Rapid and Highly Sensitive Detection of Leishmania by Combining Recombinase Polymerase Amplification and Solution-Processed Oxide Thin-Film Transistor Technology.

Nucleic acid detection is widely used to identify infectious diseases and ensure food safety. However, conventional PCR-based techniques are time consuming. Thus, this study aims to combine recombinase polymerase amplification (RPA), which enables the rapid amplification of even trace amounts of nucleic acid fragments within 10-40 min at 37-42 °C, and solution-processed oxide thin-film transistor (TFT) technology, which exhibits high detection sensitivity, to detect Leishmania. A single-stranded anti-probe was incorporated into the RPA primer to facilitate effective hybridization between the RPA product and the immobilized probe on the solution-processed oxide TFT. The RPA-amplified product carrying an anti-probe enabled specific binding to the chip surface. Changes in current were monitored before and after sample incubation to identify the target nucleic acids in the samples accurately. The proposed method achieved a remarkable limit of detection of 101 copies/µL of the Leishmania HSP70 fragment within 30 min. The design of the probes on the solution-processed oxide TFT surface and the anti-probe simplified the detection of other target nucleic acids, eliminating the need to denature DNA double-strands for specific binding during nucleic acid detection. Thus, the novel method offers the advantage of requiring minimal reagent resources and eliminates the need for complex procedures.

Fabrication and Characterizations of Axial View Liquid Electrode Plasma Atomic Emission Spectrometry for the Sensitive Determination of Trace Zinc, Cadmium, and Lead.

Axial view liquid electrode plasma-atomic emission spectrometry (axial view LEP-AES) was proposed and fabricated successfully in this work. The emission spectra from Zn, Cd, Pb, Ca, and K were applied for characterization and optimization. Comparing with conventional radial view LEP-AES devices, the newly designed axial view-LEP provided better sensing ability toward trace heavy metals. Moreover, pulsed voltage discharge was found to be advantageous over continuous discharge under the same discharge time for detection. The optimized parameters facilitate the limit of detection to achieve 0.24, 0.051, and 0.85 µg L-1 for Zn, Cd, and Pb, respectively. Furthermore, the axial view LEP-AES possessed excellent reproducibility and good durability. The real sample tests using two different certified reference water samples revealed the great potential of the axial view LEP-AES as a novel practical elemental analysis tool.

Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B1 Using Submicroliter Samples.

Microfluidic paper-based analytical devices (µPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A µPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed µPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 µL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B1 (AFB1), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

DOCK11 and DENND2A play pivotal roles in the maintenance of hepatitis B virus in host cells.

Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

Organic Ion-associate Phase Extraction/Back-microextraction for the Preconcentration and Determination of Lithium Using 2,2,6,6-Tetramethyl-3,5-heptanedione by Liquid Electrode Plasma Atomic Emission Spectrometry and GF-AAS in Environmental Water.

We developed an ion-associate phase (IAP)-extraction/acid back-extraction system for the preconcentration and atomic spectrometric determination of lithium trace amounts in water. The chelating reagent for lithium also works as a constituent of the extraction phase. The lithium in a 10 mL sample solution was converted through a chelate complex reaction with 2,2,6,6-tetramethyl-3,5-heptanedione (HDPM). The addition of a benzyldimethyltetradecylammonium ion caused the formation of IAP suspension in the solution. Centrifugation of the solution led to the isolation of a liquid organic phase and the lithium complex was extracted as the upper phase from the centrifuge tube. After the aqueous phase was removed, lithium was back-extracted with a 400 µL nitric acid solution from the IAP. The acid phase was measured using liquid-electrode-plasma atomic-emission-spectrometry (LEP-AES) or graphite-furnace atomic-absorption spectroscopy (GF-AAS). The detection limits were 0.02 mg/L for LEP-AES and 0.02 µg/L for GF-AAS. This system was applied to the determination of environmental water. The HDPM in the organic phase was reusable.

Developing Conductive Highly Ordered Zinc Oxide Nanorods by Acetylacetonate-Assisted Growth.

