Centrifugal Microfluidic Device Research Articles

A sequential liquid dispensing method in a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of foodborne pathogens.

This study proposes a sequential liquid dispensing method using a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of nucleic acid targets across multiple samples in a single operation. A pair of passive valves integrated into each microchamber enabled the liquid to fill towards the center of rotation against the centrifugal force, facilitating the complete removal of air inside the microchamber. Liquid manipulation can be achievable without any surface coating of the device by exploiting the inherent hydrophobicity of the polymer. Furthermore, design guidelines for the optimization of microfluidic devices are clarified. Consequently, our proposed method allows direct liquid dispensing into the reaction chambers without cross-contamination while simultaneously metering the sample/reagent volume for the colorimetric loop-mediated isothermal amplification (LAMP) reaction. In addition, we demonstrated the simultaneous detection of four foodborne pathogens (Salmonella spp., Vibrio parahaemolyticus, Campylobacter spp., and norovirus genogroup II (GII)) across four samples in a centrifugal microfluidic device within 60 min. Furthermore, the device exhibited high quantitation (R 2 > 0.98) of the DNA concentration in the sample. Our proposed method enables a more compact design by eliminating the need for metering chambers and offers a point-of-care testing platform with high simplicity as it operates at a constant rotational speed.

Development and validation study of compact biophotonic platform for detection of serum biomarkers

The application of advances in personalized medicine requires the support of in vitro diagnostic techniques aimed at the accurate, fast, sensitive, and precise determination of selected biomarkers. Herein, a novel optical centrifugal microfluidic device is developed for clinical analysis and point-of-care diagnostics. Based on compact disc technology, the integrated biophotonic system enables multiple immunoassays in miniaturized mode. The disposable microfluidic discs are made in cyclic olefin copolymer (COP), containing arrays of immobilized probes. In the developed approach, up to six patient samples can each be tested simultaneously. A portable instrument (<2 kg) controls the assay and the high-sensitive reproducible optical detection in transmission mode. Also, the instrument incorporates specific functionalities for personalized telemedicine.The device (analytical method, disc platform, reader, and software) has been validated to diagnose IgE-mediated drug allergies, such as amoxicillin and penicillin G. The total and specific IgE to β-lactam antibiotics were determined in human serum from patients (25 μL). The excellent analytical performances (detection limit 0.24 ng/mL, standard deviation 7–20 %) demonstrated that the developed system could have the potential for a broader impact beyond the allergy field, as it applies to other IVD tests.

Development and validation study of compact biophotonic platform for detection of serum biomarkers

The application of advances in personalized medicine requires the support of in vitro diagnostic techniques aimed at the accurate, fast, sensitive, and precise determination of selected biomarkers. Herein, a novel optical centrifugal microfluidic device is developed for clinical analysis and point-of-care diagnostics. Based on compact disc technology, the integrated biophotonic system enables multiple immunoassays in miniaturized mode. The disposable microfluidic discs are made in cyclic olefin copolymer (COP), containing arrays of immobilized probes. In the developed approach, up to six patient samples can each be tested simultaneously. A portable instrument (< 2 kg) controls the assay and the high-sensitive reproducible optical detection in transmission mode. Also, the instrument incorporates specific functionalities for personalized telemedicine.The device (analytical method, disc platform, reader, and software) has been validated to diagnose IgE-mediated drug allergies, such as amoxicillin and penicillin G. The total and specific IgE to β-lactam antibiotics were determined in human serum from patients (25 μL). The excellent analytical performances (detection limit 0.24 ng/mL, standard deviation 7-20%) demonstrated that the developed system could have the potential for a broader impact beyond the allergy field, as it applies to other IVD tests.

Photocatalytic colorimetry and fluorescence dual-signal biosensor for instant determination of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a photocatalytic colorimetry and fluorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery.

