Fabrication of graphene oxide-based pretreatment filter and Electrochemical-CRISPR biosensor for the field-ready cyanobacteria monitoring system.

Microcystis aeruginosa (M. aeruginosa) cause the eutrophication of lakes and rivers. To effectively control the overgrowth of M. aeruginosa, a suitable measurement method should be required in the aquatic fields. To address this, we developed a field-ready cyanobacterial pretreatment device and an electrochemical clustered regularly interspaced short palindromic repeats (EC-CRISPR) biosensor. The cyanobacterial pretreatment device consists of a syringe, glass bead, and graphene oxide (GO) bead. Then, the M. aeruginosa dissolved in the freshwater sample was added to fabricated filter. After filtration, the purified gene was loaded onto a CRISPR-based electrochemical biosensor chip to detect M. aeruginosa gene fragments. The biosensor was composed of CRISPR/Cpf1 protein conjugated with MXene on an Au microgap electrode (AuMGE) integrated into a printed circuit board (PCB). This AuMGE/PCB system maximizes the signal-to-noise ratio, which controls the working and counter electrode areas requiring only 3 µL samples to obtain high reliability. Using the extracted M. aeruginosa gene with a pre-treatment filter, the CRISPR biosensor showed a limit of detection of 0.089 pg/µl in fresh water. Moreover, selectivity test and matrix condition test carried out using the EC-CRISPR biosensor. These handheld pre-treatment kit and biosensors can enable field-ready detection of CyanoHABs.

Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification.

It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.

Facile and foldable point-of-care biochip for nucleic acid based-colorimetric detection of murine norovirus in fecal samples using G-quadruplex and graphene oxide coated microbeads.

Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.

Discrimination and isolation of the virus from free RNA fragments for the highly sensitive measurement of SARS-CoV-2 abundance on surfaces using a graphene oxide nano surface.

It is highly important to sensitively measure the abundance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on various surfaces. Here, we present a nucleic acid-based detection method consisting of a new sample preparation protocol that isolates only viruses, not the free RNA fragments already present on the surfaces of indoor human-inhabited environments, using a graphene oxide-coated microbead filter. Wet wipes (100 cm2), not cotton swabs, were used to collect viruses from environmental surfaces with large areas, and viruses were concentrated and separated with a graphene oxide-coated microbead filter. Viral RNA from virus was recovered 88.10 ± 8.03% from the surface and free RNA fragment was removed by 99.75 ± 0.19% from the final eluted solution. When we tested the developed method under laboratory conditions, a 10-fold higher viral detection sensitivity (Detection limit: 1 pfu/100 cm2) than the current commercial protocol was observed. Using our new sample preparation protocol, we also confirmed that the virus was effectively removed from surfaces after chemical disinfection; we were unable to measure the disinfection efficiency using the current commercial protocol because it cannot distinguish between viral RNA and free RNA fragments. Finally, we investigated the presence of SARS-CoV-2 and bacteria in 12 individual negative pressure wards in which patients with SARS-CoV-2 infection had been hospitalized. Bacteria (based on 16 S DNA) were found in all samples collected from patient rooms; however, SARS-CoV-2 was mainly detected in rooms shared by two patients.

Electrospun Nanofibers Embedded with Copper Oxide Nanoparticles to Improve Antiviral Function.

Many studies on anti-bacterial/antiviral surfaces have been conducted to prevent epidemic spread worldwide. Several nanoparticles such as those composed of silver and copper are known to have antiviral properties. In this study, we developed copper oxide (CuO) nanoparticle-incorporated nanofibers to inactivate or remove viruses. The CuO nanoparticle-incorporated nanofiber was fabricated with a hydrophobic polymer-polyvinylpyrrolidone (PVP)-using electrospinning, and CuO nanoparticles were exposed from the PVP polymer surface by etching the nanofiber with oxygen plasma. The fabrication conditions of electrospinning and oxygen plasma etching were investigated by scanning electron microscopy (SEM), and field emission transmission electron microscopy (FETEM)/ energy dispersive spectrometry (EDS). H1N1 virus was utilized as the target sample and quantified by RT-qPCR. The antiviral efficacy of CuO nanoparticle-incorporated nanofibers was compared against bare CuO nanoparticles. Overall, 70% of the viruses were inactivated after CuO nanoparticle-incorporated nanofibers were incubated with 10² pfu/mL of H1N1 virus solution for 4 h. This indicates that the developed CuO nanoparticle-incorporated nanofibers have noticeable antiviral efficacy. As the developed CuO nanoparticle-incorporated nanofibers exerted promising antiviral effects against H1N1 virus, it is expected to benefit global health by preventing epidemic spread.

Improvement of Heat Sink Effect Using Zinc Oxide Nanostructure.

Heat sinks that dissipate heat effectively play a significant role in devices with high-precision temperature control, such as thermal cyclers for polymerase chain reaction (PCR). This study was carried out to develop a heat sink with a high thermal conductivity to dissipate heat effectively. To increase the surface area of the heat sink, zinc oxide (ZnO) nanostructures were fabricated on an aluminum plate. ZnO nanostructures were fabricated by hydrothermal method and confirmed by scanning electron microscopy and X-ray diffraction. With the increase in the concentration of the precursors, the length of the nanorods increased, and with longer reaction time, nanostructures connected with higher stability and larger surface area. Thermal conductivity is increased by ZnO nanostructures and is affected by the concentration of precursors and the reaction time. Thermal conductivity of an optimal ZnO-coated Al plate is 2 times higher than that of a bare one. This technology can be applied to portable PCR devices to reduce weight, size, and power consumption.