Highly ordered vertically grown zinc oxide nanorods (ZnO NRs) were synthesized on ZnO-coated SiO2/Si substrate using zinc acetylacetonate hydrate as a precursor via a simple hydrothermal method at 85 °C. We used 0.05 M of ZnO solution to facilitate the growth of ZnO NRs and the immersion time was varied from 0.5 to 4 h. The atomic force microscopy revealed the surface roughness of ZnO seed layer used to grow the ZnO NRs. The morphology of vertically grown ZnO NRs was observed by field emission scanning electron microscopy. X-ray diffraction examination and transmission electron microscopy confirmed that the structure of highly ordered ZnO NRs was crystalline with a strong (002) peak corresponded to ZnO hexagonal wurtzite structure. The growth of highly ordered ZnO NRs was favorable due to the continuous supply of Zn2+ ions and chelating agents properties obtained from the acetylacetonate-derived precursor during the synthesis. Two-point probe current-voltage measurement and UV-vis spectroscopy of the ZnO NRs indicated a resistivity and optical bandgap value of 0.44 Ω.cm and 3.35 eV, respectively. The photoluminescence spectrum showed a broad peak centered at 623 nm in the visible region corresponded to the oxygen vacancies from the ZnO NRs. This study demonstrates that acetylacetonate-derived precursors can be used for the production of ZnO NRs-based devices with a potential application in biosensors.

Black tea polyphenol theaflavin as promising antioxidant and potential copper chelator.

BACKGROUND: In this work, we investigated the antioxidant and copper chelating abilities of theaflavin, a polyphenol responsible for astringency, color, and sensation in black tea. Using voltammetric techniques, the analyses were conducted with disposable electrochemical printed carbon chips in conjunction with a portable hand-held potentiostat. RESULTS: Voltammograms of theaflavin showed five separate oxidation peaks, corresponding to the oxidation of five individual functional groups. Electroanalytical data indicated that, after interaction with copper, theaflavin had higher antioxidant potential and was a better copper chelator than epigallocatechin gallate, a major polyphenol present in green tea and a well-known antioxidant. This could be attributed to the extra fused ring and larger number of OH groups in theaflavin. CONCLUSIONS: Our findings introduce another natural compound as a potential nutraceutical in oxidation- and copper-modulated illnesses. This simple and fast approach would also be highly pertinent to the inspection of the health benefits of natural food products. To the best of our knowledge, this is the first report of the electrochemical analysis of Cu (II) chelation with theaflavin. © 2020 Society of Chemical Industry.

Competitive non-SELEX for the selective and rapid enrichment of DNA aptamers and its use in electrochemical aptasensor.

The SELEX (Systematic Evolution of Ligands by EXponential enrichment) method has been used successfully since 1990, but work is still required to obtain highly specific aptamers. Here, we present a novel approach called 'Competitive non-SELEX' (and termed as 'SELCOS' (Systematic Evolution of Ligands by COmpetitive Selection)) for readily obtaining aptamers that can discriminate between highly similar targets. This approach is based on the theoretical background presented here, in which under the co-presence of two similar targets, a specific binding type can be enriched more than a nonspecifically binding one during repetitive steps of partitioning with no PCR amplification between them. This principle was experimentally confirmed by the selection experiment for influenza virus subtype-specific DNA aptamers. Namely, the selection products (pools of DNA aptamers) obtained by SELCOS were subjected to a DEPSOR-mode electrochemical sensor, enabling the method to select subtype-specific aptamer pools. From the clonal analysis of these pools, only a few rounds of in vitro selection were sufficient to achieve the surprisingly rapid enrichment of a small number of aptamers with high selectivity, which could be attributed to the SELCOS principle and the given selection pressure program. The subtype-specific aptamers obtained in this manner had a high affinity (e.g., KD = 82 pM for H1N1; 88 pM for H3N2) and negligible cross-reactivity. By making the H1N1-specific DNA aptamer a sensor unit of the DEPSOR electrochemical detector, an influenza virus subtype-specific and portable detector was readily constructed, indicating how close it is to the field application goal.

Polyphenols Modulate Alzheimer's Amyloid Beta Aggregation in a Structure-Dependent Manner.