Dielectrophoretic separation/classification/focusing of microparticles using electrified lab-on-a-disc platforms

BackgroundSeparation, classification, and focusing of microparticles are essential issues in microfluidic devices that can be implemented in two categories: using labeling and label-free methods. Label-free methods differentiate microparticles based on their inherent properties, including size, density, shape, electrical conductivity/permittivity, and magnetic susceptibility. Dielectrophoresis is an advantageous label-free technique for this objective. Besides, centrifugal microfluidic devices exploit centrifugal forces to move liquid and particles. The simultaneous combination of dielectrophoretic and centrifugal forces exerted on microparticles still needs to be scrutinized more to predict their trajectories in such devices. ResultsAn integrated system utilizing two categories in microfluidics is proposed: dielectrophoretic manipulation of microparticles and centrifugal-driven microfluidics, followed by a numerical analysis. In this regard, we assumed a rectangular microchannel with internal unilateral planar electrodes equipped with three equal-sized outlets placed radially on a centrifugal platform where microparticles flow toward the disc's outer edge. The effect of different coordinate-based parameters, including radial and lateral distances (X and Y offsets)/tilting angles toward the radius direction (α), on the particles' movement was investigated. Additionally, the effect of operational parameters, including applied voltage, the microchannel width, the number of enabled electrodes, the diameter of particles, and the configuration of electrodes, were analyzed, and the distributions of particles toward the outlets were monitored. It was found that enhanced particle focusing becomes possible at lower rotation speeds and higher electric field values. Furthermore, the proposed centrifugal-DEP system's efficiency for classifying red blood cells/platelets and Live/Dead yeast cells systems was evaluated. SignificanceOur integrated system is introduced as a novel method for focusing and classifying various microparticles with no need for sheath flows, having the ability to conduct particles at desired routes and focusing width. Furthermore, the system effectively separates various bioparticles and offers ease of operation and high-efficiency throughput over conventional dielectrophoretic devices.

Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics.

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

An integrated centrifugal microfluidic strategy for point-of-care complete blood counting

Centrifugal microfluidics holds the potential to revolutionize point-of-care (POC) testing by simplifying laboratory tests through automating fluid and cell manipulation within microfluidic channels. This technology can facilitate blood testing, the most frequent clinical test, at the POC. However, an integrated centrifugal microfluidic device for complete blood counting (CBC) has not yet been fully realized. To address this, we propose an integrated portable system comprising a centrifuge and a hybrid microfluidic disc specifically designed for CBC analysis at the POC. The disc enables the implementation of various spin profiles in different stages of CBC to facilitate in-situ cell separation, solution metering and mixing, and differential cell counting. Furthermore, our system is coupled with a custom script that automates the process and ensures precise quantification of cells using light and fluorescent images captured from the detection chamber of the disc. We demonstrate a close correlation between the proposed method and the hematology analyzer, considered the gold standard, for quantifying hematocrit (R2 = 0.99), white blood cell count (R2 = 0.98), white blood cell differential count (granulocyte/agranulocyte; R2 = 0.89), red blood cell count (R2 = 0.97), and mean corpuscular volume (R2 = 0.94). The integration of our portable system offers significant advantages, enabling more accessible and affordable CBC testing at the POC. Considering the simplicity, affordability (∼$250 capital cost and <$2 operational cost per test), as well as low power consumption (>100 tests using a typical 24 V/10 Ah battery), this system has the potential to enhance healthcare delivery, particularly in resource-limited settings and remote areas where access to traditional laboratory facilities is limited.

Room temperature roll-to-roll additive manufacturing of polydimethylsiloxane-based centrifugal microfluidic device for on-site isolation of ribonucleic acid from whole blood

