HYL1-CLEAVAGE SUBTILASE 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition.

The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.

Improved sensing characteristics of dual-gate transistor sensor using silicon nanowire arrays defined by nanoimprint lithography.

This work describes the construction of a sensitive, stable, and label-free sensor based on a dual-gate field-effect transistor (DG FET), in which uniformly distributed and size-controlled silicon nanowire (SiNW) arrays by nanoimprint lithography act as conductor channels. Compared to previous DG FETs with a planar-type silicon channel layer, the constructed SiNW DG FETs exhibited superior electrical properties including a higher capacitive-coupling ratio of 18.0 and a lower off-state leakage current under high-temperature stress. In addition, while the conventional planar single-gate (SG) FET- and planar DG FET-based pH sensors showed the sensitivities of 56.7 mV/pH and 439.3 mV/pH, respectively, the SiNW DG FET-based pH sensors showed not only a higher sensitivity of 984.1 mV/pH, but also a lower drift rate of 0.8% for pH-sensitivity. This demonstrates that the SiNW DG FETs simultaneously achieve high sensitivity and stability, with significant potential for future biosensing applications.

Tear-off patterning: a simple method for patterning nitrocellulose membranes to improve the performance of point-of-care diagnostic biosensors.

This article describes a new method, referred to as "tear-off patterning," for patterning nitrocellulose (NC) membranes in order to fabricate NC-based point-of-care (POC) diagnostic devices. Paper-based microfluidic sensors usually employ hydrophobic barrier coatings such as paraffin wax on either paper or membranes. Herein, complex patterns were fabricated by stamping the target area with dimethyl sulfoxide before tearing off the stamped area. Fluid flow and morphological analyses were performed in order to characterize the patterned membranes. Furthermore, the myoglobin and creatine kinase-MB levels in human serum were measured simultaneously using a dual-fluidic-channel-patterned NC membrane in order to confirm the usefulness of the patterning method for fabricating POC biosensors. The proposed method for patterning NC membranes offers clear advantages, such as the ability to fabricate complex designs and patterns without a hydrophobic barrier after protein immobilization in a laboratory and in a simple, low-cost manner. We believe that this method can be used to develop various POC diagnostic biosensors at the research and development stage and can help improve the performance and features of POC diagnostic devices.

A three-line lateral flow assay strip for the measurement of C-reactive protein covering a broad physiological concentration range in human sera.

The lateral flow assay (LFA) strip sensor possesses many advantages as a diagnostic device, including the capabilities of rapid, one-step assay performance, and high throughput production. A major limitation of the sensor, however, is its difficulty in measuring a broad concentration range of target proteins, including C-reactive protein (CRP), due to the "hook effect." In this study, we report the use of a three-line LFA strip sensor, adding an antigen line to the conventional two-line LFA sensor, for detecting CRP within a broad concentration range in human sera. We introduced an antigen line between test and control lines in the LFA sensor. The antigen line was formed by dispensing a CRP antibody solution followed by a CRP solution in nitrocellulose membrane. All other conditions were identical to those applied to the conventional LFA strip sensor. The CRP level in test samples was generated by data processing from the intensities of three lines. The strip sensor measured a linear detection range of CRP concentration from 1 ng/mL to 500 µg/mL within 10 min, with a calculated detection range of 0.69 ng/mL-1.02 mg/mL. Using the developed three-line LFA sensor, 50 clinical samples were measured at a detection range of 0.4-84.7 µg/mL. This novel and easy-to-use CRP sensor can be a useful tool for rapid, sensitive, and cost-effective detection of a broad physiological concentration range of CRP capabilities that are vital for various diagnostic applications.

Skeletal Muscle Troponin I (TnI) in Animal Fat Tissues to Be Used as Biomarker for the Identification of Fat Adulteration.

In this study, the existence of skeletal muscle troponin I (smTnI), well-known as a muscle protein in fat tissues, and the utilization of smTnI as a biomarker for the identification of fat adulteration were investigated. A commercial antibody (ab97427) specific to all of animals smTnI was used in this study. Fat and meat samples (cooked and non-cooked) of pork and beef, and chicken considered as representative meats were well minced and extracted by heating and non-heating methods, and the extracts from fat and meat tissues were probed by the antibody used in both enzyme-linked immunosorbent assay (ELISA) and immunoblot. The antibody exhibited a strong reaction to all meat and fat extracts in ELISA test. On the other hand, the results of immunoblot analsis revealed a 23 kDa high intensity band corresponding to the molecular weight of smTnI (23786 Da). These results demonstrate that the existence of smTnI in all animal fat tissues. Since there are monoclonal antibodies specific to each species smTnI, smTnI in fat tissues could be used as a biomarker to identify or determine animal species adulterated in meat products. Therefore, an analytical method to identify fraudulent fat adulteration can be developed with an antibody specific to each species smTnI.

An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum.

Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay.

A highly rapid, one-step immunoassay of high sensitivity C-reactive protein (hsCRP) using a biosensor with a vertical flow immunoassay (VFA) was developed. The VFA biosensor was primarily composed of a sample pad, conjugate pad, FTH film and nitrocellulose (NC) membrane, which were all vertically stacked upon one another. Anti-hsCRP and secondary antibodies were consecutively immobilized on the NC membrane at the position below the holes. Gold nanoparticles (AuNPs) conjugated with another anti-hsCRP antibody were encapsulated in the conjugation pad. Various assay conditions, including the size of the hole and the sample volume, were optimized. Under optimized conditions, hsCRP concentrations from 0.01 to 10 µg mL(-1) were detected within 2 min. In comparison with a lateral flow assay (LFA) system, the VFA sensor showed a gradual increase of signal in a concentration-dependent manner without a hook effect in the tested range.

A dual gold nanoparticle conjugate-based lateral flow assay (LFA) method for the analysis of troponin I.

For signal amplification without an additional operation step in a gold nanoparticle (AuNP)-based lateral flow assay (LFA), a new and simple method utilizing two AuNP-antibody conjugates was developed. The 1st conjugate was the AuNP immobilized with an anti-troponin I antibody and blocked with bovine serum albumin (BSA), and the 2nd conjugate was the AuNP immobilized with an anti-BSA antibody and blocked with human serum albumin. The two conjugates were encapsulated in different pads, respectively. A scheme of the LFA system is described in the part A of first figure. The size of the two conjugates was very critical in the detection sensitivity of troponin I. When 10nm for the 1st and 40 nm for the 2nd were used, the detection sensitivity increased about a 100-fold compared to the conventional LFA. We could detect as low as 0.01 ng/mL troponin I in 10 min using the dual AuNP conjugate-based LFA, which was successfully applied in the analysis of serum samples of patients with myocardial infarction.