MMPs) and PMPs by its two termini that can hybridize to single-strand DNA probes on microparticles, forming “MMPs-GR-5-PMPs”. When Pb2+ is present, it cleaves the GR-5, resulting in an increasing number of free PMPs. To count it, the solution was loaded into a capillary-driven microfluidic device consisting of a magnetic separator that removes “MMPs-GR-5-PMPs”. After that, the free PMPs continue to flow until being trapped by the nozzle with a diameter of 8 μm, forming a visual bar by PMP accumulation with a growing length proportional to the concentration of the lead ion. With this method, a 2.12 nM (0.44 ppb) limit of detection (LOD), together with high selectivity (>20,000-fold) against other metal ions including Nickel (Ni2+), Copper (Cu2+), Cadmium (Cd2+), Calcium (Ca2+), Barium (Ba2+) and Mercury (Hg2+) has been achieved. In addition, the tolerance towards different pH and water hardness and the compatibility with tap water have been demonstrated, indicating its feasibility for rapid screening of water safety.

Second, to extend the broader application of the portable microfluidic platform toward other heavy metal ions present in the water sample, the detection of cadmium ion (Cd2+), another toxic metal ion widely found in water, soil, and food, has been conducted. To improve the analytical performance, an enzyme-free signal amplification strategy, catalytic hairpin assembly (CHA), was employed to amplify the single strand released from cadmium-dependent DNAzyme. Cd2+-dependent DNAzyme (Cd16) will be cleaved in the presence of Cd2+ to release single-stranded DNA that can trigger catalytic hairpin assembly (CHA) with two hairpins, H1 and H2, as building blocks. The H1H2 complexes, products after Cd2+-mediated CHA, can link MMPs and PMPs to form “MMPs-H1H2-PMPs” sandwich structures. To provide a visual readout to quantify particle connection, the solution was loaded into the microfluidic chip where the magnetic separator first removes MMPs and connected PMPs. Free PMPs can continue to flow until accumulated at the particle dam. The device has a detection limit of 11.3 nM for Cd2+, selectivity >200-fold for other metal ions, high tolerance to the interferences presented in the water, and high recovery (>80%) in tap water.

Third, although a number of portable devices with user-friendly interfaces have been developed, one of the concerns becomes that the materials used for reactions in those platforms should be freshly prepared and the pipetting, mixing, and washing steps involved are inevitably required prior to conducting measurement, which is impractical for the general public who lack professional training and make it far away from “ready-to-use” state. To address it, a fully integrated platform consisting of three chambers in the macro-scale for automatic sample metering, reaction, and separation, respectively, and a micro-scale trapping channel for PMPs accumulation as signaling were proposed. The reaction substances including DNAzymes, MMPs, and PMPs with modified probes were preloaded into respective macro-scale chambers and freeze-dried. DNAzymes will be cleaved with the presence of target metal ions, resulting in disconnection between MMPs and PMPs, while the “MMPs-DNAzyme-PMPs” sandwich structure will be formed when target metal ions are absent. To sequentially control the sampling and reaction procedures, the capillary valves were employed. The limit of detection of 103.1 nM, 69.5 nM, and 793.6 nM as well as high selectivities of 100-fold, 200-fold and 20-fold against interferences, were obtained towards Cu(Ⅱ), Pb(Ⅱ) and Ag(Ⅰ) respectively. Moreover, by integrating three identical channels in parallel, simultaneous detection of the above-mentioned heavy metal ions in both fresh water and tap water samples was also investigated with high recovery rate ranging from 43.8% to 128%.

Finally, to explore its potential use for cancer diagnosis, the microchip with the capability of providing visual, quantitative detection was combined with the microneedle patch with minimally invasive sampling characteristics for the diagnosis of malignant melanoma. Tissue biopsy, as one of the traditional diagnosis approaches, is highly dependent on immunohistochemistry to indicate the occurrence of cancer cells and provide quantitative results to evaluate the efficacy of therapy, which are painful for the patient. Using the microneedle patch to directly attach it to the tumor site and extract the S100A1 protein that has been reported to be highly expressed in malignant melanoma tissues and low in benign melanoma tissues can substantially reduce the patient's painfulness and improve sensitivity. After that, the concentration of S100A1 extracted from interstitial skin fluid (ISF) will be detected based on two antibody-conjugated microparticles, namely, MMPs and PMPs, to form “MMPs-S100A1-PMPs” structure and loaded into the microchip with particle dam where the trapping distance can be correlated with the expression level of S100A1 in the original tumor, thereby providing the information for diagnosis and prognosis of melanoma.

In summary, the microfluidic platform with a distance-based readout has enabled visual and quantitative measurement of heavy metal ions, and a fully integrated platform has been developed aiming for easy handling by unschooled end-users. Moreover, the possibility of integrating the portable microfluidic platform with the microneedle patch for on-site sampling has also been explored and demonstrated, advancing what has been frequently noted as the point-of-care diagnosis. It is believed that the proposed microchip with distance-based visual quantification capability has great potential to be used for environmental, food, and health monitoring.