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Abstract 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.

Abstract 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.

Economical mutation detection method with high analytical and clinical sensitivity is necessary for early cancer diagnosis and screening. In this study, a novel 3D‐nanoplasmonic‐based multiplex mutation assay chip is developed to detect epidermal growth factor receptor (EGFR) mutations. This assay kit comprises a 3D‐nanoplasmonic substrate immobilized with capture probes and primer–probe sets for recombinase polymerase amplification, wild‐type inhibition, and fluorescence detection, enabling multiplex detection of EGFR exon 19 deletions, exon 20 insertions, and exon 21 L858R point mutations. The strategy facilitates the detection of all deletions and insertions within the target region with extremely high analytical sensitivity, detecting as low as 1 × 10−9% mutation frequency, implying three copies/reactions and 100 zM. The synergistic effects of plasmon‐enhanced fluorescence from the 3D‐nanoplasmonic substrate and wild‐type inhibitor contribute to this high analytical sensitivity. Moreover, the developed chip exhibits 100% accuracy in the clinical testing of plasma samples from normal individuals and patients with benign lung tumor and malignant lung tumor. With high sensitivity and multiplexing capabilities, this assay operates at a low reaction temperature (around 37 °C) and requires a short processing time, ≈70 min post‐cell‐free DNA extraction. These features make the chip a valuable tool for easy and widespread cancer screening.

Abstract Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

Abstract Development of coating technologies for electrochemical sensors that consistently exhibit antifouling activities in diverse and complex biological environments over extended time is vital for effective medical devices and diagnostics. Here, we describe a micrometer-thick, porous nanocomposite coating with both antifouling and electroconducting properties that enhances the sensitivity of electrochemical sensors. Nozzle printing of oil-in-water emulsion is used to create a 1 micrometer thick coating composed of cross-linked albumin with interconnected pores and gold nanowires. The layer resists biofouling and maintains rapid electron transfer kinetics for over one month when exposed directly to complex biological fluids, including serum and nasopharyngeal secretions. Compared to a thinner (nanometer thick) antifouling coating made with drop casting or a spin coating of the same thickness, the thick porous nanocomposite sensor exhibits sensitivities that are enhanced by 3.75- to 17-fold when three different target biomolecules are tested. As a result, emulsion-coated, multiplexed electrochemical sensors can carry out simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, antigen, and host antibody in clinical specimens with high sensitivity and specificity. This thick porous emulsion coating technology holds promise in addressing hurdles currently restricting the application of electrochemical sensors for point-of-care diagnostics, implantable devices, and other healthcare monitoring systems.

Abstract 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.

Abstract Objective We aimed to compare the adaptive immune response in individuals with or without prior SARS-CoV-2 infections following the administration of mRNA-based COVID-19 vaccines. Methods A total of 54 participants with ages ranging from 37 to 56 years old, consisting of 23 individuals without a history of SARS-CoV-2 infection (uninfected group) and 31 individuals with prior infection of SARS-CoV-2 (infected group) who have received two doses of mRNA SARS-CoV-2 vaccines were enrolled in this study. We measured the IFN-γ level upon administration of BNT162b2 (PF) or mRNA-1273 (MO) by QuantiFERON SARS-CoV-2. The production of neutralizing antibodies was evaluated by a surrogate virus neutralization assay, and the neutralizing capacity was assessed by a plaque reduction neutralization test (PRNT50). The immune response was compared between the two groups. Results A significantly higher level of IFN-γ (p

Abstract The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 µL of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

Abstract Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin. Graphical Abstract

