Development of a self-contained microfluidic chip and an internet-of-things-based point-of-care device for automated identification of respiratory viruses.

The COVID-19 pandemic greatly impacted the in vitro diagnostic market, leading to the development of new technologies such as point-of-care testing (POCT), multiplex testing, and digital health platforms. In this study, we present a self-contained microfluidic chip integrated with an internet-of-things (IoT)-based point-of-care (POC) device for rapid and sensitive diagnosis of respiratory viruses. Our platform enables sample-to-answer diagnostics within 70 min by automating RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescence detection. The microfluidic chip is designed to store all the necessary reagents for the entire diagnostic assay, including a lysis buffer, a washing buffer, an elution buffer, and a lyophilized RT-LAMP cocktail. It can perform nucleic acid extraction, aliquoting, and gene amplification in multiple reaction chambers without cross-contamination. The IoT-based POC device consists of a Raspberry Pi 4 for device control and data processing, a CMOS sensor for measuring fluorescence signals, a resistive heater panel for temperature control, and solenoid valves for controlling the movement of on-chip reagent solutions. The proposed device is portable and features a touchscreen for user control and result display. We evaluated the performance of the platform using 11 clinical respiratory virus samples, including 5 SARS-CoV-2 samples, 2 influenza A samples, and 4 influenza B samples. All tested clinical samples were accurately identified with high specificity and fidelity, demonstrating the ability to simultaneously detect multiple respiratory viruses. The combination of the integrated microfluidic chip with the POC device offers a simple, cost-effective, and scalable solution for rapid molecular diagnosis of respiratory viruses in resource-limited settings.

Cognitively Driven Autonomous Flow Chemistry for Producing On-Demand Perovskite Quantum Dots Via Advanced Closed-Loop Feedback Control.

Recent developments in the synthesis of hybrid organic-inorganic halide perovskite quantum dots (HP-QDs) through compositional adjustments have highlighted their potential applications in the fields of photovoltaics and light sources due to their unique optoelectronic properties. However, traditional methods to fine-tune their composition involve repetitive, labor-intensive, and costly processes. Herein, the utilization of a continuous flow chemistry approach is developed, in combination with a Proportional-Integral (PI) feedback control system as an effective method for producing on-demand methylammonium lead bromoiodide (MAPbBrx I3-x ) HP-QDs. The PI feedback control allows for real-time optimization of the flow rates of halide precursor solutions (halide PSs), enabling the precise tuning of the emission wavelength of HP-QDs. HP-QDs having an emission wavelength of 550 and 650 nm are synthesized through a blue-shifted and red-shifted algorithm, respectively, from any arbitrary reaction condition within 400 s. The iterative process through the PI feedback control produces the target HP-QDs with short rise time and low overshoot. The proposed automatic flow chemistry system integrated with a universal and accessible control algorithm of PI can generate the target HP-QDs with high accuracy, stability, and robustness, demonstrating a significant advancement in constructing an autonomous flow chemistry synthetic system.

Transjugular approach in aspiration thrombectomy and angioplasty of a thrombosed straight arteriovenous graft compared to the direct hemodialysis access approach.

PURPOSE: To evaluate the efficacy and outcome of the transjugular approach in endovascular recanalization of a thrombosed straight arteriovenous graft (AVG) compared to those of the direct hemodialysis access approach (conventional approach). MATERIALS AND METHODS: We retrospectively assessed patients who underwent aspiration thrombectomy and percutaneous transluminal angioplasty for thrombosed straight AVG performed at a single institution between October 2006 and October 2021. A total of 138 thrombosed AVGs in 83 patients (39 male and 44 females) were divided into the transjugular approach group (Group A) and the conventional approach group (Group B). Technical and clinical success, postintervention primary patency, cumulative patency, and periprocedural complications were compared. RESULTS: There was no statistical difference in demographic data between groups A and B. The technical success rate of group A and B was 96.4% (80/83) and 98.2% 54/55, respectively (p > 0.05). The mean procedure time was 61.4 min (Group A) and 70.5 min (Group B) (p > 0.05). There was no statistically significant difference between the two groups in postintervention primary patency. The cumulative patency of Groups A and B was 911.9 days (range 122-6277) and 1062.3 days (range 72-2302 days), respectively (p > 0.05). One patient in Group B experienced a major graft rupture. Pseudoaneurysm formation at the sheath insertion site occurred in two patients in Group B. No cases of stenosis or thrombosis of the IJV or hematoma at the puncture site were observed in Group A. CONCLUSION: The transjugular approach is as safe and effective as the conventional approach for aspiration thrombectomy and percutaneous transluminal angioplasty of thrombosed straight AVGs.