Some polyphenols, which are common natural compounds in fruits, vegetables, seeds, and oils, have been considered as potent inhibitors of amyloid beta (Aß) aggregation, one critical pathogenic event in Alzheimer's disease (AD). However, the mechanisms by which polyphenols affect aggregation are not fully understood. In this study, we aimed to investigate the effect of two classes of polyphenols (flavonoids and stilbenes) on the self-assembly of Aß_42, in particular, how this relates to structure. We found that the flavonoids gallocatechin gallate (GCG) and theaflavin (TF) could completely inhibit Aß aggregation, while two stilbenes, resveratrol and its glucoside derivative piceid, could also suppress Aß aggregation, but to a much lesser extent. Intriguingly, resveratrol accelerated the formation of Aß fibrils before its decreasing effect on fibrillation was detected. Atomic force microscopy (AFM) images showed a huge mass of long and thin Aß fibrils formed in the presence of resveratrol. Although the morphology was the same in the presence of piceid, the fibrils were sparse in the presence of picead. In the presence of flavonoids, Aß morphology was unchanged from prior to incubation (0 h), in agreement with amyloid beta kinetics analysis using thioflavin-T fluorescence assay. The electrochemical data showed a higher ability of GCG and TF to interact with Aß than resveratrol and piceid, which could be attributed to the presence of more aromatic rings and hydroxyl groups. In addition, the two flavonoids exhibited a similar propensity for Aß aggregation, despite having some differences in their structure. However, in the case of stilbenes, the addition of a glucoside at C-7 slightly decreased anti-Aß aggregation property compared to resveratrol. These findings contribute to a better understanding of the essential structural features of polyphenols required for inhibiting Aß aggregation, and the possible mechanisms for modulating aggregation.

'Head-to-Head' mRNA display for the translation of multi-copied proteins with a free C-terminus.

With the development of various methods for affinity-based selection of proteins such as phage display, ribosomal display, and mRNA display, the progress in this field has been gradually shifting to function-based selection, such as through single-molecule observation, genetic selection, and compartmentalization technologies. In this vein, we present an opposite link mode of mRNA display termed as a 'Head-to-Head' (H2H) link. The key technique in H2H, formation of a covalent bond between O6-benzylguanine (BG) and O6-alkylguanine-DNA alkyltransferase (AGT), was demonstrated to be workable in H2H ligation, where mRNA is linked to a nascent AGT via a BG-DNA linker, resulting in a "(C-terminus) protein-BG-DNA linker-mRNA (5'-terminus)" conjugate. Thus, a head (N-terminus) to head (5'-terminus) linkage is formed. Among the advantages of H2H, the generation of multi-copied proteins is the most promising and was proven to be possible owing to the restored stop codon, which had been intentionally removed in the conventional mRNA display. Another advantage is obviously having a free C-terminus of the protein, which can be used for modifications such as C-terminal methylation, α-amidation, and others, which occur in nature. A superior merit of H2H is that it makes it possible to use a single construct commonly in mRNA display (affinity-based) and compartmentalization technologies (function-based) without requiring complicated construct changes.

Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning.

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM). The key idea is combination of support vector machine (SVM) learning for individual exosome particles and their interpretation by principal component analysis (PCA). We observed exosomes derived from three different cancer cells on SiO2/Si, 3-aminopropyltriethoxysilane-modified-SiO2/Si, and TiO2 substrates by AFM. Then, 14-dimensional feature vectors were extracted from AFM particle data, and classifiers were trained in 14-dimensional space. The prediction accuracy for host cells of test AFM particles was examined by the cross-validation test. As a result, we obtained prediction of exosome host cells with the best accuracy of 85.2% for two-class SVM learning and 82.6% for three-class one. By PCA of the particle classifiers, we concluded that the main factors for prediction accuracy and its strong dependence on substrates are incremental decrease in the PCA-defined aspect ratio of the particles with their volume.

Quantitative determination of total cesium in highly active liquid waste by using liquid electrode plasma optical emission spectrometry.

A sensitive analytical method for determination of total cesium (Cs) in highly active liquid waste (HALW) by using modified liquid electrode plasma optical emission spectrometry (LEP-OES) is developed in this study. The instrument is modified to measure radioactive samples in a glove box. The effects of important factors, including pulsed voltage sequence and nitric acid concentration, on the emission of Cs are investigated. The limit of detection (LOD) and limit of quantification (LOQ) are 0.005 mg/L and 0.02 mg/L, respectively. The achieved LOD is one order lower than that of recently developed spectroscopic methods using liquid discharge plasma. The developed method is validated by subjecting a simulated HALW sample to inductively coupled plasma mass spectrometry (ICP-MS). The recoveries obtained from a spike-and-recovery test are 96-102%, implying good accuracy. The method is successfully applied to the quantification of Cs in a real HALW sample at the Tokai reprocessing plant in Japan. Apart from dilution and filtration of the HALW sample, no other pre-treatment process is required. The results agree well with the values obtained using gamma spectrometry. The developed method offers a reliable technique for rapid analysis of total Cs in HALW samples.