Background: The coronavirus disease (COVID-19) has been a worldwide concern since 2019. Vaccines are predicted to be crucial in preventing further outbreaks. The development and kinetics of immune responses determine the efficacy of COVID-19 vaccines. Methods: We measured interferon-gamma (IFN-γ) levels upon administering homologous adenovirus vector-based (ChAdOx1-S [AZ], Ad26.COV2.S [JAN]), mRNA-based (BNT162b2 [PF]; mRNA-1273 [MO]), and heterologous (AZ/PF) vaccines in healthy Korean individuals using two IFN-γ release assays: the Covi-FERON ELISA and T-SPOT Discovery SARS-CoV-2 assay. B cell responses were evaluated by assessing the production of neutralizing antibodies by surrogate virus neutralization assay. The immune response among the vaccine groups was compared after adjusting the vaccination dose and interactions between each group. Results: AZ triggered the highest T cell response after the first dose but showed instability after the second. PF and MO yielded stable and higher increments of T and B cell responses compared to AZ. MO yielded a higher immune response than PF. JAN yielded T and B cell responses at lower levels than the other vaccines. The booster dose triggered significant increases in the T and B cell responses and is therefore needed to protect against SARS-CoV-2 given the possibility of waning immune responses. Conclusion: Administering two doses of mRNA vaccines provides the most effective results among the administered vaccines in triggering the immune response specific to SARS-CoV-2 in healthy Korean individuals. Administration of booster doses demonstrated a significant increase in the immune response and may provide longer protection against SARS-CoV-2.

Abstract Since COVID-19 and flu have similar symptoms, they are difficult to distinguish without an accurate diagnosis. Therefore, it is critical to quickly and accurately determine which virus was infected and take appropriate treatments when a person has an infection. This study developed a dual-mode surface-enhanced Raman scattering (SERS)-based LFA strip that can diagnose SARS-CoV-2 and influenza A virus with high accuracy to reduce the false-negative problem of the commercial colorimetric LFA strip. Furthermore, using a single strip, it is feasible to detect SARS-CoV-2 and influenza A virus simultaneously. A clinical test was performed on 39 patient samples (28 SARS-CoV-2 positives, 6 influenza A virus positives, and 5 negatives), evaluating the clinical efficacy of the proposed dual-mode SERS-LFA strip. Our assay results for clinical samples show that the dual-mode LFA strip significantly reduced the false-negative rate for both SARS-CoV-2 and influenza A virus.

In this study, we evaluated the effectiveness of the bivalent mRNA COVID-19 vaccines against the Omicron variant in individuals with or without prior SARS-CoV-2 infection history. We assessed the SARS-CoV-2-specific neutralizing antibody in serum samples by surrogate virus neutralizing assay (sVNT) and determined the serum’s neutralizing capacity against the Omicron BA.5 by a plaque reduction neutralizing test (PRNT50). The results of the sVNT assay demonstrate a higher percentage of inhibition of the serum samples from the infected group than from the uninfected group (p = 0.01) before the bivalent vaccination but a similarly high percentage of inhibition after the vaccination. Furthermore, the results of the PRNT50 assay demonstrate a higher neutralizing capacity of the serum samples against Omicron BA.5 in the infected group compared to the uninfected group, both before and after the bivalent vaccine administration (p < 0.01 and p = 0.02 for samples collected before and after the bivalent vaccination, respectively). A higher neutralizing capacity of the serum samples against BA.5 following bivalent vaccination compared to those before vaccination suggests the efficacy of bivalent mRNA COVID-19 vaccines in triggering an immune response against the Omicron variant, particularly BA.5, regardless of infection history.

Rapid detection of antiviral resistant strains is important for effective clinical treatment. Here the authors develop an antibody which specifically binds to multidrug-resistant influenza virus and demonstrated detection in a number of different systems using the antibody.