Feasibility and Safety of a Technique Intended to Place the Catheter Tip in the Right Atrium without Abutment Against the Cardiac Wall during Implantation of the Totally Implantable Venous Access Port.

Purpose: To assess the safety and feasibility of intentionally positioning the catheter tip in the right atrium (RA) without an abutment during implantation of a totally implantable venous access port (TIVAP). Materials and Methods: We enrolled 330 patients who had undergone TIVAP implantation between January and December 2016 and postoperative chest CT. The TIVAP was placed using the single-incision technique to access the axillary vein directly from the incision line. To position the catheter tip in the RA without abutment, blood return was checked before cutting. Catheter length and complications were evaluated by retrospectively reviewing medical images and records. Results: All patients achieved successful catheter tip positioning without abutment or dysfunction. The median tip position was 15.3 mm distal to the cavoatrial junction (CAJ) on fluoroscopy and 6 mm distal to the CAJ on CT. Catheter tips migrated a median of 10.4 mm cephalically on CT compared to fluoroscopy. Thromboses were detected in the RA and superior vena cava in one patient each. Conclusion: Intentional catheter tip positioning in the RA without abutment is a safe and feasible technique with a low incidence of thrombosis and no observed dysfunction.

Quantification of polystyrene microsphere attachment probability at the oil‒water interface using a microfluidic platform.

This study investigates the effects of interparticle interactions and wettability on the particle attachment efficacy to the oil‒water interface. Three types of PS particles with different surface functional groups were examined at varying salt concentrations and the number of particles injected into the interface. Based on the microfluidic method and the surface coverage measurement, we found that the two contributing factors significantly influenced particle attachment efficiency to the interface, while the wettability factor has a major contribution. This research contributes to the understanding of physicochemical aspects of particle assembly at fluid interfaces and can offer strategies for forming tailored structures with desired interfacial properties.

Fabrication of Partially Etched Polystyrene Nanoparticles.

Non-spherical polymer nanoparticles (NPs) have gained attention in various fields, but their fabrication remains challenging. In this study, we present a simple protocol for synthesizing partially etched polystyrene (PS) nanoparticles through emulsion polymerization and chemical etching. By adjusting the degree of crosslinking, we selectively dissolve the weakly crosslinked portions of the particles, resulting in partially etched PS NPs with increased surface area. These partially etched NPs are evaluated for their use as solid surfactants in Pickering emulsions, where they demonstrate significantly improved emulsion stability compared to intact spherical NPs. Our results contribute to the field of nanoparticle shape control and provide insights into developing novel materials for various applications, particularly in the area of solid surfactant usage. Additionally, the importance of conducting cellular toxicity studies using these partially etched NPs for future work is also emphasized.

Function of the Speech Recognition of the Smartphone to Automatically Operate a Portable Sample Pretreatment Microfluidic System.

We proposed a portable sample pretreatment microsystem, which can be automatically operated through speech recognition in a smartphone app. The proposed sample pretreatment microsystem consists of a microfluidic chip, an air router, pressure and vacuum lines with air pump motors, six 3-way solenoid valves, and a microcontroller with a Bluetooth module. The command of a human voice conducted the whole process of DNA extraction from pathogenic bacterial samples. Thus, manual interference during the DNA extraction is eliminated, preventing any potential infection from human touch. The palm-sized sample pretreatment microsystem can be run by a portable battery or a conventional smartphone charger. Genomic DNA ofSalmonella typhimuriumwas purified on a chip in less than 1 min with an extraction efficiency of 70 ± 5%.