Polymer-based lab-on-a-disc (LoaD) devices for isolating ribonucleic acid (RNA) from whole blood samples have gained considerable attention for accurate biomedical analysis and point-of-care diagnostics. However, the mass production of these devices remains challenging in manufacturing cost and sustainability, primarily due to the utilization of a laser cutter or router computer numerical control (CNC) machine for engraving and cutting plastics in the conventional prototyping process. Herein, we reported the first energy-efficient room-temperature printing-imprinting integrated roll-to-roll manufacturing platform for mass production of a polydimethylsiloxane (PDMS)-based LoaD to on-site isolate ribonucleic acid (RNA) from undiluted blood samples. We significantly reduced energy consumption and eliminated thermal expansion variations between the mold, substrate, and resists by accelerating the PDMS curing time to less than 10 min at room temperature without using heat or ultraviolet radiation. The additive manufacturing technology was applied to fabricate a multi-depth flexible polymer mold that integrated macro (2 mm) and micro-sized (500 μm) features, which overcomes the economic and environmental challenges of conventional molding techniques. Our integrated R2R platform was enabled to print adhesion-promoting films at the first printing unit and continuously in-line imprint with a high replication accuracy (99%) for high-volume manufacturing of a new centrifugal microfluidic chip with an enhancement of mixing performance by integrating an efficient mixing chamber and serpentine micromixer. This research paved the way for scalable green manufacturing of large-volume polymer-based microfluidic devices, often required in real-world sample-driven analytical systems for clinical bioanalysis.

Application of the fuzzy proportional integral differential (PID) temperature control algorithm in a liver function test system based on a centrifugal microfluidic device

Clinical laboratory examinations frequently include biochemical analysis of the liver. The presence of alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum can be used to identify liver damage. In this study, a centrifugal microfluidic-based clinical biochemical detection system was developed for the detection of liver function markers. Using the centrifugal microfluidic chip and centrifugal force on the chip, separation of blood cells and serum was performed. The extraction and mixing of quantitative serum and diluent were completed under the chip design of microchannels and microchambers. The lyophilized reagent beads in the chip interacted with the combined solution. The Fuzzy PID algorithm regulates the power of the heating film to deliver the ideal reaction temperature. In accordance with Beer–Lambert, the rate of change in the absorbance of the reaction solution at 340 nm of the light source was measured and a standard curve for the relationship between concentration and rate of change in absorbance was constructed. The system is portable, quick, and simple to use because it uses a centrifugal microfluidic chip instead of the conventional detection and analysis approach. In the future, it is anticipated that the system will have several applications in the detection of highly integrated on-chip point-of-care devices.

Cell-free fetal DNA (cffDNA) extraction from whole blood by using a fully automatic centrifugal microfluidic device based on displacement of magnetic silica beads

The purpose of this research was to design a fully automated centrifugal microfluidic system (Lab-on-a-Disk) for isolating cell free fetal DNAs (cffDNAs) from whole blood. To achieve this goal, magnetic silica beads were used, such that after attaching cffDNA to them, they were transferred between chambers by using external fixed magnets. All the standards and required steps for cffDNA extraction including plasma separation, adding proteinase K, lysis buffer, binding buffer, washing buffer, and elution buffer were considered in this designed disk. To evaluate the function of the disk, the collected samples were tested from several aspects. First, the purity of extracted plasma from whole blood was investigated which included hemoglobin test, hemocytometer, etc. This disk could extract 1.3 mL pure plasma from 3 mL blood with 45% hematocrit. The results of the extracted plasma showed 99% purity. Finally, the cffDNAs were examined by using a male fetal gender identification kit and real-time PCR machine. The results indicated the correct function of the disk in extracting cffDNAs in samples of 10 Landa from cycle 34 onwards. Compared to the clinical method, the disk not only was able to extract cffDNA in 20 min but also it led to less buffer consumption since the disk only required 1 mL plasma for extraction of cffDNAs.

Microfiber generation on centrifugal microfluidic platforms using fluidic barriers

Fluidic barriers within centrifugal microfluidic devices represent a novel concept for creating emulsions and microstructures by leveraging the density variations among different liquids. This study focuses on characterizing this approach for a specific application: the generation of microfibers, specifically calcium alginate microfibers (Ca-alginate). Our investigation incorporates both experimental and numerical simulation approaches. We examined how the microfiber diameter responds to variations in polymer concentration, rotation speed, and micronozzle size. Notably, we observed the transition of droplets into microfibers when the rotational speed ranged between 600 rpm and 1300 rpm. The findings indicate that reducing the concentration of the sodium-alginate solution from 3 % to 1 % and decreasing the micronozzle diameter from 0.3 mm to 0.15 mm led to a reduction in microfiber diameter by up to 18.4 % and 47.9 %, respectively. The generated grooved microfibers in this study can be used as scaffolds for tissue growth and regeneration in tissue engineering.

