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Aastha Kukreja,1 Eun-Kyung Lim,2–4 Byunghoon Kang,1 Yuna Choi,2 Taeksu Lee,1 Jin-Suck Suh,2,3 Yong-Min Huh,2,3 Seungjoo Haam1,31Department of Chemical and Biomolecular Engineering, College of Engineering, 2Department of Radiology, College of Medicine, Yonsei University, Seoul, Republic of Korea; 3YUHS-KRIBB Medical Convergence Research Institute, Seoul, Republic of Korea; 4BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of KoreaAbstract: In this study, dextran-encrusted magnetic nanoclusters (DMNCs) were synthesized using a one-pot solution phase method for detection of atherosclerosis by magnetic resonance imaging. Pyrenyl dextran was used as a surfactant because of its electron-stabilizing effect and its amphiphilic nature, rendering the DMNCs stable and water-dispersible. The DMNCs were 65.6±4.3 nm, had a narrow size distribution, and were superparamagnetic with a high magnetization value of 60.1 emu/g. Further, they showed biocompatibility and high cellular uptake efficiency, as indicated by a strong interaction between dextran and macrophages. In vivo magnetic resonance imaging demonstrated the ability of DMNCs to act as an efficient magnetic resonance imaging contrast agent capable of targeted detection of atherosclerosis. In view of these findings, it is concluded that DMNCs can be used as magnetic resonance imaging contrast agents to detect inflammatory disease.Keywords: magnetic nanocrystal, magnetic resonance imaging, atherosclerosis, macrophages, dextran

During the period 1971–4, Torulopsis glabrata formed 42% of yeasts isolated from 5677 clinical specimens from patients with cancers, diabetes mellitus, chronic renal failure, pregnancy or major surgical operations complicated by mycotic infections. The significance of T. glabrata in these patients is discussed. [ABSTRACT FROM PUBLISHER]

The growing risk of bacterial food poisoning due to global warming has necessitated the development of methods for accurate detection of food-poisoning bacteria. Despite extensive efforts to develop enhanced bacterial-capture methods, challenges associated with the release of the captured bacteria have limited the sensitivity of bacterial detection. In this study, thermo-responsive intelligent 3D nanostructures to improve food-poisoning bacterial analysis performance were fabricated by introducing a thermo-responsive polymer onto an urchin-like 3D nanopillar substrate (URCHANO). A co-polymer of methacryloyl glycinamide and benzyl acrylate (MNAGA-Bn 5%) was introduced as a thermo-responsive co-polymer onto URCHANO using an electron-transfer atom-transfer radical-polymerization method to fabricate Thermo-URCHANO. A temperature-related analysis of the surface properties of Thermo-URCHANO revealed a hydrophobic-to-hydrophilic transition at 37 °C, which facilitated the release of bacteria captured within the nanostructure. In a one-pot analysis to capture and analyze various food-poisoning bacteria in kitchenware (gloves and aprons) and food items (eggs and sausages), mimicking real-life environments, specimens collected using Thermo-URCHANO showed lower C t values than those collected with uncoated URCHANO, indicating greater bacterial detection. This method could effectively release captured bacteria through temperature changes, improving extraction efficiency during swab collection. While Thermo-URCHANO needs further optimization, it is expected to enhance bacterial analysis performance and sensitivity.