Development of nucleocapsid-specific monoclonal antibodies for SARS-CoV-2 and their ELISA diagnostics on an automatic microfluidic device.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has threatened public health globally, and the emergence of viral variants has exacerbated an already precarious situation. To prevent further spread of the virus and determine government action required for virus control, accurate and rapid immunoassays for SARS-CoV-2 diagnosis are urgently needed. In this study, we generated monoclonal antibodies (mAbs) against the SARS-CoV-2 nucleocapsid protein (NP), compared their reactivity using an enzyme-linked immunosorbent assay (ELISA), and selected four mAbs designated 1G6, 3E10, 3F10, and 5B6 which have higher reactivity to NP and viral lysates of SARS-CoV-2 than other mAbs. Using an epitope mapping assay, we identified that 1G6 detected the C-terminal domain of SARS-CoV-2 NP (residues 248-364), while 3E10 and 3F10 bound to the N-terminal domain (residues 47-174) and 3F10 detected the N-arm region (residues 1-46) of SARS-CoV-2 NP. Based on the epitope study and sandwich ELISA, we selected the 1G6 and 3E10 Abs as an optimal Ab pair and applied them for a microfluidics-based point-of-care (POC) ELISA assay to detect the NPs of SARS-CoV-2 and its variants. The integrated and automatic microfluidic system could operate the serial injection of the sample, the washing solution, the HRP-conjugate antibody, and the TMB substrate solution simply by controlling air purge via a single syringe. The proposed Ab pair-equipped microsystem effectively detected the NPs of SARS-CoV-2 variants as well as in clinical samples. Collectively, our proposed platform provides an advanced protein-based diagnostic tool for detecting SARS-CoV-2.

Effectiveness of mechanical recanalization for intraluminal occlusion of totally implantable venous access ports.

PURPOSE: To evaluate if the mechanical injection of saline is effective in restoring patency of a totally implantable venous access port (TIVAP) with an intraluminal occlusion. MATERIALS AND METHODS: From January 2017 to June 2019, 64 cases of dysfunction of a TIVAP were referred to interventional radiology. Among these, 16 cases showed normal function of the TIVAP, 19 cases the showed the appearance of a fibroblastic sheath, and 29 cases showed intraluminal occlusion. Mechanical recanalization was performed for intraluminal occlusion of the TIVAP with an indeflator and a 20G non-coring needle. Linograms were performed in all recanalized cases. The success or failure of recanalization and the pressure of the indeflator were recorded. Linograms were evaluated for breakage or migration of catheters. Medical records were retrospectively reviewed. RESULTS: Among the 29 intraluminal occlusion cases, 24 cases (82.7%) were recanalized by mechanical recanalization via an indeflator. The pressure of the indeflator ranged from 29 to 220 psi (median: 118 psi). Linograms revealed breakage of the catheter of the TIVAP in two failed cases. The median interval from implantation to dysfunction was 405 days (range: 43-1723 days). The median interval from last use to dysfunction was 8 days (mean: 15.4 days; range: 1-119 days). The median re-occlusion free period after successful mechanical recanalization was 100.5 days (range: 6-859 days). CONCLUSION: In the absence of an available thrombolytic agent, mechanical injection of saline was a tolerable alternative method for restoring occluded catheters and sustaining the function of catheters. Because breakage of the catheter can occur during mechanical recanalization of a TIVAP, a linogram should follow the procedure.

Developing a Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Seven Respiratory Viruses including SARS-CoV-2.

