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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.

With the growth of drug-facilitated crimes, prevention has become increasingly important. Although various drug detection technologies exist, most focus on postconsumption detection. However, the prevention of drug-facilitated crimes requires technology for the quick and easy detection of amphetamine-type stimulants (ATSs) before ingestion. Herein, drug screening kits (DSKs) were developed for the simple detection of ATSs in drinks. The DSKs consisted of polydiacetylene nanofiber-based paper sensors fabricated by electrospinning with 10,12-pentacosadiynoic acid (PCDA) and PCDA-dopamine as sensing materials that can bind ATSs via hydrogen bonding and π-π interactions. Dropping a drink on the DSK provided an immediate visual indication of the presence of ATSs. When ATSs were present in the drink, the color of the DSK clearly changed from blue to red, with the increase in red intensity being more than twofold greater than that observed when water alone was tested. Notably, the result could be confirmed by the naked eye without any analytical instrumentation. A color change indicating the presence of ATSs was successfully observed in various alcoholic and nonalcoholic drinks. These results indicate the potential of DSKs for preventing drug-facilitated crimes caused by unwanted drug intake.

Ovarian cancer is the fifth leading cause of cancer-related deaths among women. In particular, it is a high cause of mortality among women in industrialized countries. Human epididymis protein 4 (HE4) has recently emerged as a serological biomarker for the diagnosis of ovarian cancer. Herein, we report the ultrasensitive detection of HE4 using a gold (Au) nanoplate (NPl)-based surface-enhanced Raman scattering (SERS) immunoassay, wherein a capture antibody-immobilized Au NPl acts as an immune substrate and detection antibody-immobilized Au nanoparticles (NPs) serve as immunoprobes. The presence of the target biomarker (HE4) results in the formation of a sandwich structure of Au NPls and NPs, providing strong SERS signals. The developed method allows us to detect HE4 at low concentrations of 10–17 M. The selective detection of HE4 was verified using the Au NPl SERS immunoassay. We anticipate that the current approach could be helpful for the early diagnosis of ovarian cancer and eventually applied for diverse biomarker detection.

Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies., 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.

For the development of immunoassays into sophisticated analyte-sensing methods, it is a priority to suppress nonspecific binding in immunoassays. Herein, we report a one-step surface coating method that can not only optimally immobilize antibodies but also suppress nonspecific binding. Zwitterionic dopamine (

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.

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.

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.

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

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.

Atomically flat surfaces of single-crystalline Au nanoplates can maximize the functionality of biomolecules, thus realizing extremely high-performance biosensors. Here, we report both highly specific and supersensitive detection of C-reactive protein (CRP) by employing atomically flat Au nanoplates. CRP is a protein biomarker for inflammation and infection and can be used as a predictive or prognostic marker for various cardiovascular diseases. To maximize the binding capacity for CRP, we carefully optimized the Au nanoplate-Cys3-protein G-anti-CRP structure by observing atomic force microscopy (AFM) images. The optimally anti-CRP-immobilized Au nanoplates allowed extremely specific detection of CRP at the attomolar level. To confirm the binding of CRP onto the Au nanoplate, we assembled Au nanoparticles (NPs) onto the CRP-captured Au nanoplate by sandwich immunoreaction and obtained surface-enhanced Raman scattering (SERS) spectra and scanning electron microscopy (SEM) images. Both the SERS and SEM results showed that we completely eliminated the nonspecific binding of Au NPs onto the optimally anti-CRP-immobilized Au nanoplate. Compared with the anti-CRP-immobilized rough Au film and the randomly anti-CRP-attached Au nanoplate, the optimally anti-CRP-immobilized Au nanoplate provided a highly improved detection limit of 10

To prevent the global transmission of mutant viruses and minimize the damage caused by mutant virus infection, the accurate identification of newly emerged mutant viruses should be a priority. The key problem in mutant virus identification is that the selective detection of a mutant virus in the biological environment, where small amounts of mutant virus and copious amounts of wild-type virus coexist, is difficult. Herein, we report specific and ultrasensitive detection of oseltamivir-resistant (pH1N1/H275Y mutant) virus using functional Au nanoparticles (NPs). The functional Au NPs were prepared by modifying the surfaces of Au NPs with oseltamivir hexylthiol (OHT) and malachite green isothiocyanate (MGITC) simultaneously. OHT is an excellent receptor for the pH1N1/H275Y mutant virus because it has a 250-fold higher binding affinity for the pH1N1/H275Y mutant virus than for the wild-type virus. MGITC is a Raman reporter that provides a distinctive surface-enhanced Raman scattering (SERS) signal. The SERS signal of MGITC on Au NPs allows us to detect pH1N1/H275Y mutant viruses sensitively and quantitatively. The functional Au NPs enable naked-eye and SERS dual-mode detection of mutant viruses. Only in the presence of the pH1N1/H275Y mutant virus, the functional Au NPs aggregate, and the color of the NPs changes from red to purple. This allows us to detect mutant viruses with the naked eye. Furthermore, the aggregated Au NPs can provide strong SERS signals of MGITC. By measuring the SERS signals, we could detect the pH1N1/H275Y mutant virus with a detection limit of 10 PFU. Importantly, the pH1N1/H275Y mutant virus could be detected by using the functional Au NPs even in a mixture of mutant and wild-type viruses with a ratio of 1/100. This result suggests that the present method might be employed for the diagnosis of oseltamivir-resistant virus and for further research, including mutant virus analysis and drug development.

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).

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.