Development of a high-throughput centrifugal microsystem for enzyme-linked immunosorbent assay to detect SARS-CoV-2

Enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic and quantification tool for protein biomarkers in biomedical research and industrial quality control. However, this technique has prolonged assay durations, along with requiring expensive equipment and trained technicians, which limits its on-site applications. In this study, we report a novel centrifugal microfluidic device that can perform the conventional ELISA assay in a high-throughput (HTP) and automatic manner. The centrifugal microfluidic microsystem incorporates four innovative features: (1) a rapid and easy dispenser structure with aliquoting microchannels, which can accurately fill the solution in 40 reaction chambers with just one injection, replacing the labor-intensive aliquoting process, (2) ultra-fast reagent loading into and discharging from 40 reaction chambers simultaneously within 15 sec through the combination of a siphon valve and centrifugal force control, (3) the exact and automatic handling of a small volume (only 30 µL per reaction) of the designated solution such as a sample, a washing solution, an HRP-antibody solution, and a TMB solution, (4) a miniaturized ELISA diagnostic system, consisting of a solution injector, a simple spindle motor, and a UV–vis absorbance detector for the point-of-care testing (POCT). Using the HTP centrifugal microfluidic device with the in-house POCT ELISA analyzer, we could automatically detect 40 samples of SARS-CoV-2 per run in 75 min, significantly reducing the time, cost, and labor compared with the conventional ELISA assay. SARS-CoV-2 was diagnosed with a limit-of-detection (LOD) of 10.2 ng/mL. In addition, clinical samples as well as the SARS-CoV-2 variants were successfully confirmed on the POCT ELISA analyzer.

Rapid detection of live bacteria in water using nylon filter membrane-integrated centrifugal microfluidics

Water is one of the most indispensable elements for human beings. People can live without food for a couple of weeks but cannot live without water for a couple of days. Unfortunately, drinking water is not always safe around the world; in many areas, the water for drinking could be contaminated with various microbes. However, the total viable microbe count in water still relies on culture-based methods in laboratories. Therefore, in this work, we report a novel, simple, and highly efficient strategy to detect live bacteria in water via a nylon membrane-integrated centrifugal microfluidic device. A handheld fan and a rechargeable hand warmer were utilized as the centrifugal rotor and the heat resource for reactions, respectively. The bacteria in water can be rapidly concentrated >500-fold by our centrifugation system. After incubation with water-soluble tetrazolium-8 (WST-8), the color change of the nylon membranes can be visually interpreted directly by the naked eye or recorded with a smartphone camera. The whole process can be finished in 3 h, and the detection limit can reach 102 CFU/mL. The detection range ranges from 102 CFU/mL to 105 CFU/mL. The cell counting results of our platform are highly positively correlated with the results of cell counting by the conventional lysogeny broth (LB) agar plate approach or the commercial 3 M Petrifilm™ cell counting plate. Our platform provides a convenient and sensitive strategy for rapid monitoring. We highly anticipate that this platform can improve water quality monitoring in resource-poor countries in the near future.

Rapid microfluidics prototyping through variotherm desktop injection molding for multiplex diagnostics.

In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6% attained for the measured widths of 100 μm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 μL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts.

Integrated membranes within centrifugal microfluidic devices: a review.