Cas12j, a hypercompact and efficient Cas protein, has potential for use in CRISPR diagnostics, but has not yet been used because the trans-cleavage activity of Cas12j is veiled. Here, the trans-cleavage behavior of Cas12j1, 2, and 3 variants and evaluate their suitability for nucleic acid detection is unveiled. The target preferences and mismatch specificities of the Cas12j variants are precisely investigated and the optimal Cas12j reaction conditions are determined. As a result, the EXP-J assay for miRNA detection by harnessing the robust trans-cleavage activity of Cas12j on short ssDNA is developed. The EXP-J method demonstrates exceptional detection capabilities for miRNAs, proving that Cas12j can be a pivotal component in molecular diagnostics. Furthermore, the translational potential of the EXP-J assay is validated by detecting oncogenic miRNAs in plasma samples from lung cancer patients. This investigation not only elucidates the trans-cleavage characteristics of Cas12j variants, but also advances the Cas12j-based diagnostic toolkit., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Gastric cancer (GC) is recognized as the fifth most prevalent malignant tumor worldwide. It is characterized by diverse clinical symptoms, treatment responses, and prognoses. In GC prognosis, the promotion of epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, thereby triggering the dissemination of tumor cells. This study proposes a nucleic acid amplification circuit-based hydrogel (NACH) assay for identifying exosomal miRNA derived from metastatic GC. The NACH assay employs the rolling circle amplification method and targets miRNA-21, a tumor-related oncogene, and miRNA-99a, which promotes EMT. Specific amplification probes for each target are immobilized within the hydrogel, enabling a streamlined, one-step amplification reaction. The NACH assay exhibits a detection limit of 1 fm for miRNA-21 and miRNA-99a, thereby enabling rapid and highly sensitive on-site detection. Performance evaluation using exosomal miRNA extracted from cell culture media, mouse plasma, and human plasma revealed fluorescence intensity patterns similar to those obtained in qRT-PCR. Furthermore, deploying a custom-developed portable fluorometer for the NACH assay allows for diagnostic performance assessment and point-of-care testing using clinical samples from GC patients. These findings emphasize the potential of the NACH assay to be used as a robust tool for the genetic diagnosis of GC based on exosome detection., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Extracellular vesicles (EVs) have emerged as valuable biological materials for treating intractable diseases. Extensive studies are conducted on EVs derived from various cellular sources. In this study, EVs derived from Lactobacillus reuteri (L. reuteri), a probiotic, exhibit remarkable cancer therapeutic efficacy when administered orally is reported. These L. reuteri-derived EVs (REVs) demonstrate stability in the gastrointestinal tract and exert significant anti-tumor effects. Using A549 cells and murine models, we confirmed that REVs mediate their therapeutic effects by modulating apoptotic signaling pathways. Furthermore, the combination of REV with drugs enhances tumor ablation and induces immunogenic cell death. In a mouse model, oral administration of REVs encapsulating indocyanine green followed by photothermal therapy led to complete tumor elimination within 32 days. REVs represent a promising biological therapeutic platform for cancer treatment, either independently or in combination with other therapies, depending on the treatment objectives., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Natural polyphenols found in health supplements and drinks have antioxidant and anti-inflammatory properties. In particular, to determine the beneficial qualities of antioxidant drinks and beverages, consumers demand precise quantification of the total amount of polyphenols as on-site detection. Herein, we developed a new concept of portable beads suitable for the field detection available: colorimetric quantification of polyphenols equipped with color converting software applications in a smartphone or tablet PC. The yellowish beads contain ferric ions to react with polyphenol to produce blackish metal-phenolic complexes. It is simple to perform the detection procedure: dipping the beads in the analytical sample and out-taking a photo-converting into RGB color values and quantification of the existed polyphenol. The overall process was completed within 5 min. Compared with the Folin-Ciocalteu assay, which is a representative optical sensor kit for total phenolic content, the bead-based sensor showed a better limit of detection of 0.0415 mM for tannic acid and comparable sensing capability for a polyphenol-containing plant extract and brewed tea. The beads conserved the shape and sensitivity after months of storage or under environmental interference such as a change in the temperature., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 μg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Breast cancer (BC) is a major global health problem, with ≈20-25% of patients overexpressing human epidermal growth factor receptor 2 (HER2), an aggressive marker, yet access to early detection and treatment varies across countries. A low-cost, equipment-free, and easy-to-use polydiacetylene (PDA)-based colorimetric sensor is developed for HER2-overexpressing cancer detection, designed for use in low- and middle-income countries (LMICs). PDA nanoparticles are first prepared through thin-film hydration. Subsequently, hydrophilic magnetic nanoparticles and HER2 antibodies are sequentially conjugated to them. The synthesized HER2-MPDA can be concentrated and separated by a magnetic field while inheriting the optical characteristics of PDA. The specific binding of HER2 antibody in HER2-MPDA to HER2 receptor in HER2-overexpressing exosomes causes a blue-to-red color change by altering the molecular structure of the PDA backbone. This colorimetric sensor can simultaneously separate and detect HER2-overexpressing exosomes. HER2-MPDA can detect HER2-overexpressing exosomes in the culture medium of HER2-overexpressing BC cells and in mouse urine samples from a HER2-overexpressing BC mouse model. It can selectively isolate and detect only HER2-overexpressing exosomes through magnetic separation, and its detection limit is found to be 8.5 × 10 8  particles mL -1 . This colorimetric sensor can be used for point-of-care diagnosis of HER2-overexpressing BC in LMICs., (© 2023 Wiley‐VCH GmbH.)

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections., (© 2023. The Author(s).)

