Microfluidic Particle Accumulation Enabling Visual Quantification of Analytes for Point-of-Care Testing

通過微流控顆粒積累實現分析物的可視化定量即時檢測

Student thesis: Doctoral Thesis

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Award date26 Apr 2022

Abstract

Point-of-care testing (POCT), as an emerging and promising detection method, has been focused on developing portable, reusable, and effective miniaturized platform and aiming at major advances in healthcare system and analytical process. Compared with the traditional method, POCT doesn’t required professional operators, huge cost of instrumentations and consumables. It’s integral and particularly acquired in the health care system for the diagnosis and monitoring of diseases. This simplified testing process is not only friendly to clinicians/physicians but also end-users to provide timely diagnostic information for better decisions of diagnosis and treatment. POCT devices are assumed to be used on-site or at home for patient care. Among them, microfluidic devices, assumed as state-of-the-art POCT devices, have the advantages of low cost, ease of use, rapid and accurate results, small volume sample consumption, lower detection limit, and instrument-free operation. In this work, visualized, user-friendly, and quantified microfluidic chips were designed for lead ion (Pb2+), SARS-CoV-2 antibody and alpha-fetoprotein (AFP) biomarker POCT. The obtained results demonstrate that the microfluidic chip platforms not only offer a promising POCT tool to complement standard serological assays and but also make healthcare diagnostics easier, cheaper, and faster.

First, lead ions (Pb2+) have been a severe threat to the environment and public health. It is especially a primary concern in drinking water since lead can accumulate in organs or tissues after chronic exposure, resulting in neurological, reproductive, cardiovascular, and developmental disorders. Nevertheless, traditional analytical techniques including atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS) and capillary electrophoresis all required costly instruments or complicated procedures, which are inconvenient, time-consuming and not suitable for end-users. To provide a preventive measure for lead intoxication, here a sensitive visual quantification of lead in drinking water was reported and whole blood on a microfluidic device. GR-5 DNAzyme can be cleaved by Pb2+, releasing an oligonucleotide T that triggers catalytic assembly of hairpin complex H1H2 connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). After loading the particle solution into a capillary-driven microfluidic device, MMPs-H1H2-PMPs are first attracted and removed by a magnetic separator, and the remaining free PMPs continue flowing along the microchannel until accumulating at a particle dam. As such, more lead ions cause shorter PMP accumulation quantifiable by the naked eye. The method achieved a limit of detection (LOD) of 246 pM, and is extremely selective (> 40,000 folds to other metal ions), and highly tolerant to acidity/basicity (6 ‒ 8.5) and water hardness (55 ‒ 318.3 mg/L). More importantly, high recovery rate (> 78%) in tap water and LOD of 2.57 nM for lead level in whole blood were achieved, demonstrating a visual quantification method for screening water safety and lead intoxication with user-friendly interface. As far as we know, this work obtained one of the lowest LOD of lead ions in drinking water and whole blood assays.

Second, the current coronavirus disease 2019 (COVID-19) pandemic has severely impacted the public health and global economy. According to World Health Organization (WHO), at the end of March 2021, there are globally more than 132 million confirmed cases and about 2.88 million confirmed deaths. Regretfully, the virus's spreading continues to grow rapidly and has not yet been validly controlled. To slow down it, policies were made to close borders and apply mandatory isolations/quarantines to constrain people movements for preventing further spread of the disease in the community. Nevertheless, such arrangement hamstrings the economies and causes fears and panic. While quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most reliable method to detect viral genes of SARS-CoV-2. Serological tests for specific antiviral antibodies are also important since they identify false negative qRT-PCR responses and track the effectivity of the patient’s immune system with fighting the infection, which is potentially helpful for plasma transfusion therapies. Moreover, many COVID-19 vaccines have been deployed, but the acquired immunogenicity varies and the decays overtime. Antibody tests allowing quantification of immunogenicity and its decaying is urgently needed. Here, a decentralized, instrument-free fingerprick blood device was reported to enable quantification of immunogenicity of COVID-19 vaccines via direct visual inspection. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are designed to bind to neutralizing antibodies against SARS-CoV-2. After loading to a microfluidic chip, the MMPs-antibody-PMPs is removed by a permanent magnet, while the free PMPs continue flowing until being trapped at a particle dam. Thus, the amount of antibody is inversely proportional to the PMP accumulation length readable by the naked eye. This method achieved a LOD of 18.1 ng/mL, high selectivity (>2000 folds to other antibody), and compatible with serum and fingerprick whole blood. Moreover, the quantitative results from 91 volunteers reveals much higher level of antibody arisen by mRNA vaccine than that by inactivated virus, and significant decay is observed after 1.5 month, demonstrating the practicality of home-use monitoring of immune status after vaccination. To the best of our knowledge, it is the first to achieve the quantified detection of SARS-CoV-2 antibody detection by microfluidics.

Third, hepatocellular carcinoma (HCC) is the fifth most common cancer and third most significant cause of cancer mortality in the world, with 5-year survival rates at a mere 7% in patients. The Alpha-fetoprotein (AFP) biomarker level at diagnosis was an independent risk predictor associated with pathological grade, progression, and survival. Nevertheless, the current method for AFP detection are ELISA and protein microarray, which are complicated and time-consuming, not suitable for end-users. This work aims at providing an AFP biomarker test that is sensitive, quantitative, suitable for point-of-care testing, for conducting large-scale surveying without increasing medical burden. Based on the microfluidic device for lead ion and SRAS-CoV-2 antibody detection, a platform was carried out for AFP detection. The method exhibited sensitive test mode (60-min incubation) with a quite low limit of detection (LOD) of 15.8 ng/mL and rapid test mode (5-min incubation) with a low LOD of 34.3 ng/mL in undiluted serum samples. The LOD meets the requirement of the clinical analysis criteria for HCC early diagnosis, demonstrating this method has great potential of clinical healthcare applications.

In summary, combined the technology of microfluidic chips with the advantages of simplicity, cost-effectiveness, portable testing, visualization and qualitative detection, the proposed methods are quite valuable explorations in the development of personal healthcare and clinical sample detection for point-of-care testing (POCT) in biomedical and health care systems.