Abstract This work reports on a rapid diagnostic platform for the detection of Plasmodium falciparum lactate dehydrogenase (PfLDH), a representative malaria biomarker, using a microfluidic microplate-based immunoassay. In this study, the microfluidic microplate made it possible to diagnose PfLDH with a small volume of sample (only 5 μL) and short time (

Abstract A fluorescent fullerene nanoparticle (NP)-based lateral flow immunochromatographic assay (LFIA) was developed for the rapid and quantitative detection of C-reactive protein (CRP) in serum. The polyclonal CRP-antibody-conjugated fullerene NPs were simply prepared by 1-ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride coupling after carboxylation of fluorescent fullerene NPs. By applying the CRP-antibody-conjugated fullerene NPs to a lateral flow test strip, quantitative analysis of CRP in serum was possible at a concentration range of 0.1–10 ng/ml within 15 min. We anticipate that this novel fluorescent fullerene NP-based LFIA can be useful for the rapid and accurate sensing of biological and chemical species, contributing to the disease diagnosis and prognosis, environmental monitoring, and food safety.

The rapid diagnosis of SARS-CoV-2 is an essential aspect in the detection and control of the spread of COVID-19. We evaluated the accuracy of the rapid antigen test (RAT) using samples from the nasal cavity and nasopharynx based on sample collection timing and viral load. We enrolled 175 patients, of which 71 patients and 104 patients had tested positive and negative, respectively, based on real time-PCR. Nasal cavity and nasopharyngeal swab samples were tested using STANDARD Q COVID-19 Ag tests (Q Ag, SD Biosensor, Korea). The sensitivity of the Q Ag test was 77.5% (95% confidence interval [CI], 67.8–87.2%) for the nasal cavity and 81.7% (95% [CI, 72.7–90.7%) for the nasopharyngeal specimens. The RAT results showed a substantial agreement between the nasal cavity and nasopharyngeal specimens (Cohen’s kappa index = 0.78). The sensitivity of the RAT for nasal cavity specimens exceeded 89% for E and RdRp < 25. The Q Ag test performed fairly well, especially in the early DSO when a high viral load was present, and the nasal cavity swab can be considered an alternative site for the rapid diagnosis of COVID-19.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

In this data article, we developed a Au nanowire injector (Au NWI) for directly delivering plasmid into the 1-cell stage of the mouse embryos designed to successfully attach and detach the plasmid on the Au NWI, highly minimizing physical and chemical damage on the embryos. This data presents that a Au NWI system does not induce detrimental damages on development of embryos and efficiently express the green fluorescence protein in vitro. The data provided herein is in association with the research article related to reduce the occurrence of mosaicism by a Au NWI,” Suppressing Mosaicism by Au Nanowire Injector-driven Direct Delivery of Plasmids into Mouse Embryos” (Park et al., 2017 [1]).

Well-ordered bioreceptors on atomically flat Au surfaces can be a high-performance biosensor. Cardiac troponin I proteins (cTnIs) have been regarded as a specific biomarker for acute myocardial infarction (AMI). Here, we report the accurate detection of cTnIs using an aptamer-immobilized Au nanoplate platform. The single-crystalline and atomically flat Au nanoplate was characterized by atomic force microscopy. For the precise detection of cTnI, we immobilized an aptamer that can strongly bind to cTnI onto an atomically flat Au nanoplate. Using the aptamer-immobilized Au nanoplate, cTnIs were successfully detected at a concentration of 100 aM (2.4 fg/mL) in buffer solution. Furthermore, cTnIs in serum could be identified at a concentration of 100 fM (2.4 pg/mL). The total assay time was ~7 h. Importantly, the aptamer-immobilized Au nanoplate enabled us to diagnose AMI patients accurately, suggesting the potential of the present method in the diagnosis of AMI.

Antibiotic-resistant bacteria present a global threat because the infections they cause are difficult to treat. Therefore, it is highly important to develop advanced methods for the identification of antibiotic resistance gene in the virulent bacteria. Here, we report the development of novel nanoprobes for fluorescence in situ hybridization (FISH) and the application of the nanoprobe to the detection of ampicillin-resistant Escherichia coli. The nanoprobe for FISH was synthesized by the modified sol–gel chemistry and the synthesized nanoprobe provided strong fluorescent signals and pH stability even under natural light condition. For the double-identification of bacteria species and ampicillin-resistance with a single probe in situ, the nanoprobes were conjugated to the two kinds of biotinylated probe DNAs; one for E. coli-species specific gene and the other for a drug-resistant gene. By using the nanoprobe-DNA conjugants, we successfully detected the ampicillin-resistant E. coli through the FISH technique. This result suggests the new insight into light stable FISH application of the nanoprobe for a pathogenic antibiotic-resistance bacterium.