A new classification of injury patterns of the medial patellofemoral ligament after acute lateral patella dislocation detected using magnetic resonance imaging studies.

INTRODUCTION: Acute lateral patellar dislocation is a very common condition in orthopedics, especially among adolescents and physically active patients. To evaluate distinct medial patellofemoral ligament (MPFL) injury patterns and the associated knee pathology after acute lateral patellar dislocation (ALPD) using magnetic resonance imaging (MRI) studies, which is essential for the development of treatment protocols. MATERIALS AND METHODS: MRI images of 74 ALPD patients were taken between January 2015 to December 2016. Images were evaluated using standardized protocols. RESULTS: The prevalence of MPFL injury following ALPD was 97.3% (72/74 patients). Among the 72 patients with MPFL, the prevalence of Type Ⅰ injury was 26.4% (19/72). Since only bone marrow edema and a partial tear were showed on MRI of these patients, conservative treatment was given. Tear of the MPFL occurred at the patellar attachment (Type Ⅱa) in 16 patients (16/72, 22.2%), at the middle area of the ligament (Type Ⅱb) in 5 patients (5/72, 6.9%), and at the femoral attachment (Type Ⅱc) in 27 patients (27/72, 37.5%). For Type Ⅱ injuries, all patients had the surgery to reconstruct the MPFL. The prevalence of Type Ⅲ MPFL injury was 6.9% (5/72) after the surgery. CONCLUSION: MPFL injury of is a common sequel following ALPD. We assessed the distinct injury pattern and associated pathology of MPFL using MRI studies. A good understanding of the injury pattern and associated knee pathology of MPFL is essential in managing patients with ALPD, especially if surgical intervention is considered.

Short-Term Outcomes of Open Reduction and Internal Fixation for Sanders Type III Calcaneal Fractures With and Without Bone Grafts.

Calcaneal fractures, often caused by a fall from a height, are the most common injuries encountered by orthopedic surgeons. Currently, open anatomic reduction and internal fixation (ORIF) is considered a valuable treatment of displaced intraarticular fractures of the calcaneus; however, the need for bone grafting in the treatment is still controversial. Therefore, in the present study, we investigated the outcomes of 2 methods (with and without bone grafting) used for the surgical treatment of Sanders type III calcaneal fractures. From January 2013 to September 2015, 57 cases (55 patients) with displaced Sanders type III calcaneal fractures (53 unilateral and 2 bilateral) were enrolled. The patients were divided into 2 groups: group I was treated by ORIF with bone grafting (n = 28) and group II was treated by ORIF without bone grafting (n = 29). The radiologic evaluation included Böhler's angle, Gissane's angle, and the height and width of the calcaneum. In addition, the American Orthopaedic Foot and Ankle Society questionnaires and visual analog scale were completed by the patients. During the follow-up period, no differences were found in the outcome measures (Böhler's angle, p = .447; Gissane's angle, p = .599; calcaneal height, p = .065; calcaneal width p = .077; and American Orthopaedic Foot and Ankle Society questionnaires, p = .282) with or without bone grafting. The only difference between the 2 groups was the occurrence of postoperative pain (p = .024 and p = ≤ .05), which was greater in the patients who had undergone bone grafting. We have provided evidence that bone grafting with internal fixation in the treatment of intraarticular calcaneal fractures failed to improve the restoration of Böhler's angle or Gissane's angle. No statistically significant difference was found in the short-term outcomes between the 2 methods used for the surgical treatment of Sanders type III calcaneal fractures.

Matrix metalloproteinase-2 and -9 expression correlated with angiogenesis in human adenomyosis.

BACKGROUND: Adenomyosis is an important cause of dysmenorrhea and infertility for women all over the world, however, the pathogenesis has not been completely elucidated. The purpose of the study was to investigate the role of matrix metalloproteinases (MMPs) and their relation to angiogenesis in human adenomyosis. METHODS: Adenomyotic endometrial specimens were removed by hysterectomy from 68 women with adenomyosis. The control group consisted of 26 normal endometrial specimens. Immunohistochemistry was used to demonstrate expression of MMP-2, -9, vascular endothelial growth factor (VEGF) and microvessel density (MVD). Staining intensity was analyzed by computerized image analysis system. RESULTS: In both eutopic and ectopic endometrium of adenomyosis, the expression of MMP-2, -9 as well as VEGF was significantly greater than in normal endometrium (p < 0.05). MVD was higher in ectopic endometrium than eutopic endometrium with or without adenomyosis (p < 0.05). In adenomyosis, a positive correlation was observed between VEGF expression and MMP-2 (p < 0.001, r = 0.583) as well as MMP-9 expression (p = 0.002,r = 0.490). A positive correlation was also found between MVD and MMP-2 (p < 0.001,r = 0.589) or MMP-9 expression (p < 0.001,r = 0.589). CONCLUSION: Our results indicate that the elevation of MMP-2, -9 expression may have an important role in the development of adenomyosis, probably through contributing to invasion of endometrial tissues into the myometrium and angiogenesis in adenomyotic implants.