Background and Objectives: The coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a pandemic even in 2022. As the initial symptoms of COVID-19 overlap with those of infections from other respiratory viruses, an accurate and rapid diagnosis of COVID-19 is essential for administering appropriate treatment to patients. Currently, the most widely used method for detecting respiratory viruses is based on real-time polymerase chain reaction (PCR) and includes reverse-transcription real-time quantitative PCR (RT-qPCR). However, RT-qPCR assays require sophisticated facilities and are time-consuming. This study aimed to develop a real-time quantitative loop-mediated isothermal amplification (RT-qLAMP) assay and compare its analytical performance with RT-qPCR. Materials and Methods: A total of 315 nasopharyngeal swabs from patients with symptoms of respiratory infections were included in this study. A primary screening of the specimens was performed using RT-qPCR. RNA/DNA from standard strains for respiratory viruses and heat-inactivated preparations of standard strains for SARS-CoV-2 were used to evaluate the accuracy and target specificity of the RT-qLAMP assay. Results: We successfully developed an RT-qLAMP assay for seven respiratory viruses: respiratory syncytial virus (RSV) A, RSV B, adenovirus, influenza (Flu) A (H1N1 and H3N2), Flu B, and SARS-CoV-2. RT-qLAMP was performed in a final reaction volume of 9.6 µL. No cross-reactivity was observed. Compared with the RT-PCR results, the sensitivity and specificity of the RT-qLAMP assay were 95.1% and 100%, respectively. The agreement between the two methods was 97.1%. The median amplification time to RT-qLAMP positivity was 22:34 min (range: 6:80-47:98 min). Conclusions: The RT-qLAMP assay requires a small number of reagents and samples and is performed with an isothermal reaction. This study established a fast, simple, and sensitive test that can be applied to point-of-care testing devices to facilitate the detection of respiratory viruses, including SARS-CoV-2.

A surfactant-free approach: Novel one-step ultrasonic nebulizer spray method to generate amphiphilic Janus particles.

HYPOTHESIS: A solvent evaporation-induced phase separation method, which is based on the preferential partitioning of two or more immiscible materials after solvent evaporation on providing heat, has been one of the main strategies for synthesis of Janus particles (JPs). Considering this approach, it should be possible to synthesize surfactant free-JPs in continuous flow by the ultrasonic nebulizer spray method. EXPERIMENTS: Two polymers, polystyrene and polymethylmethacrylate, were dissolved in dichloromethane, and droplets of a precursor solution generated by an ultrasonic nebulizer were then conveyed through a borosilicate glass cylinder with two heating zones. The solvent evaporation-induced phase separation occurred in a single flow process, which resulted in the preferential partitioning of two incompatible polymers in the droplets, leading to the formation of the spherical bicompartmental JPs. FINDINGS: The successful fabrication of spherical JPs was observed at high polymer concentrations (1.5 and 2.0 wt%), and at elevated temperature (40-75 °C). The fluorescent compartmentalization of JPs was confirmed. Furthermore, the interfacial arrangement of JPs at oil-water interface was studied. A detailed explanation of theoretical prediction of interfacial configurations of JPs was provided. Lastly, the generated JPs were proved as Pickering stabilizers at the oil-water interface.

The Impact of Socioeconomic Status on Mortality in Patients with Hepatocellular Carcinoma: A Korean National Cohort Study.

Background/Aims: We studied the impact of socioeconomic status (SES) on mortality in hepatocellular carcinoma patients and analyzed the effect of SES on initial treatment allocation. Methods: A cohort study was conducted using data from the National Health Insurance Service- National Sample Cohort of Korea. A total of 3,032 hepatocellular carcinoma patients who were newly diagnosed between January 2003 and December 2013 were included. Income level was categorized as Medical Aid and ≤30th, 31st-70th, or >70th percentile as an SES indicator. Results: The proportion of Medical Aid was 4.3%. The highest risks of all-cause mortality associated with Medical Aid were evident in the transcatheter arterial chemoembolization group (fully adjusted hazard ratio [HR], 2.40; 95% confidence interval [CI], 1.25 to 4.58), the other treatments group (fully adjusted HR, 2.86; 95% CI, 1.85 to 4.41), and the no treatment group (fully adjusted HR, 2.69; 95% CI, 1.79 to 4.04) but not in the curative treatment group. An association between the lower-income percentile and higher liver cancer-specific mortality was also observed, except in the curative treatment group. The association between income percentile and all-cause mortality was nonlinear, with a stronger association in the lower-income percentiles than in the higher income percentiles (p-value for nonlinear spline terms <0.05). Conclusions: Patients in the lower SES group, especially patients not eligible for curative treatment, had an increased risk of mortality. In addition, the association between SES and the risk for mortality was stronger in the lower-income percentile than in the moderate to higher income percentiles.