The simultaneous infection with a tripledemic-simultaneous infection with influenza A pH1N1 virus (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV)-necessitates the development of accurate and fast multiplex diagnostic tests. The coronavirus disease 2019 (COVID-19) pandemic has emphasized the importance of virus detection. Field-effect transistor (FET)-based immuno-biosensors have a short detection time and do not require labeling or polymerase chain reaction. This study demonstrates the rapid, sensitive detection of influenza A pH1N1, SARS-CoV-2, and RSV using a multiplex immunosensor based on a dual-gate oxide semiconductor thin-film transistor (TFT), a type of FET. The dual-gate oxide TFT was modified by adjusting both top and bottom gate insulators to improve capacitive coupling to approximately 120-fold amplification, exhibiting a high pH sensitivity of about 10 V/pH. The dual-gate oxide TFT-based immunosensor detected the target proteins (hemagglutinin (HA) protein of Flu, spike 1 (S1) protein of SARS-CoV-2, and fusion protein of RSV) of each virus, with a limit of detection of approximately 1 fg/mL. Cultured viruses in phosphate-buffered saline or artificial saliva and clinical nasopharynx samples were detected in 1-μL sample volumes within 60 s. This promising diagnosis could be potentially as point-of-care tests to facilitate a prompt response to future pandemics with high sensitivity and multiplexed detection without pretreatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer-derived exosomes. Therefore, a microfluidic chip-based magnetically labeled exosome isolation system (MEIS-chip) was developed as a lab-on-a-chip platform for human epidermal growth factor receptor 2 (HER2)-positive cancer diagnosis and monitoring. Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2-overexpressing and common exosomes using immunoaffinity. MNC-bonded exosomes were separated into different exits according to their magnetization degrees. The MEIS-chip efficiently separated HER2-overexpressing exosomes from common exosomes that did not contain disease-related information. The simultaneous separation of HER2-and non-HER2-overexpressing exosomes provided a means of analyzing high-purity HER2-overexpressing exosomes while minimizing the contribution of non-target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real-time changes in HER2 expression. These findings support the application of MEIS-chip for cancer diagnosis and treatment monitoring via effective exosome isolation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, user-friendly nucleic acid testing that involves simple but efficient RNA extraction. Here, we present a charge-shifting polyplex as an RNA extraction carrier for advanced diagnosis of infectious viral diseases. The polyplex comprises poly(2-(dimethylamino) ethyl acrylate) (pDMAEA) electrostatically conjugated with RNA. The pDMAEA film can rapidly dissolve in the viral RNA solution, promoting immediate binding with RNA to form the polyplex, which enables the efficient capture of a substantial quantity of RNA. Subsequently, the captured RNA can be readily released by the quick hydrolysis of pDMAEA at the onset of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), streamlining the entire process from RNA extraction to analysis. The developed method requires only 5 min of centrifugation and enables the detection of RNA in a one-pot setup. Moreover, the proposed method is fully compatible with high-speed qRT-PCR kits and can identify clinical samples within 1 h including the entire extraction to detection procedure. Indeed, the method successfully detected influenza viruses, SARS-CoV-2, and their delta and omicron variants in 260 clinical samples with a sensitivity of 99.4% and specificity of 98.9%. This rapid, user-friendly polyplex-based approach represents a significant breakthrough in molecular diagnostics.

Introduction: Effective smoking cessation programmes are essential for assisting smokers in quitting, indirectly lowering mortality and morbidity associated with smoking. Numerous studies have indicated positive outcomes when using mindfulness treatment (MT) to treat psychological or behavioural health issues. Although to date, no study has looked at the effectiveness of online MT for quitting smoking while addressing mental health, particularly among the Asian population. Therefore, this study compares the efficiency of online MT to traditional counselling therapy (CT) in aiding smoking cessation programmes while also addressing mental health., Materials and Methods: A randomised control study with a two-group, single-blind design and baseline evaluation was selected. Social media sites were used to advertise for participants, who were then admitted after meeting the requirements. Participants who met the eligibility requirements were randomly split into two groups. Each group received a total of three sessions of online therapy (MT or CT), once every two weeks, as well as one phone call per week as reinforcement. At the beginning and end of the intervention, participants completed questionnaires (1st week and 5th week). Generalized Estimating Equation (GEE) statistical analysis was used to analyse all the variables., Results: The MT group experienced a statistically significant decrease in cigarette consumption (β: -3.50, 95% Wald CI: - 4.62, -2.39) compared to the CT group over time. Furthermore, the MT group demonstrated significant improvements in their scores for the AAQ-2, anxiety, stress, depression and mindfulness compared to the CT group., Conclusion: Online MT is more successful at assisting smokers in lowering their daily cigarette intake and supporting their mental health during the smoking cessation process. Further longitudinal comparisons of the effectiveness of online MT should be undertaken using online platforms in future studies.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Most nucleic acid biosensors employ nucleic acid-processing enzymes to bind, degrade, splice, synthesize, and modify nucleic acids. Utilizing their unique substrate preference, binding mode, and catalytic activity is of great importance in designing nucleic acid biosensors. Combination with DNA-processing enzymes enables them to transform into a new generation of molecular diagnostics tools with enhanced selectivity and sensitivity and reduced reaction time. Here, we report an isothermal amplification strategy by coemploying a structure-specific endonuclease (flap endonuclease 1, FEN1) and a strand-displacing DNA polymerase ( Bst DNA polymerase) to detect long RNA targets. This approach couples the FEN1-driven invasive cleavage reaction with toehold-mediated rolling circle amplification (iFEN-tRCA), enabling the highly selective and rapid detection of long RNA targets and offering a detection limit below 10 pM within 1 h. We used two targets, such as human epidermal growth factor receptor 2 (HER2, encoded by ERBB2 ) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP, encoded by PPP1R1B ), associated with prognosis or response to anticancer therapy. We demonstrated the feasibility and quantitative capability of the iFEN-tRCA assay by assessing the expression of two RNA transcripts ( ERBB2 and PPP1R1B ) with total RNA extracts purified from human breast cancer cells. Therefore, we envision that the developed assay will provide a suitable prognostic and diagnostic tool for identifying appropriate patients for HER2-targeted therapy and predicting the clinical outcome and occurrence of metastasis relapse in breast cancer.

