Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR).

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

A portable smartphone-based colorimetric sensor that utilizes dual amplification for the on-site detection of airborne bacteria.

Over the past few years, infections caused by airborne pathogens have spread worldwide, infecting several people and becoming an increasingly severe threat to public health. Therefore, there is an urgent need for developing airborne pathogen monitoring technology for use in confined environments to enable epidemic prevention. In this study, we designed a colorimetry-based bacterial detection platform that uses a clustered regularly interspaced short palindromic repeat-associated protein 12a system to amplify signals and a urease enzyme to induce color changes. Furthermore, we have developed a smartphone application that can distinguish colors under different illumination conditions based on the HSV model and detect three types of disease-causing bacteria. Even synthetic oligomers of a few picomoles of concentration and genomic DNA of airborne bacteria smaller than several nanograms can be detected with the naked eye and using color analysis systems. Furthermore, in the air capture model system, the bacterial sample generated approximately a 2-fold signal difference compared with that in the control group. This colorimetric detection method can be widely applied for public safety because it is easy to use and does not require complex equipment.

Conductive Thread-Based Immunosensor for Pandemic Influenza A (H1N1) Virus Detection.

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.

Polydiacetylene-based hydrogel beads as colorimetric sensors for the detection of biogenic amines in spoiled meat.

Ingesting large quantities of biogenic amines (BAs), which are released from spoiled foods, can have adverse side effects on the human body. Herein, we developed a colorimetric sensor using polydiacetylene (PDA)-based hydrogel beads that change color upon binding with BAs, thereby conveniently checking whether food is spoiled due to improper storage and distribution. The colorimetric sensor is fabricated by mixing PDA liposomes with an alginate solution. PDA undergoes a color change from blue to red when exposed to various external stimuli. In addition, alginate bestows the hydrogel with a three-dimensional porous structure, affording a large surface area. The PDA-based hydrogel beads can visually confirm the presence of BAs in solution or vapor form. Cadaverine and propylamine were rapidly detected with distinct color changes in the solution and vapor phases, respectively. The spoilage of pork meat at room temperature could be detected after two days as a 40.84% red chromatic shift.

Isothermal amplification-mediated lateral flow biosensors for in vitro diagnosis of gastric cancer-related microRNAs.

MicroRNAs (miRNAs) are important diagnostic and prognostic biomarkers for various tumors. Currently, many diagnostic systems have been developed to detect miRNAs, but simple techniques for detecting miRNAs are still required. Recently, we reported that the expression of miRNA-135b is upregulated in gastric epithelial cells during gastric inflammation and carcinogenesis. Our aim was to develop an in vitro diagnostic platform to analyze the expression of gastric cancer-related biomarkers in the blood. The diagnostic platform comprised an isothermal amplification-based lateral flow biosensor (IA-LFB) that enables easy diagnosis of gastric cancer through visual observation. In this platform, trace amounts of biomarkers are isothermally amplified through rolling circle amplification (RCA), and the amplified product is grafted to the LFB. The performance of the IA-LFB was confirmed using RNAs extracted from in vitro and in vivo models. The platform could detect target miRNAs within 3 h with excellent sensitivity and selectivity. In particular, the IA-LFB could detect the overexpression of gastric cancer-related markers (miRNA-135b and miRNA-21) in RNAs extracted from the blood of patients with various stages (stages 1-4) of gastric cancer compared to that in healthy volunteers. Therefore, IA-LFB is a simple and sensitive in vitro diagnostic system for detecting gastric cancer-related biomarkers and can contribute to the early diagnosis and prognosis monitoring of gastric cancer. Furthermore, this technology can be applied to systems that can detect multiple biomarkers related to various diseases (such as infectious and genetic diseases).

miRNA sensing hydrogels capable of self-signal amplification for early diagnosis of Alzheimer's disease.

Alzheimer's disease (AD), one of the leading senile disorders in the world, causes severe memory loss and cognitive impairment. To date, there is no clear cure for AD. However, early diagnosis and monitoring can help mitigate the effects of this disease. In this study, we reported a platform for diagnosing early-stage AD using microRNAs (miRNAs) in the blood as biomarkers. First, we selected an appropriate target miRNA (miR-574-5p) using AD model mice (4-month-old 5XFAD mice) and developed a hydrogel-based sensor that enabled high-sensitivity detection of the target miRNA. This hydrogel contained catalytic hairpin assembly (CHA) reaction-based probes, leading to fluorescence signal amplification without enzymes and temperature changes, at room temperature. This sensor exhibited high sensitivity and selectivity, as evidenced by its picomolar-level detection limit (limit of detection: 1.29 pM). Additionally, this sensor was evaluated using the plasma of AD patients and non-AD control to validate its clinical applicability. Finally, to use this sensor as a point-of-care-testing (POCT) diagnostic system, a portable fluorometer was developed and verified for feasibility of application.