Centrifugal microfluidics has evolved into a sophisticated technology capable of enabling the exploration of fundamental questions in such fields as protein analysis, environmental monitoring, and live cell handling. These microdevices also hold unique potential for translating promising academic research into many real-world scenarios, with several products already available on the market. Yet, in order to fully realize this potentially transformative technology, there remains an outstanding need to incorporate simple to operate world-to-chip interfaces alongside the integration and automation of complex workflows. This requires cost-effective and versatile materials that are, ideally, already commercially available. Membranes not only meet these exigencies, they are also capable of enhancing the inherent advantages of microdevices when thoughtfully combined. This review provides an overview of the importance of these two technologies and the manifold benefits upon their unification. The fundamental principles governing fluid flow with centrifugal actuation, as well as within porous membranes, are briefly covered in addition to a comment on their relative advantages compared to classical microdevices and porous media. The major subtypes in membrane composition, preparation, and microfluidic integration strategies are next discussed in detail, along with their relativistic capabilities and drawbacks. This is followed by recent examples in the literature displaying the enormous versatility membranes have already demonstrated within microfluidic devices, highlighting recent centrifugal microdevices wherever possible. Finally, recommendations for areas where the incorporation of these materials still face challenges, as well as possible new avenues for exploration, are also provided.

Fast detection of ß2 microglobulin in patient blood by a handhold centrifugal microfluidic device

Elevating blood ß2 microglobulins (ß2 M) level is one of the indicators of diseases such as diabetes, kidney failure, and tumors. A centrifugal microfluidic chip was designed to conduct latex immunoagglutination assays for testing ß2 M. A handhold centrifuge was assembled to drive solution mixing in the microfluidic chip. An office scanner recorded the binding of ß2 M to the latex beads induced agglutination, reducing reliance on expensive scientific equipment and avoiding environmental light interference. The results revealed that the degree of beads agglutination was proportional to the grayscale value changes (∆G). The ß2 M testing can be accomplished using a 1.25 µL blood sample, and the testing time is 10 min. The limit of detection is 0.0335 mg/L. The recovery rate calculated by the measured and spiked ß2 M concentration ratio is 91.8–111.8%. The centrifugal chip was applied to test 50 patients’ samples. The confusion matrix analyzed the rate of false positives, false negatives, true positives, and true negatives for the ß2 M chip-based test, showing the sensitivity of the ß2 M chip is 77.3%, the specificity is 82.1%, and the efficiency is 80%. The Area Under the Curve analysis shows that the cutoff value (∆G = 1.011) of ∆G in predicting ß2 M elevating has a sensitivity of 100% (95% CI: 92.87% −100%) with the specificity of 92% (95% CI:81.6–96.85%).

Alpha-2-macroglobulin as a novel diagnostic biomarker for human bladder cancer in urinary extracellular vesicles.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p<0.0001). There was no significant difference in whole urine a2M levels between patients with BC and controls (p=0.317). We observed that compared to classical differential centrifugation, ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method for uEV protein analysis. We expect that our approach for EV analysis will provide an efficient route for the identification of clinically meaningful uEV-based biomarkers for cancer diagnosis.

Serially diluting centrifugal microfluidics for high-throughput gold nanoparticle synthesis using an automated and portable workstation

We report a novel high-throughput (HTP) synthetic platform for gold (Au) nanoparticles (NPs), consisting of a HTP centrifugal microfluidic device and a portable automatic workstation. The proposed platform is capable of performing 60 different reactions, and can produce a variety of Au NP types on a single device in one run without any human interference. In the microdevice, a zigzag aliquoting microchannel was designed to facilitate the loading of each reactant in a single injection shot. Besides, we developed serially diluting microfluidic structures by pairing the ascorbic acid (AA) solution-loaded microchannel with the water-loaded microchannel, and gradually varying the depths of the two microchannels in reverse, thereby generating the serially diluted concentration-gradient of the AA solution. The morphology of the Au NPs in the triplicate experiments changed from spherical to triangle/ quadrangular/ polyhedron to star shapes in the end, as the concentration of the reducing agent AA increased. Among them, the hexapod star morphology of Au NP was the most effective substrate for surface-enhanced Raman spectroscopy (SERS). In addition, we constructed a portable automatic workstation, which enables on-demand injection of the designated solution onto a microfluidic centrifugal device, the programmed chip rotation, and the multiplex NP synthesis on a chip in a completely automatic manner. Therefore, we could obtain the desired nanomaterials using a HTP automatic screening platform that is cost-, time-, and labor-effective.

