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.

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%.

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.

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.

Quantification of colorimetric isothermal amplification on the smartphone and its open-source app for point-of-care pathogen detection.

The increasing risk of infectious pathogens, especially in the under-developed countries, is demanding the development of point-of-care (POC) nucleic acid testing in the low-resource setting conditions. Here, we describe a methodology for colorimetric quantitative analysis of nucleic acid using an easy-to-build smartphone-based platform, offering low-cost, portability, simplicity in operation, and user-friendliness. The whole system consists of a hand-held box equipped with a smartphone, a film heater, a white LED, a loop-mediated isothermal amplification (LAMP) chip, and a DC converter, and all the processes were powered by a portable battery of 5 V. Upon the amplification of the target gene by an Eriochrome Black T-mediated LAMP reaction, the color of the LAMP reaction was changed from violet to blue that was real-time recorded by a smartphone camera. To keep track of the progress of the color change, we developed a novel mobile app in which a hue value was accepted as an indicator for color transition and for determining the threshold time of the amplification reaction. A calibration curve could be generated by plotting the logarithm of the known concentration of the DNA templates versus the threshold time, and it can be used to predict the copy number of nucleic acids in the test samples. Thus, the proposed mobile platform can inform us of not only qualitative but also quantitative results of the pathogens. We believe that this advanced colorimetric approach and the mobile app can expand the potentials of the smartphone for the future POCT system in the bio-diagnostic fields.

Nucleic acid diagnostics on the total integrated lab-on-a-disc for point-of-care testing.

Since the emergence of the lab-on-a-chip technology in 1979, a variety of microfluidic devices have been developed and utilized for chemical and biological applications. Among the microfluidic devices, the centrifugal microfluidic device or lab-on-a-disc (LOAD) has advanced remarkably due to simple operation by the rotation, total integration, and high-throughput capability. Moreover, the centrifugal microdevices do not need complex tubing and pumping systems, which render them ideal for point-of-care testing (POCT) system. Owing to these characteristics, the centrifugal microdevices have been extensively used for bio-diagnostics. In particular, molecular diagnostics, which are regarded as an essential method for definite determination of the targets related with diseases, have been widely applied on the LOAD. In this review paper, we focus on the molecular diagnostics on the LOAD. The steps for the molecular diagnostics such as cell lysis, genome purification, gene amplification, amplicon detection, and data analysis can be performed individually or totally on the LOAD. Future directions of the LOAD in the fields of bio-diagnostics is to realize POCT for U-healthcare monitoring. In this context, the latest LOAD strategies for molecular diagnostics are summarized in this review paper, which would provide an insight for future POCT platform.

Calpain-dependent Beclin1 cleavage stimulates senescence-associated cell death in HT22 hippocampal cells under the oxidative stress conditions.

Oxidative damage in neurons including glutamate excitotoxicity has been linked to increasing numbers of neuropathological conditions. Under these conditions, cells trigger several different cellular responses such as autophagy, apoptosis, necrosis and senescence. However, the connection between these responses is not well understood. In this study, we found that the 60-kDa BECN1 was specifically degraded to a 40-kDa fragment in hippocampal HT22 cells treated with 5 mM glutamate. Increased BECN1 cleavage was specifically associated with a decrease in cell viability under oxidative stress. Interestingly, this BECN1 cleavage was specifically inhibited by a calpain inhibitor ALLN but was not affected by other protease inhibitors. Also, the BECN1 cleavage was not detected in calpain-4-deficient cell lines. Furthermore, calpain cleaved BECN1 at a specific site between the coiled-coil domain and Bcl2 homology 3 domain, which is associated with the anti-apoptotic protein Bcl-2. Moreover, some cellular senescence markers, including ß-galactosidase, p21, p27Kip1, p53 and p16INK4A, increased proportionally to those of BECN1 cleaved fragments. These results suggest that calpain-mediated BECN1 cleavage under oxidative conditions is specifically associated with cell death induced by cellular senescence.

Co-Expression Network Analysis of AMPK and Autophagy Gene Products during Adipocyte Differentiation.

Autophagy is involved in the development and differentiation of many cell types. It is essential for the pre-adipocytes to respond to the differentiation stimuli and may contribute to reorganizing the intracellulum to adapt the morphological and metabolic demands. Although AMPK, an energy sensor, has been associated with autophagy in several cellular processes, how it connects to autophagy during the adipocyte differentiation remains to be investigated. Here, we studied the interaction between AMPK and autophagy gene products at the mRNA level during adipocyte differentiation using public-access datasets. We used the weighted-gene co-expression analysis to detect and validate multiple interconnected modules of co-expressed genes in a dataset of MDI-induced 3T3-L1 pre-adipocytes. These modules were found to be highly correlated with the differentiation course of the adipocytes. Several novel interactions between AMPK and autophagy gene products were identified. Together, it is possible that AMPK-autophagy interaction is temporally and locally modulated in response to the differentiation stimuli.

Systematic characterization of autophagy-related genes during the adipocyte differentiation using public-access data.

Autophagy contributes to reorganizing intracellular components and forming fat droplets during the adipocyte differentiation. Here, we systematically describe the role of autophagy-related genes and gene sets during the differentiation of adipocytes. We used a public dataset from the European Nucleotide Archive from an RNA-seq experiment in which 3T3-L1 cells were induced by a differentiation induction medium, total RNA was extracted and sequenced at four different time points. Raw reads were aligned to the UCSC mouse reference genome (mm10) using HISAT2, and aligned reads were summarized at the gene or exon level using HTSeq. DESeq2 and DEXSeq were used to model the gene and exon counts and test for differential expression and relative exon usage, respectively. After applying the appropriate transformation, gene counts were used to perform the gene set and pathway enrichment analysis. Data were obtained, processed and annotated using R and Bioconductor. Several autophagy-related genes and autophagy gene sets, as defined in the Gene Ontology, were actively regulated during the course of the adipocyte differentiation. We further characterized these gene sets by clustering their members to a few distinct temporal profiles. Other potential functionally related genes were identified using a machine learning procedure. In summary, we characterized the autophagy gene sets and their members to biologically meaningful groups and elected a number of genes to be functionally related based on their expression patterns, suggesting that autophagy plays a critical role in removal of some intracellular components and supply of energy sources for lipid biogenesis during adipogenesis.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 ß) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.