A 3D printed size-tunable flow-focusing droplet microdevice to produce cell-laden hydrogel microspheres.

Flow-focusing droplet generators have been extensively employed for the generation of monodisperse droplets. However, the droplet device is usually designed with an application-specific performance that includes prescribed droplet size and generation frequency. To achieve an ideal device, cost- and time-inefficient iterations for the chip design and fabrication are usually needed. In this study, we take an advantage of 3D printing technology to rapidly prototype the droplet device that enables the facile control of the droplet size as well as the droplet generation frequency. Our device was designed with a screw-and-nut combination and the gap height (hg) between the dispersed phase outlet and the orifice could be easily and finely controlled by rotating the head of the threaded screw. The hg values can be precisely adjusted from 0 to 2000 µm supported by 20 designated control teeth on the screw head, which enable us to produce droplets in different sizes or in the same size with different generation frequencies. The proposed 3D printed device was employed to synthesize a variety of Ca-alginate microspheres containing A549 cells. The facile assembly of the screw-and-nut components allows us to prepare the droplet generator in a simple yet effective manner, and the size controllability of the droplets resulted in the production of various sizes of A549 cell-encapsulated microspheres, which can be used for drug screening.

Biosynthesis and applications of iron oxide nanocomposites synthesized by recombinant Escherichia coli.

Recombinant Escherichia coli (E. coli) strain that produces phytochelatin (PC) and/or metallothionein (MT) can synthesize various metal nanoparticles (NPs) by reducing metal ions. Here we report in vivo biosynthesis of iron oxide nanocomposites (NCs) using recombinant E. coli. We designed a strategy of biosynthesizing iron oxide NCs by first internalizing chemically synthesized iron oxide NPs, followed by the reduction of added metal ions on the surface of internalized NPs by PC and/or MT in E. coli. For this, chemically synthesized Fe3O4 NPs were internalized by recombinant E. coli, and then, Au and Ag ions were added for the biosynthesis of AuFe3O4 and AgFe3O4 NCs, respectively. The NCs synthesized were analyzed by transmission electron microscopy, UV-vis spectrophotometry, and X-ray diffractometry to characterize their shape, optical property, and crystallinity. The Fe3O4 NPs in the biosynthesized NCs allowed easy purification of the biosynthesized NCs by applying a magnetic field. The AuFe3O4 NCs were used for enzyme-linked immunosorbent assay to detect prostate-specific antigen protein, while AgFe3O4 NCs were utilized for the antimicrobial application with low minimum inhibitory concentration. As recombinant E. coli can uptake and reduce various NPs and metal ions, biosynthesis of a wide range of NCs as new nanomaterials will be possible for diverse applications. KEY POINTS: • AuFe3O4 and AgFe3O4 nanocomposites were synthesized by recombinant E. coli. • Escherichia coli synthesized different iron oxide NCs depending on the metal ions to be added. • Biosynthesized AuFe3O4 NC was used for ELISA and AgFe3O4 NC for antimicrobial tests.

Latent Class Analysis for Repeatedly Measured Multiple Latent Class Variables.

