Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection.

Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance.

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.

Ultrafast Real-Time PCR in Photothermal Microparticles.

As the turnaround time of diagnosis becomes important, there is an increasing demand for rapid, point-of-care testing (POCT) based on polymerase chain reaction (PCR), the most reliable diagnostic tool. Although optical components in real-time PCR (qPCR) have quickly become compact and economical, conventional PCR instruments still require bulky thermal systems, making it difficult to meet emerging needs. Photonic PCR, which utilizes photothermal nanomaterials as heating elements, is a promising platform for POCT as it reduces power consumption and process time. Here, we develop a photonic qPCR platform using hydrogel microparticles. Microparticles consisting of hydrogel matrixes containing photothermal nanomaterials and primers are dubbed photothermal primer-immobilized networks (pPINs). Reduced graphene oxide is selected as the most suitable photothermal nanomaterial to generate heat in pPIN due to its superior light-to-heat conversion efficiency. The photothermal reaction volume of 100 nL (predefined by the pPIN dimensions) provides fast heating and cooling rates of 22.0 ± 3.0 and 23.5 ± 2.6 °C s-1, respectively, enabling ultrafast qPCR within 5 min only with optical components. The microparticle-based photonic qPCR facilitates multiplex assays by loading multiple encoded pPIN microparticles in a single reaction. As a proof of concept, four-plex pPIN qPCR for bacterial discrimination are successfully demonstrated.

Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles.

In recent decades "saliva" has emerged as an important non-invasive biofluid for diagnostic purposes in both human and animal health sectors. However, with the rapid evolution of molecular detection technologies, the limitation has been the lack of an efficient method for the facile amplification of target RNA from such a complex matrix. Herein, we demonstrate the novel application of hydrogel microparticles of primer-immobilized networks (PIN) for direct quantitative reverse transcription PCR (dirRT-qPCR) of viral RNA from saliva samples without prior RNA purification. Each of these highly porous PIN particles operates as an independent reactor. They filter in micro-volumes of the analyte solution. Viral RNA is captured and converted to complementary DNA (cDNA) through the RT step using covalently incorporated RT primers. The PIN with cDNA of the viral target will be ready for subsequent highly specific qPCR. Preceded by heat-treatment for viral lysis, we were able to conduct PIN dirRT-qPCR with 95% efficiency of the matrix (M) gene for influenza A virus (IAV) and 5' untranslated region (5' UTR) for chicken coronavirus spiked into saliva samples. The addition of reverse transcriptase enzyme (RTase) and 10% dilution of the matrix improved the assay sensitivity considerably. PIN particles' compatibility with microfluidic PCR chip technology has significantly reduced total sample processing time to 50 min, instead of an average of 120 min that are normally used by other assays. We anticipate this technology will be useful for other viral RNA targets by changing the incorporated RT primer sequences and can be adapted for onsite diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00065-0.

Novel human skin surface antimicrobial peptide quantification method using a skin patch test chamber: A pilot study.

INTRODUCTION: Antimicrobial peptides (AMPs) on the skin surface are related to the innate immunity of the skin in preventing external infection. Skin rinsing and tape stripping (TS) are acceptable methods for analyzing AMPs on the skin surface but have limitations, such as causing skin damage. In this study, we proposed a noninvasive method to measure AMPs on the skin surface with minimal skin damage. METHODS: Using the patch test assay, we aimed to analyze the skin surface human ß-defensin (hBDs) levels without damaging the skin barrier. The concentrations of hBDs on the skin surface were evaluated through the skin patch testing of 13 healthy subjects, and hBD-1 concentrations were compared with those obtained using the TS method in this proof-of-concept study. In addition, changes in skin physiology and concentration of hBDs under 1% sodium lauryl sulfate stimulation were monitored in 14 healthy subjects (8 young and 6 elderly subjects) for 150 h. RESULTS: The correlation between the two methods had a Pearson's coefficient of 0.640, and skin patch analysis led to a relatively less impaired barrier with no significant increase in transepidermal water loss after analysis. Age-specific comparisons suggested that higher skin surface hBD-2 concentrations were present in the young group as compared with the elderly group. Skin surface expression of hBD-2 after skin barrier disruption was also higher in the young group. CONCLUSION: Our findings show that skin patch analysis is a convenient method to analyze hBDs on the skin surface. hBDs are factors of innate immunity that can be used as an index to predict a decreased chemical immune response of skin due to aging.