Wearable devices are becoming widespread in a wide range of applications, from healthcare to biomedical monitoring systems, which enable continuous measurement of critical biomarkers for medical diagnostics, physiological health monitoring and evaluation. Especially as the elderly population grows globally, various chronic and acute diseases become increasingly important, and the medical industry is changing dramatically due to the need for point-of-care (POC) diagnosis and real-time monitoring of long-term health conditions. Wearable devices have evolved gradually in the form of accessories, integrated clothing, body attachments and body inserts. Over the past few decades, the tremendous development of electronics, biocompatible materials and nanomaterials has resulted in the development of implantable devices that enable the diagnosis and prognosis through small sensors and biomedical devices, and greatly improve the quality and efficacy of medical services. This article summarizes the wearable devices that have been developed to date, and provides a review of their clinical applications. We will also discuss the technical barriers and challenges in the development of wearable devices, and discuss future prospects on wearable biosensors for prevention, personalized medicine and real-time health monitoring.

Au nanostructures (Au NSs) have been considered promising materials for applications in fuel cell catalysis, electrochemistry, and plasmonics. For the fabrication of high-performance Au NS-based electronic or electrochemical devices, Au NSs should have clean surfaces and be directly supported on a substrate without any mediating molecules. Herein, we report the vapor-phase synthesis of Au NSs on a fluorine-doped tin oxide (FTO) substrate at 120 °C and their application to the electrocatalytic methanol oxidation reaction (MOR). By employing AuCl as a precursor, the synthesis temperature for Au NSs was reduced to under 200 °C, enabling the direct synthesis of Au NSs on an FTO substrate in the vapor phase. Considering that previously reported vapor-phase synthesis of Au NSs requires a high temperature over 1000 °C, this proposed synthetic method is remarkably simple and practical. Moreover, we could selectively synthesize Au nanoparticles (NPs) and nanoplates by adjusting the location of the substrate, and the size of the Au NPs was controllable by changing the reaction temperature. The synthesized Au NSs are a single-crystalline material with clean surfaces that achieved a high methanol oxidation current density of 14.65 mA/cm2 when intimately supported by an FTO substrate. We anticipate that this novel synthetic method can widen the applicability of vapor-phase synthesized Au NSs for electronic and electrochemical devices.

We designed a high-sensitivity magnetic resonance imaging contrast agent that could be used to diagnose diseases. First, magnetic nanocrystals were synthesized by a thermal decomposition method on an organic solvent to obtain a high magnetism and methoxy poly(ethylene glycol)-poly(lactic acid) as an amphiphilic polymer using the ring-opening polymerization method to stably disperse the magnetic nanocrystals in an aqueous phase. Subsequently, the magnetic nanoclusters simultaneously self-assembled with methoxy poly(ethylene glycol)-poly(lactic acid) using the nano-emulsion method to form magnetic nanoclusters. Because their shape was similar to a raspberry, they were named PEGylated magnetic nano-assemblies. The PEGylated magnetic nano-assemblies were dispersed stably in the aqueous phase with a uniform size of approximately 65–70 nm for an extended period (0 days: 68.8 ± 5.1 nm, 33 days: 69.2 ± 2.0 nm, and 44 days: 63.2 ± 5.6). They exhibited both enough of a magnetic resonance (MR) contrast effect and biocompatibility. In an in vivo study, the PEGylated magnetic nano-assemblies provided a high contrast effect for magnetic resonance images for a long time after one treatment, thereby improving the diagnostic visibility of the disease site.