Background: Bacterial peritonitis remains a significant cause of mortality in patients on peritoneal dialysis (PD). Early detection of causative organisms and targeted antimicrobial treatment allow for better clinical outcomes. This study compares bacterial growth results from peritoneal dialysate in the BACTEC blood culture system vs. conventional culture., Materials and Methods: We conducted a prospective study on 46 patients with 63 consecutive episodes of suspected PD peritonitis between August 2020 and August 2021. PD dialysate was simultaneously sent to the laboratory in both BACTEC and sterile bottles. BACTEC bottles were incubated in the BD BACTEC FX system for 5 days. PD effluent transported from the sterile bottles was centrifuged at 3,000 rpm for 15 minutes; the supernatant was inoculated into cooked-meat broth for enrichment. Both incubation methods were extended to 14 days if microorganisms were seen on the Gram-stained smear. Recovery of isolated micro-organisms and time to detection (TTD) were compared., Results: 26 episodes of suspected PD peritonitis based on clinical criteria were identified during the study period. The sensitivity of the BACTEC and the conventional culture methods was 50% and 42.3%, respectively (p = 0.45). Seven samples had partial concordance or discordant results. McNemar's χ 2 -test revealed no statistical difference between either method (p = 0.45). TTD was 18.9 ± 24.4 hours via the BACTEC method vs. 37 ± 16.5 hours in conventional cultures (p = 0.014)., Conclusion: The comparable sensitivities and similar yield in identifying pathogens could be due to the enrichment medium and prolonged incubation period. The shorter TTD for the BACTEC method could facilitate earlier confirmation of bacteriological diagnosis and subsequent treatment strategies.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.

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)., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 10 3 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

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., (Copyright © 2022. Published by Elsevier B.V.)

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Background/aims: Surgeons and endoscopists have started to use endoscopically inserted double pigtail stents (DPTs) in the management of upper gastrointestinal (UGI) leaks, including UGI anastomotic leaks. We investigated our own experiences in this patient population., Methods: From March 2017 to June 2020, 12 patients had endoscopic internal drainage of a radiologically proven anastomotic leak after UGI surgery in two tertiary UGI centers. The primary outcome measure was the time to removal of the DPTs after anastomotic healing. The secondary outcome measure was early oral feeding after DPT insertion., Results: Eight of the 12 patients (67%) required only one DPT, whereas four (33%) required two DPTs. The median duration of drainage was 42 days. Two patients required surgery due to inadequate control of sepsis. Of the remaining 10 patients, nine did not require a change in DPT before anastomotic healing. Nine patients were allowed oral fluids within the 1st week and a soft diet in the 2nd week. One patient was allowed clear oral feeds on the 8th day after DPT insertion., Conclusion: Endoscopic internal drainage is becoming an established minimally invasive technique for controlling anastomotic leak after UGI surgery. It allows for early oral nutritional feeding and minimizes discomfort from conventional external drainage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected humans worldwide for over a year now. Although various tests have been developed for the detection of SARS-CoV-2, advanced sensing methods are required for the diagnosis, screening, and surveillance of coronavirus disease 2019 (COVID-19). Here, we report a surface-enhanced Raman scattering (SERS)-based immunoassay involving an antibody pair, SERS-active hollow Au nanoparticles (NPs), and magnetic beads for the detection of SARS-CoV-2. The selected antibody pair against the SARS-CoV-2 antigen, along with the magnetic beads, facilitates the accurate direct detection of the virus. The hollow Au NPs exhibit strong, reproducible SERS signals, allowing sensitive quantitative detection of SARS-CoV-2. This assay had detection limits of 2.56 fg/mL for the SARS-CoV-2 antigen and 3.4 plaque-forming units/mL for the SARS-CoV-2 lysates. Furthermore, it facilitated the identification of SARS-CoV-2 in human nasopharyngeal aspirates and diagnosis of COVID-19 within 30 min using a portable Raman device. Thus, this assay can be potentially used for the diagnosis and prevention of COVID-19., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)