Microfluidic device for one-step detection of breast cancer-derived exosomal mRNA in blood using signal-amplifiable 3D nanostructure.

Metastasis attributed to approximately 90% of cancer-related deaths; hence, the detection of metastatic tumor-derived components in the blood assists in determining cancer recurrence and patient survival. Microfluidic-based sensors facilitate analysis of small fluid volumes and represent an accurate, rapid, and user-friendly method of field diagnoses. In this study, we have developed a microfluidic chip-based exosomal mRNA sensor (exoNA-sensing chip) for the one-step detection of exosomal ERBB2 in the blood by integrating a microfluidic chip and 3D-nanostructured hydrogels. The exoNA-sensing chip is a vacuum-driven power-free microfluidic chip that can accurately control the flow of trace fluids (<100 µL). The sensing part of the exoNA-sensing chip includes 3D-nanostructured hydrogels capable of detecting ERBB2 and a reference gene by amplifying a fluorescent signal via an enzyme-free catalytic hairpin assembly reaction at room temperature. This hydrogel offers a detection limit of 58.3 fM with good selectivity for target sequences. The performance of the exoNA-sensing chip was evaluated by testing in vitro and in vivo samples and was proven to be effective for cancer diagnosis and liquid biopsies.

Using Phytochemicals to Investigate the Activation of Nicotine Detoxification via Upregulation of CYP2A6 in Animal Models Exposed Tobacco Smoke Condensate by Intratracheal Instillation.

This study examined the efficacy of standardized Smilax china L. root extract (SSCR) containing chlorogenic acid on detoxifying nicotine from tobacco smoke condensate (TSC) in vitro and in vivo. Chlorogenic acid is an identified bioactive component in SSCR by ultraperformance liquid chromatography/photodiode array/electrospray ionization/mass spectroscopy (UPLC/PDA/ESI/MS). HepG2 liver cells and A549 lung cells were carried for measuring ROS and antioxidant enzymes. Sprague-Dawley rats were treated with nicotine by intratracheal instillation (ITI). Cell viabilities by pretreatments of 5, 12.5, and 25, 50 µg SSCR/mL ranged from 41 to 76% in HepG2 and 65 to 95% in A549. Pretreatments of SSCR inhibited TSC-mediated production of reactive oxygen species (ROS) by 8 and 10% in HepG2 and A549 cells, respectively. However, the expression of CAT, SOD1, and AOX1 was downregulated by SSCR in the both cells. The highest conversion of cotinine was observed at 50 µg/mL of SSCR after 120 min of incubation. SSCR upregulated CYP2A6 3-fold in A549 cells regardless of TSC cotreatment. When Sprague-Dawley rats were treated with nicotine by ITI or subjected to SSCR administration for 14 days, the levels of cotinine in urine increased in SSCR treatment only. The cellular level of antioxidant capacity at 10 or 100 mg/kg body weight/day of SSCR treatment was 1.89 and 1.86 times higher than those of nicotine-control. Results suggest that the intake of SSCR can detoxify nicotine by elevating nicotine conversion to cotinine and antioxidant capacity.

Silver nanoparticles enhance wound healing in zebrafish (Danio rerio).

Silver nanoparticles (AgNPs) were successfully synthesized by a chemical reduction method, physico-chemically characterized and their effect on wound-healing activity in zebrafish was investigated. The prepared AgNPs were circular-shaped, water soluble with average diameter and zeta potential of 72.66 nm and -0.45 mv, respectively. Following the creation of a laser skin wound on zebrafish, the effect of AgNPs on wound-healing activity was tested by two methods, direct skin application (2 µg/wound) and immersion in a solution of AgNPs and water (50 µg/L). The zebrafish were followed for 20 days post-wounding (dpw) by visual observation of wound size, calculating wound healing percentage (WHP), and histological examination. Visually, both direct skin application and immersion AgNPs treatments displayed clear and faster wound closure at 5, 10 and 20 dpw compared to the controls, which was confirmed by 5 dpw histology data. At 5 dpw, WHP was highest in the AgNPs immersion group (36.6%) > AgNPs direct application group (23.7%) > controls (18.2%), showing that WHP was most effective in fish immersed in AgNPs solution. In general, exposure to AgNPs induced gene expression of selected wound-healing-related genes, namely, transforming growth factor (TGF-ß), matrix metalloproteinase (MMP) -9 and -13, pro-inflammatory cytokines (IL-1ß and TNF-α) and antioxidant enzymes (superoxide dismutase and catalase), which observed differentiation at 12 and 24 h against the control; but the results were not consistently significant, and many either reached basal levels or were down regulated at 5 dpw in the wounded muscle. These results suggest that AgNPs are effective in acceleration of wound healing and altered the expression of some wound-healing-related genes. However, the detailed mechanism of enhanced wound healing remains to be investigated in fish.

The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes.

Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood-brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4-16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.