Research on stage-sequential shifts across multiple latent classes can be challenging in part because it may not be possible to observe the particular stage-sequential pattern of a single latent class variable directly. In addition, one latent class variable may affect or be affected by other latent class variables and the associations among multiple latent class variables are not likely to be directly observed either. To address this difficulty, we propose a multivariate latent class analysis for longitudinal data, joint latent class profile analysis (JLCPA), which provides a principle for the systematic identification of not only associations among multiple discrete latent variables but sequential patterns of those associations. We also propose the recursive formula to the EM algorithm to overcome the computational burden in estimating the model parameters, and our simulation study shows that the proposed algorithm is much faster in computing estimates than the standard EM method. In this work, we apply a JLCPA using data from the National Longitudinal Survey of Youth 1997 in order to investigate the multiple drug-taking behavior of early-onset drinkers from their adolescence, via young adulthood, to adulthood.

An internet of things-based point-of-care device for direct reverse-transcription-loop mediated isothermal amplification to identify SARS-CoV-2.

Rapid and accurate testing tools for SARS-CoV-2 detection are urgently needed to prevent the spreading of the virus and to take timely governmental actions. Internet of things (IoT)-based diagnostic devices would be an ideal platform for point-of-care (POC) screening of COVID-19 and ubiquitous healthcare monitoring for patients. Herein, we present an advanced IoT-based POC device for real-time direct reverse-transcription-loop mediated isothermal amplification assay to detect SARS-CoV-2. The diagnostic system is miniaturized (10 cm [height] × 9 cm [width] × 5.5 cm [length]) and lightweight (320 g), which can be operated with a portable battery and a smartphone. Once a liquid sample was loaded into an integrated microfluidic chip, a series of sample lysis, nucleic amplification, and real-time monitoring of the fluorescent signals of amplicons were automatically performed. Four reaction chambers were patterned on the chip, targeting As1e, N, E genes and a negative control, so multiple genes of SARS-CoV-2 could be simultaneously analyzed. The fluorescence intensities in each chamber were measured by a CMOS camera upon excitation with a 488 nm LED light source. The recorded data were processed by a microprocessor inside the IoT-based POC device and transferred and displayed on the wirelessly connected smartphone in real-time. The positive results could be obtained using three primer sets of SARS-CoV-2 with a limit of detection of 2 × 101 genome copies/µL, and the clinical sample of SARS-CoV-2 was successfully analyzed with high sensitivity and accuracy. Our platform could provide an advanced molecular diagnostic tool to test SARS-CoV-2 anytime and anywhere.

A handheld-type total integrated capillary electrophoresis system for SARS-CoV-2 diagnostics: Power, fluorescence detection, and data analysis by smartphone.

A micro-capillary electrophoresis (µCE) system is one of the widely adopted techniques in the molecular diagnostics and DNA sequencing due to the benefits of high resolution, rapid analysis, and low reagent consumption, but due to the requirements of bulky high-power suppliers and an expensive laser-induced fluorescence detector module, the conventional set-up of µCE system is not adequate for point-of-care (POC) molecular diagnostics. In this study, we constructed a miniaturized and integrated µCE system which can be manipulated by a smartphone. The smartphone not only powers two boost converters and an excited laser, but also controls the relay for the power switch. Moreover, the complementary metal-oxide-semiconductor (CMOS) camera of the smartphone was used for detecting the fluorescence signal of amplicons amplified with reverse transcription-polymerase chain reaction (RT-PCR). We also developed a web-based application so that the raw data of the recorded fluorescence intensity versus the running time can display typical capillary electropherograms on the smartphone. The total size of the hand-held µCE system was 9.6 cm [Width] × 22 cm [Length] × 15.5 cm [Height], and the weight was ∼1 kg, which is suitable for POC DNA testing. In the integrated smartphone-associated µCE system, we could accurately analyze two genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely N gene and S gene along with two bracket ladders in 6 min to identify SARS-CoV-2. Such an advanced µCE platform can be applied for a variety of on-site molecular diagnostics fields with user-friendliness.

Prediction of acute rejection in kidney transplanted patients based on the point-of-care isothermal molecular diagnostics platform.