Electrochemical Immunoassay Using Open Circuit Potential Detection Labeled by Platinum Nanoparticles.

In this work, a simple electrochemical immunoassay based on platinum nanoparticles (PtNPs) using open circuit potential (OCP) detection was developed. The detection of human chorionic gonadotropin hormone (hCG) as a model analyte, was demonstrated by direct electrical detection of PtNPs in hydrazine solution using OCP measurement without any application of either potential or current to the system. Disposable screen-printed carbon electrodes (SPCEs) were utilized for the development of our immunosensor, which required a sample volume as small as 2 µL. After preparation of a sandwich-type immunosystem, hydrazine solution was dropped on the electrode's surface, which was followed immediately by electrical detection using OCP. The change of the OCP signal originated from electrocatalytic oxidation of the hydrazine on PtNPs. Under the optimal conditions of a pH of 6.0 and a hydrazine concentration of 1 mM, a detection limit of 0.28 ng mL-1 and a linearity of 0-10 ng mL-1 were obtained. The PtNP-based OCP method is a simpler electrochemical detection procedure than those obtained from other electrochemical methods and has an acceptable sensitivity and reproducibility. The simplicity of the detection procedure and the cost-effectiveness of the disposable SPCE illustrate the attractive benefits of this sensor. Moreover, it could be applied to a simplified and miniaturized diagnostic system with minimal user manipulation.

Peptide aptamer-modified single-walled carbon nanotube-based transistors for high-performance biosensors.

Biosensors employing single-walled carbon nanotube field-effect transistors (SWCNT FETs) offer ultimate sensitivity. However, besides the sensitivity, a high selectivity is critically important to distinguish the true signal from interference signals in a non-controlled environment. This work presents the first demonstration of the successful integration of a novel peptide aptamer with a liquid-gated SWCNT FET to achieve highly sensitive and specific detection of Cathepsin E (CatE), a useful prognostic biomarker for cancer diagnosis. Novel peptide aptamers that specifically recognize CatE are engineered by systemic in vitro evolution. The SWCNTs were firstly grown using the thermal chemical vapor deposition (CVD) method and then were employed as a channel to fabricate a SWCNT FET device. Next, the SWCNTs were functionalized by noncovalent immobilization of the peptide aptamer using 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker. The resulting FET sensors exhibited a high selectivity (no response to bovine serum albumin and cathepsin K) and label-free detection of CatE at unprecedentedly low concentrations in both phosphate-buffered saline (2.3 pM) and human serum (0.23 nM). Our results highlight the use of peptide aptamer-modified SWCNT FET sensors as a promising platform for near-patient testing and point-of-care testing applications.

High yield matrix-free ionization of biomolecules by pulse-heating ion source.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been widely used for biomolecular analysis. However, with conventional MALDI, it is difficult to analyse low-molecular-weight compounds because of the interference of matrix ion signals. Here, we report a matrix-free on-chip pulse-heating desorption/ionization (PHDI) method for a wide range of biomolecules ranging from low molecular-weight substances such as glycine (75.7 Da) to large species such as α-lactalbumin (14.2 kDa). Compared with the conventional MALDI, the matrix-free PHDI method affords high yields of singly charged ions with very less fragmentation and background using only one-pulse without light (laser). We believe that this new technique for matrix-free biomolecules analysis would overcome the limitations of the conventional MALDI.

Comprehensive single-cell transcriptome analysis reveals heterogeneity in endometrioid adenocarcinoma tissues.

Single cell transcriptome analysis of a cancer tissue can provide objective assessment of subtype population or the activation of each of various microenvironment component cells. In this study, we applied our newly developed technique of single cell analysis to the myometrial infiltration side (M-side) and the endometrial side (E-side) of a human endometrioid adenocarcinoma with squamous differentiation tissues. We also analyzed spherogenic cultures derived from the same tissue to identify putative regulators of stemness in vivo. Cancer cells in the E-side were highly malignant compared with those in the M-side. Many cells on the E-side were positive for spheroid-specific tumorigenesis-related markers including SOX2. In addition, there were higher numbers of epithelial-to-mesenchymal transition (EMT) cells in the E-side compared with the M-side. This study identified a site containing cells with high malignant potential such as EMT and cancer stem-like cells in cancer tissues. Finally, we demonstrate that established endometrioid adenocarcinoma subtype classifiers were variably expressed across individual cells within a tumor. Thus, such intratumoral heterogeneity may be related to prognostic implications.