Zirconia Toughened Alumina-Based Separator Membrane for Advanced Alkaline Water Electrolyzer.

Hydrogen is nowadays considered a favorable and attractive energy carrier fuel to replace other fuels that cause global warming problems. Water electrolysis has attracted the attention of researchers to produce green hydrogen mainly for the accumulation of renewable energy. Hydrogen can be safely used as a bridge to successfully connect the energy demand and supply divisions. An alkaline water electrolysis system owing to its low cost can efficiently use renewable energy sources on large scale. Normally organic/inorganic composite porous separator membranes have been employed as a membrane for alkaline water electrolyzers. However, the separator membranes exhibit high ionic resistance and low gas resistance values, resulting in lower efficiency and raised safety issues as well. Here, in this study, we report that zirconia toughened alumina (ZTA)-based separator membrane exhibits less ohmic resistance 0.15 Ω·cm2 and low hydrogen gas permeability 10.7 × 10-12 mol cm-1 s-1 bar-1 in 30 wt.% KOH solution, which outperforms the commercial, state-of-the-art Zirfon® PERL separator. The cell containing ZTA and advanced catalysts exhibit an excellent performance of 2.1 V at 2000 mA/cm2 at 30 wt.% KOH and 80 °C, which is comparable with PEM electrolysis. These improved results show that AWEs equipped with ZTA separators could be superior in performance to PEM electrolysis.

The Structural Effect of Electrode Mesh on Hydrogen Evolution Reaction Performance for Alkaline Water Electrolysis.

Alkaline water electrolysis (AWE) is a mature water electrolysis technology that can produce green hydrogen most economically. This is mainly attributed to the use of Ni-based materials that are easy to process and inexpensive. The nickel-based meshes with various structures such as woven mesh and expanded mesh are widely used as electrode in the AWE due to its common availability and easy fabrication. However, the morphological effect of meshes on hydrogen evolution reaction (HER) performance has not been studied. Here a new parameter to determine the structural effect of mesh on HER performance was first proposed. The key factors of the parameter were found to be the strand width, pore width and the strand surface area. The woven mesh with the ratio of pore width to strand width that converges to 1 showed the lowest the overpotential. The expanded mesh with the higher the structural surface area exhibited the lowest the overpotential. This study will help to choose an optimal structure for the mesh with the HER electrode.

Highly Selective Multiplex Quantitative Polymerase Chain Reaction with a Nanomaterial Composite Hydrogel for Precise Diagnosis of Viral Infection.

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.

An NIR dual-emitting/absorbing inorganic compact pair: A self-calibrating LRET system for homogeneous virus detection.

Many conventional optical biosensing systems use a single responsive signal in the visible light region. This limits their practical applications, as the signal can be readily perturbed by various external environmental factors. Herein, a near-infrared (NIR)-based self-calibrating luminescence resonance energy transfer (LRET) system was developed for background-free detection of analytes in homogeneous sandwich-immunoassays. The inorganic LRET pair was comprised of NIR dual-emitting lanthanide-doped nanoparticles (LnNPs) as donors and NIR-absorbing LnNPs as acceptors, which showed a narrow absorption peak (800 nm) and long-term stability, enabling stable LRET with a built-in self-calibrating signal. Screened single-chain variable fragments (scFvs) were used as target avian influenza virus (AIV)-binding antibodies to increase the LRET efficiency in sandwich-immunoassays. The compact sensor platform successfully detected AIV nucleoproteins with a 0.38 pM limit of detection in buffer solution and 64 clinical samples. Hence, inorganic LnNP pairs may be effective for self-calibrating LRET systems in the background-free NIR region.

Highly sensitive and multiplexed one-step RT-qPCR for profiling genes involved in the circadian rhythm using microparticles.