In this study, we proposed an advanced point-of-care molecular diagnostic technology to evaluate the acute rejection (AR) in kidney transplanted patients. On the contrary to the conventional PCR method, we developed a colorimetric loop mediated isothermal amplification (LAMP) for quantitative analysis of the six biomarkers related to AR (CD3ϵ, IP-10, Tim-3-HAVCR2, CXCL9, PSMB9, C1QB) with a reference gene (18S rRNA). Using urinary cDNA samples of transplanted patients, it turned out that three biomarkers among six, namely IP-10, Tim-3-HAVCR2 and C1QB, have significant discrepancy in quantity between the stable graft (STA) patient and the AR patient. The AR prediction model using these three biomarkers was established, which could estimate the immune-rejection in the patients with 93.3% of accuracy. For the point-of-care (POC) molecular diagnostics for the AR evaluation, we constructed a centrifugal microfluidic platform, in which the RNA extraction from the clinical urinary samples, the quantitative reverse-transcription (RT)-LAMP reaction, and the data analysis based on the AR prediction model could be performed in a serial order. Ten blind clinical samples were analyzed on the POC genetic analyzer, showing 100% match with the validated qPCR data. Thus, the proposed advanced molecular diagnostic platform enables us to perform the timely treatment for the transplanted patients who are suffering from the allograft failure and side effects such as infection and malignancy.

A portable centrifugal genetic analyzer for multiplex detection of feline upper respiratory tract disease pathogens.

We present a portable genetic analyzer with an integrated centrifugal disc which is equipped with a glass-filter extraction column for purifying nucleic acid (NA) and multiple reaction chambers for analyzing major feline upper respiratory tract disease (FURTD) pathogens. We targeted four kinds of FURTD including Feline herpesvirus 1 (FHV), Mycoplasma felis (MPF), Bordetella bronchiseptica (BDB), and Chlamydophila felis (CDF). The portable genetic analyzer consists of a spinning motor, two pairs of Peltier heaters, two Minco heater, fluorescent optics, a touchscreen, and software for data analysis, so loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) can be performed. The overall size of the genetic analyzer was 28 cm × 28 cm × 26 cm and the weight was 10 kg, which was deliverable for point-of-care testing (POCT). Owing to the sophisticated microchannel design and spinning program, the serial injection of the sample solution, the washing solution, and the elution solution was executed through a glass filter membrane for nucleic acid (NA) extraction, and then the cocktail with the purified genome was aliquoted into 9 reaction chambers for LAMP or PCR. The whole process for the LAMP reaction or the PCR was completed within 1.5 h. The fluorescence profiles by a scanning mode showed the matched results between the LAMP and the PCR.

High-throughput human DNA purification on a centrifugal microfluidic device for rapid forensic sex-typing.

We introduce a novel centrifugal disc for purifying nucleic acid (NA) in a high-throughput manner to perform the human sex-typing of forensic samples. The centrifugal disc was designed with double-sided etched channels to fabricate 30 extraction units, which was capable of purifying 30 forensic samples in a single run. In order to introduce the washing solution (70% ethanol) and the elution buffer for the 30 extraction units in an automatic manner, we designed the aliquoting chambers that were connected with a zigzag delivery channel. The super-hydrophobic zigzag-shaped aliquot structure plays a crucial role in automatically dividing the washing solution and the elution buffer into 30 aliquots with one injection shot. The Whatman glass filter paper was used as an NA extraction matrix and sophisticated passive valves were equipped to avoid the overflowing of these buffers to the neighboring chamber during the injection. To operate the disc, we developed a portable workstation that consists of a buffer storage system, a buffer injection system, and a spinning unit. The entire process was automatically operated by the in-house portable workstation. Genomic DNA extraction using thirty forensic samples was completed in 10 min. Using the purified genomic DNA, we performed a loop-mediated isothermal amplification (LAMP) reaction for sex-typing by targeting the human alphoid repeat sequence of the Y-chromosome and the human 18S rRNA. The combination of the high-throughput centrifugal disc for NA extraction and the LAMP reaction enables us to complete the genetic sex-typing in 30 min.