Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts

植物の生育状態を野外で早期診断できる装置を開発〜ストレスに応答して生じるmiRNAを葉から検出〜.京都大学プレスリリース. 2024-04-04.Plants are exposed to a variety of environmental stress, and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is up-regulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management

FDTD modeling of nonperiodic antenna located above metasurface using surface impedance boundary condition

This paper investigates an FDTD modeling method for precisely calculating the characteristics of a single, that is, a nonperiodic antenna located above a metasurface that consists of an infinite periodic conducting element on a flat dielectric substrate. The original FDTD method requires enormous computational resources to analyze such structures because an appropriate periodic boundary condition (PBC) is not supported, and a brute force approach has to be used for this reason. Another option is to use the array scanning method in which a single source is synthesized from a superposition of infinite phased array of point sources. In this method, some problems such as a mutual coupling between the single antenna and the metasurface, a computational error contained in a numerical integration over the Brillouin zone and so on have not been resolved yet. In order to resolve these difficulties and to reduce computational resources, a surface impedance boundary condition (SIBC) is incorporated into the FDTD method in this paper. The validity of the method is numerically confirmed by calculating an input impedance and a radiation pattern of a horizontal dipole antenna located above the metasurface

Volume discrimination of nanoparticles via electrical trapping using nanopores

Abstract Electrophoretic capture of an oversized object on a solid-state nanopore is a useful approach for single-particle analyses via post electrical and optical measurements. Here we report on nanoparticle discriminations by the volume through combining this nanopore trap method with the cross-membrane ionic current measurements. We investigated ion transport through a pore channel being partially occluded by an electrophoretically-drawn nanoparticle at the orifice. We found distinct difference in the amount of current blockage by particles of different sizes. Multiphysics simulations revealed dominant contribution of particle volume over the other properties. We also demonstrated single-particle discriminations of two different sizes in a mixture solution. The present results demonstrate that this electrical capturing is a promising technique to immobilize a target at a single particle level that concomitantly offer wealth of information concerning their volume

Solid-State Nanopore Platform Integrated with Machine Learning for Digital Diagnosis of Virus Infection

The history of viruses in human society can never be taughtwithout the history of detection and treatment. As anancient example, Egyptian mummies have been found to havescars from smallpox infection.1 There have been various viralpandemics; however, the first virus to be discovered was tobaccomosaic virus (TMV) in 1892.2 Russian microbiologist D. I.Ivanovsky found an infectious agent in the extraction liquid oftobacco plants passed through a Chamberland filter, whichfiltered out bacteria. The technology of the day could not detectthis mysterious factor, and observation had to await thedevelopment of scanning electron microscopy (SEM). Todate, various techniques have revealed the structure andproperties of numerous virus species

Electrical trapping mechanism of single-microparticles in a pore sensor

Nanopore sensing via resistive pulse technique are utilized as a potent tool to characterize physical and chemical property of single –molecules and –particles. In this article, we studied the influence of particle trajectory to the ionic conductance through a pore. We performed the optical/electrical simultaneous sensing of electrophoretic capture dynamics of single-particles at a pore using a microchannel/nanopore system. We detected ionic current drops synchronous to a fluorescently dyed particle being electrophoretically drawn and become immobilized at a pore in the optical imaging. We also identified anomalous trapping events wherein particles were captured at nanoscale pin-holes formed unintentionally in a SiN membrane that gave rise to relatively small current drops. This method is expected to be a useful platform for testing novel nanopore sensor design wherein current behaves in unpredictable manner

Detecting Single Molecule Deoxyribonucleic Acid in a Cell Using a Three-Dimensionally Integrated Nanopore

Amplification-free genome analysis can revolutionize biology and medicine by uncovering genetic variations among individuals. Here, the authors report on a 3D-integrated nanopore for electrolysis to in situ detection of single-molecule DNA in a cell by ionic current measurements. It consists of a SiO2 multipore sheet and a SiNx nanopore membrane stacked vertically on a Si wafer. Single cell lysis is demonstrated by 106 V m−1-level electrostatic field focused at the multinanopore. The intracellular molecules are then directly detected as they move through a sensing zone, wherein the authors find telegraphic current signatures reflecting folding degrees of freedom of the millimeter-long polynucleotides threaded through the SiNx nanopore. The present device concept may enable on-chip single-molecule sequencing to multi-omics analyses at a single-cell level

Shortened microsatellite d(CA)21 sequence down-regulates promoter activity of matrix metalloproteinase 9 gene

AbstractOne characteristic elements in the promoter of the matrix metalloproteinase 9 (MMP-9) gene is the d(CA) repeat. To investigate whether this element regulates the transcription of the MMP-9 gene and its enzymatic activities, we sequenced the promoter region isolated from esophageal carcinoma cell lines. TE9 cells with low MMP-9 enzymatic activity had the number of d(CA) repeats shortened from 21 to 14 or 18. TE8, TE10 and TE11 cells with high MMP-9 activities had 21 or 23 d(CA) repeats. Luciferase assays using MMP-9 promoter containing 18, 14 or 0 d(CA) repeats showed transcriptional activities which were 50, 50 or 5%, respectively, of the level achieved with promoter containing 21 d(CA) repeats. Sequence analysis of the promoter of 223 Japanese subjects revealed that most had two alleles with 20, 21 or 22 d(CA) repeats, whereas six had one or two alleles with 14, 18 or 19 d(CA) repeats. We postulate that length alteration of the d(CA) repeat causes phenotypic differences among carcinoma cells and that microsatellite instability may contribute to the polymorphism of d(CA) repeat length

ZnO/SiO2 core/shell nanowires for capturing CpG rich single-stranded DNAs

Atomic layer deposition (ALD) is capable of providing an ultrathin layer on high-aspect ratio structures with good conformality and tunable film properties. In this research, we modified the surface of ZnO nanowires through ALD for the fabrication of a ZnO/SiO2 (core/shell) nanowire microfluidic device which we utilized for the capture of CpG-rich single-stranded DNAs (ssDNA). Structural changes of the nanowires while varying the number of ALD cycles were evaluated by statistical analysis and their relationship with the capture efficiency was investigated. We hypothesized that finding the optimum number of ALD cycles would be crucial to ensure adequate coating for successful tuning to the desired surface properties, besides promoting a sufficient trapping region with optimal spacing size for capturing the ssDNAs as the biomolecules traverse through the dispersed nanowires. Using the optimal condition, we achieved high capture efficiency of ssDNAs (86.7%) which showed good potential to be further extended for the analysis of CpG sites in cancer-related genes. This finding is beneficial to the future design of core/shell nanowires for capturing ssDNAs in biomedical applications

Oxide Nanowire Microfluidic Devices for Capturing Single-stranded DNAs

Since DNA analysis is the fundamental process for most applications in biomedical fields, capturing DNAs with high efficiency is important. Here, we used several oxide nanowire microfluidic devices to capture CpG-rich single-stranded DNAs (ssDNAs) in different pH solutions. All the oxide nanowires exhibited the highest capture efficiency around pH 7 with good capture efficiency shown by each metal oxide; ZnO/ZnO core/shell NWs (71.6%), ZnO/Al2O3 core/shell NWs (86.3%) and ZnO/SiO2 core/shell NWs (86.7%). ZnO/Al2O3 core/shell NWs showed the best performance for capturing ssDNAs under varying pH, which suggests its suitability for application in diverse biological fluids. The capturing efficiencies were attributed to the interactions from phosphate backbones and nucleobases of ssDNAs to each nanowire surface. This finding provides a useful platform for highly efficient capture of the target ssDNAs, and these results can be extended for future studies of cancer-related genes in complex biological fluids