Given the growing interest in molecular diagnosis, highly extensive and selective detection of genetic targets from a very limited amount of samples is in high demand. We demonstrated the highly sensitive and multiplexed one-step RT-qPCR platform for RNA analysis using microparticles as individual reactors. Those particles are equipped with a controlled release system of thermo-responsive materials, and are able to capture RNA targets inside. The particle-based assay can successfully quantify multiple target RNAs from only 200 pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. We carried out 8-plex one-step RT-qPCR using tens of microparticles, which allowed extensive mRNA profiling. The circadian cycles were shown by the multiplex one-step RT-qPCR in human cell and human hair follicles. Reliable 24-plex one-step RT-qPCR was developed using a single operation in a PCR chip without any loss of performance (i.e., selectivity and sensitivity), even from a single hair. Many other disease-related transcripts can be monitored using this versatile platform. It can also be used non-invasively for samples obtained in clinics.

Cerium Oxide-Polysulfone Composite Separator for an Advanced Alkaline Electrolyzer.

The intermittent and volatile nature of renewable energy sources threatens the stable operation of power grids, necessitating dynamically operated energy storage. Power-to-gas technology is a promising method for managing electricity variations on a large gigawatt (GW) scale. The electrolyzer is a key component that can convert excess electricity into hydrogen with high flexibility. Recently, organic/inorganic composite separators have been widely used as diaphragm membranes; however, they are prone to increase ohmic resistance and gas crossover, which inhibit electrolyzer efficiency. Here, we show that the ceria nanoparticle and polysulfone composite separator exhibits a low area resistance of 0.16 Ω cm2 and a hydrogen permeability of 1.2 × 10-12 mol cm-1 s-1 bar-1 in 30 wt% potassium hydroxide (KOH) electrolyte, which outperformed the commercial separator, the Zirfon PERL separator. The cell using a 100 nm ceria nanoparticle/polysulfone separator and advanced catalysts has a remarkable capability of 1.84 V at 800 mA cm-2 at 30 wt% and 80 °C. The decrease in the average pore size of 77 nm and high wettability (contact angle 75°) contributed to the reduced ohmic resistance and low gas crossover. These results demonstrate that the use of ceria nanoparticle-based separators can achieve high performance compared to commercial zirconia-based separators.

In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling.

Here we report a novel method of microRNA (miRNA) profiling with particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR). To achieve target-specific reaction in a particle, the stem-loop RT primer and forward primer for each target miRNA were chemically immobilized to the particle. Target-specific cDNA synthesis proceeds with the stem-loop RT primer and then qPCR subsequently proceeds with the forward primer to rapidly achieve a quantitative result. High-fidelity multiplex assay was also accomplished in a single PCR process by loading multiple particles for each specific miRNA. The method for primer supply in the particles, involving confinement of the target-specific RT and PCR primers in the matrix of particles, led to the reduction of nonspecific reactions and improved the selectivity of the miRNA assay while minimizing labor in a multiple target assay. Specifically, this particle-based assay enabled the differentiation of mature miRNA from precursor with selectivity of 270:1 in terms of amplification speed. This advanced method also showed good discrimination among highly homologous let-7 family members, with cross-reaction rates of less than 5%. We demonstrated a very simple process of five-plex miRNA profiling in total RNA, and the measured changes in expression level were consistent with those from a conventional singleplex method.

Thermo-Responsive Polymer Capsules in Real-Time One-Step RT-PCR for Highly Multiplex RNA Analysis.

Rapid and simple detection of RNA targets is in high demand due to the growing threat of pandemic viruses. One-step real-time, reverse transcription-polymerase chain reaction (One-step RT-qPCR) using a controlled release system of thermo-responsive materials is developed in this paper to enable high-fidelity RNA analysis as suppressing by-products. The nanocapsules, consisting of upper critical solution temperature (UCST) material and PCR primers, carry or release the primers depending upon the temperature. The UCST nanocapsules are introduced into hydrogel microparticles incorporated with RT primers and then the target RNA is selectively amplified in the microparticle through one-step RT-qPCR. Severe side products are sharply subdued by separating the PCR primers from the RT process by means of the microparticles with nanocapsules. Because the one-step assay is now implemented in a single microparticle, multiple target RNAs can be analyzed in a simple RT-qPCR of multiple particles. Reliable 18-plex one-step RT-qPCR is successfully conducted within 30 min using single-color fluorescent optics. This work also explains the facile fabrication processes used for the thermo-responsive nanocapsules and hydrogel microparticles by the blending polymerization method. Extensible multiplex analysis of influenza virus demonstrates the versatile uses of this one-step RT-qPCR platform.