Density-gradient-assisted centrifugal microfluidics: an approach to continuous-mode particle separation.

Centrifugal microfluidics has been recognized as a promising pumping method in microfluidics because of its simplicity, easiness of automation, and parallel processing. However, the patterning of stripe flow in centrifugal microfluidics is challenging because a fluid is significantly affected by the Coriolis force, which produces an intrinsic secondary flow. This paper reports a technical and design strategy for centrifugal microfluidics called "density-gradient-assisted centrifugal microfluidics." The flow behavior is observed with the presence of a density gradient and without a density gradient in two concentrically traveling phase flows. As a result, clear stripe flow pattern is observed with a density difference of 0.05 g/cm3 between water and a percoll solution at a flow rate of 11.8 µl/s (7 ml/10 min) and spinning speed of 3000 rpm. In contrast, without a density gradient, it is necessary to reduce the flow rate and spinning speed to 0.1 µl/s and 1000 rpm, respectively. This paper also presents the use of a density gradient to assist in focusing resin (polystyrene) particles on the boundary of a stripe flow pattern that consists of water and percoll with different densities. Moreover, the density-based separation and sorting of particles in a mixed particle suspension is demonstrated. Polystyrene is selectively focused on the boundary, but silica particles are separated from the focused trajectory due to a difference in density. The separated particles are continuously sorted into different reservoirs with polystyrene and silica separation efficiencies of 96.5% and 98.5%, respectively. The pumping, stripe flow pattern formation, particle concentration, and sorting are simultaneously realized by applying a density gradient and centrifugal force. Therefore, this principle can realize a very simple technique for label-free particle separation by just spinning a disk device and can be applied in other applications by the use of the density-gradient assistance.

DEP-On-Go for Simultaneous Sensing of Multiple Heavy Metals Pollutants in Environmental Samples.

We describe a simple and affordable "Disposable electrode printed (DEP)-On-Go" sensing platform for the rapid on-site monitoring of trace heavy metal pollutants in environmental samples for early warning by developing a mobile electrochemical device composed of palm-sized potentiostat and disposable unmodified screen-printed electrode chips. We present the analytical performance of our device for the sensitive detection of major heavy metal ions, namely, mercury, cadmium, lead, arsenic, zinc, and copper with detection limits of 1.5, 2.6, 4.0, 5.0, 14.4, and, 15.5 µg·L-1, respectively. Importantly, the utility of this device is extended to detect multiple heavy metals simultaneously with well-defined voltammograms and similar sensitivity. Finally, "DEP-On-Go" was successfully applied to detect heavy metals in real environmental samples from groundwater, tap water, house dust, soil, and industry-processed rice and noodle foods. We evaluated the efficiency of this system with a linear correlation through inductively coupled plasma mass spectrometry, and the results suggested that this system can be reliable for on-site screening purposes. On-field applications using real samples of groundwater for drinking in the northern parts of India support the easy-to-detect, low-cost (<1 USD), rapid (within 5 min), and reliable detection limit (ppb levels) performance of our device for the on-site detection and monitoring of multiple heavy metals in resource-limited settings.

Design of dual working electrodes for concentration process in metalloimmunoassay.

Electrochemical immunosensing, particularly through a metalloimmunoassay, is a promising approach for development of point-of-care (POC) diagnostics devices. This study investigated the structure of dual working electrodes (W1 and W2), used in a silver nanoparticles-labeled sandwich-type immunoassay and silver concentration process, paying special attention to the position of W1 relative to W2. The new structures of the dual working electrodes were fabricated for efficient silver concentration and evaluated experimentally, which showed that the duration of prereduction before current measurement decreased from 480 s to 300 s by transforming the position of W1 from 1 line to 2 lines or 6 parts. The experimental results were also compared with numerical simulations based on three-dimensional diffusion, and the prereduction step almost followed the three-dimensional diffusion equation. Using numerical simulations, the ideal structures of dual working electrodes were designed based on relationships between the structures and duration of prereduction or the LOD. In the case of 36 lines at an area ratio of W1 to W1 + W2 of 1 to 10, the prereduction duration decreased to 96 s. The dual working electrodes designed in this study promise to shorten the total analysis time and lower the LOD for POC diagnostics.