MP06-17 ALPHA-2-MACROGLOBULIN IN URINARY EXTRACELLULAR VESICLES IS A NOVEL PROTEIN MARKER FOR DIAGNOSIS OF HUMAN BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-17 ALPHA-2-MACROGLOBULIN IN URINARY EXTRACELLULAR VESICLES IS A NOVEL PROTEIN MARKER FOR DIAGNOSIS OF HUMAN BLADDER CANCER Jinsung Park, Jisu Lee, Yun Hee Kang, Ji Young Mun, Hyun-Woo Oh, Yoon-Kyoung Cho, and Myung-Shin Lee Jinsung ParkJinsung Park More articles by this author , Jisu LeeJisu Lee More articles by this author , Yun Hee KangYun Hee Kang More articles by this author , Ji Young MunJi Young Mun More articles by this author , Hyun-Woo OhHyun-Woo Oh More articles by this author , Yoon-Kyoung ChoYoon-Kyoung Cho More articles by this author , and Myung-Shin LeeMyung-Shin Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary extracellular vesicles (uEVs) are considered potential biomarkers for bladder cancer (BC). However, challenges associated with the effective isolation and analyses of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we sought to identify novel uEV-derived diagnostic biomarkers for BC. METHODS: Urine samples were obtained between July 2018 and October 2020. 1) To identify optimal uEV isolation methods, three different methods (differential centrifugation, ExoLutE®, ExoDisc) were compared by various methods. 2) Through comparative analysis of uEVs obtained from BC patients pre- and post-operation using an antibody array, candidate uEV biomarkers were selected. 3) Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from 83 patients (23 control subjects and 60 BC patients) validated the clinical significance of the uEV biomarkers. RESULTS: We found that ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method than other methods (Fig 1). In the analysis of pre-and post-operative uEVs from BC patients by the ExoDisc, three proteins, namely cadherin-13, clusterin, and alpha-2-macroglobulin (A2M), were identified as potential uEV biomarkers, while A2M exhibited the most significant difference. In the validation study, the A2M expression in uEVs from BC patients was significantly higher than the control group (Fig 2A) with good diagnostic performance (Fig 2B). The uEV A2M expression in the high-grade BC was significantly higher than that in low-grade BC (Fig 2C), whereas no significant difference were detected in the whole urine samples (Fig 2D). The uEV A2M expression with a cut-off of 0.035 (highest Youden index) robustly discriminated BC patients from controls, with a sensitivity of 93.3%, a specificity of 34.8%. CONCLUSIONS: We demonstrated the potential of A2M as a novel uEV biomarker for BC diagnosis through an optimized EV isolation and analysis workflow. Source of Funding: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01060913) to Dr. Jinsung Park © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e83 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jinsung Park More articles by this author Jisu Lee More articles by this author Yun Hee Kang More articles by this author Ji Young Mun More articles by this author Hyun-Woo Oh More articles by this author Yoon-Kyoung Cho More articles by this author Myung-Shin Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...

Euler force-assisted sequential liquid release on the centrifugal microfluidic platform

The sequential release of liquid is essential for the automation of multistage biochemical analyses, such as immunoassays and nucleic acid extraction. Current methods for sequential liquid release are either too complicated owing to the bulky external control component or lack of flexibility. The Euler force can be flexibly adjusted on centrifugal platforms; this characteristic has great potential for developing interior force-triggered valves, increasing the methods for liquid manipulation in centrifugal microfluidic devices. In this study, a Euler force-assisted method is proposed to realize sequential liquid release in a convenient and flexible manner. The Euler force-assisted siphon valve proposed in this study has an ascent part of the siphon channel for priming by Euler force and a descent part for priming by capillary forces so that this siphon can be actively actuated by acceleration. With a certain channel size and angle, siphons with different lengths of the ascent channel can be triggered by different Euler forces, making the controllable release of liquid with different accelerations possible. By the simple combination of this kind of siphon, multiple unit operations can be realized, including sequential release, selective distribution, interruptible priming, and multistep immunoassay. This suggests that Euler force-assisted siphon priming can be a simple but efficient solution for complex liquid manipulation on the centrifugal microfluidic platform.