A Capacitive Micromachined Ultrasonic Transducer-Based Resonant Sensor Array for Portable Volatile Organic Compound Detection with Wireless Systems.

The development of portable volatile organic compound (VOC) sensors is essential for home healthcare and workplace safety because VOCs are environmental pollutants that may critically affect human health. Here, we report a compact and portable sensor platform based on a capacitive micromachined ultrasonic transducer (CMUT) array offering multiplex detection of various VOCs (toluene, acetone, ethanol, and methanol) using a single read-out system. Three CMUT resonant devices were functionalized with three different layers: (1) phenyl-selective peptide, (2) colloids of single-walled nanotubes and peptide, and (3) poly(styrene-co-allyl alcohol). As each device exhibited different sensitivities to the four VOCs, we performed principal component analysis to achieve selective detection of all four gases. For the simultaneous detection of VOCs using CMUT sensors, the changes in the resonant frequencies of three devices were monitored in real time, but using only a single oscillator through an electrically controlled relay to achieve compactness. In addition, by devising a wireless system, measurement results were transmitted to a smartphone to monitor the concentration of VOCs. We used multiple sensors to obtain a larger number of fingerprints for pattern recognition to enhance selectivity but interfaced these sensors with a single read-out circuit to minimize the footprint of the overall system. The compact CMUT-based sensor array based on a multiplex detection scheme is a promising sensor platform for portable VOC monitoring.

Fully Packaged Portable Thin Film Biosensor for the Direct Detection of Highly Pathogenic Viruses from On-Site Samples.

The thin film transistor (TFT) is a promising biosensor system with great sensitivity, label-free detection, and a quick response time. However, even though the TFT sensor has such advantageous characteristics, the disadvantages hamper the TFT sensor's application in the clinical field. The TFT is susceptible to light, noise, vibration, and limited usage, and this significantly limits its on-site potential as a practical biosensor. Herein, we developed a fully packaged, portable TFT electrochemical biosensor into a chip form, providing both portability through minimizing the laboratory equipment size and multiple safe usages by protecting the semiconductor sensor. Additionally, a safe environment that serves as a miniature probe station minimizes the previously mentioned disadvantages, while providing the means to properly link the TFT biosensor with a portable analyzer. The biosensor was taken into a biosafety level 3 (BSL-3) laboratory setting to analyze highly pathogenic avian influenza virus (HPAIV) samples. This virus quickly accumulates within a host, and therefore, early stage detection is critical to deterring the further spread of the deadly disease to other areas. However, current on-site methods have poor limits of detection (105-106 EID50/mL), and because the virus has low concentration in its early stages, it cannot be detected easily. We have compared the sample measurements from our device with virus concentration data obtained from a RT-PCR (virus range: 100-104 EID50/mL) and have identified an increasing voltage signal which corresponds to increasing virus concentration.

Zygotic gene activation in the chicken occurs in two waves, the first involving only maternally derived genes.

The first wave of transcriptional activation occurs after fertilisation in a species-specific pattern. Despite its importance to initial embryonic development, the characteristics of transcription following fertilisation are poorly understood in Aves. Here, we report detailed insights into the onset of genome activation in chickens. We established that two waves of transcriptional activation occurred, one shortly after fertilisation and another at Eyal-Giladi and Kochav Stage V. We found 1544 single nucleotide polymorphisms across 424 transcripts derived from parents that were expressed in offspring during the early embryonic stages. Surprisingly, only the maternal genome was activated in the zygote, and the paternal genome remained silent until the second-wave, regardless of the presence of a paternal pronucleus or supernumerary sperm in the egg. The identified maternal genes involved in cleavage that were replaced by bi-allelic expression. The results demonstrate that only maternal alleles are activated in the chicken zygote upon fertilisation, which could be essential for early embryogenesis and evolutionary outcomes in birds.

Expression of transcription factors during area pellucida formation in intrauterine chicken embryos.

Initial embryological development in avian species, consisting of cleavage and area pellucida formation, occurs prior to oviposition. In chickens, the first lineage segregation is known to occur during the last 10 hours of intrauterine development, a finding which has primarily been identified on the basis of morphological perspectives. We traced the early expression of the transcription factors NANOG, POUV and EOMES at Eyal-Giladi and Kochav (EGK) stages VI through X using in situ hybridization. At EGK.VI, NANOG and EOMES were heterogeneously expressed in a salt-and-pepper manner. From EGK.VIII to EGK.X, NANOG- or EOMES-positive cells were predominantly located in the epiblast or area opaca regions, respectively. POUV-expressing cells were found in the upper layer at EGK.VIII. After oviposition, POUV mRNA was strongly expressed in the epiblast, but weakly expressed in the hypoblast at EGK.X. Furthermore, NANOG- and POUV-negative cells were located in the subgerminal cavity where layer reduction occurs during area pellucida formation. Those cells were larger and did not seem to contribute to epithelialization until EGK.X. Our results on the spatiotemporal expression of transcription factors contribute to a greater understanding of the dynamic process of intrauterine development in chickens.

Rapid and background-free detection of avian influenza virus in opaque sample using NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay platform.

Rapid and sensitive on-site detection of avian influenza virus (AIV) is the key for achieving near real-time surveillance of AIV and reducing the risk of dissemination. However, unlike the laboratory-prepared transparent buffer solutions containing a single type of influenza virus, distinction between real- and false- positive outputs and detection of low concentrations of AIV in stool specimens or cloacal swabs are difficult. Here, we developed a rapid and background-free lateral flow immunoassay (LFA) platform that utilizes near-infrared (NIR)-to-NIR upconversion nanoparticles (UCNPs) to yield a sensor that detects AIV nucleoproteins (NPs) from clinical samples within 20 min. Ca2+ as a heterogeneous dopant ion in the shell enhanced the NIR-to-NIR upconversion photoluminescence (PL) emission without inducing significant changes in the morphology of the UCNPs. In a mixture of opaque stool samples and gold nanoparticles (GNPs), which are components of commercial AIV LFA, the background signal of the stool samples masked the absorption peak of GNPs. However, UCNPs dispersed in the stool samples still show strong emission centered at 800 nm when excited at 980 nm, which enables the NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay (NNLFA) platform to detect 10-times lower viral load than a commercial GNP-based AIV LFA. The detection limit of NNLFA for LPAI H5N2 and HPAI H5N6 viruses was 102 and 103.5 EID50/mL, respectively. Moreover, the viruses were successfully detected within dark brown-colored samples using the NNLFA but not the commercial AIV LFA. Therefore, the rapid and background-free NNLFA platform can be used for sensitive on-site detection of AIV.

Acquisition of pluripotency in the chick embryo occurs during intrauterine embryonic development via a unique transcriptional network.

BACKGROUND: Acquisition of pluripotency by transcriptional regulatory factors is an initial developmental event that is required for regulation of cell fate and lineage specification during early embryonic development. The evolutionarily conserved core transcriptional factors regulating the pluripotency network in fishes, amphibians, and mammals have been elucidated. There are also species-specific maternally inherited transcriptional factors and their intricate transcriptional networks important in the acquisition of pluripotency. In avian species, however, the core transcriptional network that governs the acquisition of pluripotency during early embryonic development is not well understood. RESULTS: We found that chicken NANOG (cNANOG) was expressed in the stages between the pre-ovulatory follicle and oocyte and was continuously detected in Eyal-Giladi and Kochav stage I (EGK.I) to X. However, cPOUV was not expressed during folliculogenesis, but began to be detectable between EGK.V and VI. Unexpectedly, cSOX2 could not be detected during folliculogenesis and intrauterine embryonic development. Instead of cSOX2, cSOX3 was maternally inherited and continuously expressed during chicken intrauterine development. In addition, we found that the pluripotency-related genes such as cENS-1, cKIT, cLIN28A, cMYC, cPRDM14, and cSALL4 began to be dramatically upregulated between EGK.VI and VIII. CONCLUSION: These results suggest that chickens have a unique pluripotent circuitry since maternally inherited cNANOG and cSOX3 may play an important role in the initial acquisition of pluripotency. Moreover, the acquisition of pluripotency in chicken embryos occurs at around EGK.VI to VIII.