Antimicrobial resistance and multidrug resistance are slower-moving pandemics than the fast-spreading coronavirus disease 2019; however, they have potential to cause a much greater threat to global health. Here, we report a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated surface-enhanced Raman scattering (SERS) assay for multidrug-resistant (MDR) bacteria. This assay was developed via a synergistic combination of the specific gene-recognition ability of the CRISPR system, superb sensitivity of SERS, and simple separation property of magnetic nanoparticles. This assay detects three multidrug-resistant (MDR) bacteria, species Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae, without purification or gene amplification steps. Furthermore, MDR A. baumannii-infected mice were successfully diagnosed using the assay. Finally, we demonstrate the on-site capture and detection of MDR bacteria through a combination of the three-dimensional nanopillar array swab and CRISPR-mediated SERS assay. This method may prove effective for the accurate diagnosis of MDR bacterial pathogens, thus preventing severe infection by ensuring appropriate antibiotic treatment.

We propose that nanogels (HLGs) prepared by simply blending an epidermal growth factor (EGF)-loaded hyaluronan (HA)-based nanoformulation and poloxamers can be efficient transdermal drug carriers. In particular, due to the thermogelling behavior of poloxamer, when the HLGs, which are liquid at room temperature, are applied to the skin's surface, they form a gel at skin temperature. First, lipid-based nanoformulations (EGF-LNs) were fabricated by the lipid thin film method and then chemically conjugated with HA on the surface of the films to prepare EGF-loaded HA-based nanoformulations (EGF-HLNs). Both EGF-LNs and EGF-HLNs exhibited a uniform size and spherical lamellar structure. The EGF-HLN was added to a poloxamer solution to form EGF-HLG, which is a liquid at room temperature and a gel at skin temperature. HLGs have been shown to be able to deliver and permeate EGF well into the skin using both in vitro and in vivo systems, thus serving as an effective transdermal delivery system. In addition, it has been confirmed that this system could be a possible implantable drug carrier. Therefore, HLGs, which are uncomplicated and easily prepared, are expected to be easily used not only in the pharmaceutical field but also in the cosmetic field.

Background: Pentasodium tetracarboxymethyl palmitoyl dipeptide-12 (PTPD-12), a newly-synthesized peptide, enhances the autophagy activity, ultimately managing inflammation. Objective: To determine the effect of a new moisturizer containing PTPD-12 as the treatment of mild-to-moderate atopic dermatitis (AD). Methods: In this double-blind, randomized, placebo-controlled trial, 43 patients with mild-to-moderate AD were randomly assigned to either the PTPD-12 or control groups. Evaluations were performed at baseline, week 2, and week 4, including SCORing Atopic Dermatitis (SCORAD) index score, corneometry, trans-epidermal water loss (TEWL), visual analog scale (VAS) for pruritus, 7-point investigator's global assessment (IGA), and collection of adverse events. Results: The PTPD-12 group showed significant improvement with respect to SCORAD score, skin hydration, TEWL, and pruritus at weeks 2 and 4 when compared with baseline. Although the control group showed significant improvement regarding the SCORAD score and skin hydration, no significant change in TEWL or pruritus was demonstrated throughout the study. The mean changes in the SCORAD index score, skin hydration, TEWL, pruritus, and number of patients with improvement in IGA were not statistically different between the two groups. Conclusion: The moisturizer with autophagy-stimulating property provides a good therapeutic option to mild-to-moderate atopic dermatitis by contributing to skin barrier restoration and control of inflammation.

Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.

We have developed a novel oseltamivir derivative (oseltamivir hexylthiol; OHT) that exhibits a higher binding affinity for Tamiflu-resistant virus (Tamiflu resistance) than for the wild-type virus (Tamiflu-susceptible virus; WT) as an antibody. First, OHT-modified gold nanoparticles (OHT-GNPs) are used in a simple colorimetric assay as nanoprobes for the Tamiflu-resistant virus. In the presence of Tamiflu-resistant virus, they show a colorimetric change from deep red to purple because of the OHT-GNP aggregation driven by strong interactions between OHT and neuraminidase (NA) on the surface of the Tamiflu-resistance. Moreover, the color gradually turns purple as the concentration of the Tamiflu-resistant virus increases, allowing the determination of the presence of the virus with the naked eye. Furthermore, an OHT-based lateral flow assay (LFA) has been developed as a rapid and easy detection device for Tamiflu resistance. It shows detection specificity for various virus concentrations of Tamiflu-resistant virus even for the mixture of WT and Tamiflu-resistant viruses, where the limit of detection (LOD) is 5 × 102 ~ 103 PFU per test (=1 × 104 PFU/mL). It has been confirmed that this platform can provide accurate information on whether a virus exhibits Tamiflu resistance, thus supporting the selection of appropriate treatments using point-of-care (POC) diagnostics.

Respiratory syncytial virus (RSV) infections are associated with severe bronchiolitis or pneumonia. Although palivizumab is used to prevent RSV infections, the occurrence of palivizumab-resistant RSV strains is increasing, and these strains pose a threat to public health. Herein, we report an antibody with affinity to the S275F RSV antigen, enabling the specific detection of palivizumab-resistant RSV strains. Experimental and simulation results confirmed the affinity of the antibody to the S275F RSV antigen. Furthermore, we developed a rapid S275F RSV antigen detection method using a split superfolder green fluorescent protein (ssGFP) that can interact with the antibody. In the presence of the mutant virus antigen, ssGFP emitted fluorescence within 1 min, allowing the rapid identification of S275F RSV. We anticipate that the developed antibody would be useful for the precise diagnosis of antiviral drug-resistant RSV strains and help treat patients with RSV infections.

Drug-facilitated crimes using date-rape drugs are on the rise. Gamma-hydroxybutyric acid (GHB), a notorious date-rape drug, is colorless, odorless, and tasteless, and therefore difficult to detect in beverages. The ingestion of GHB can severely incapacitate individuals by inducing dizziness, drowsiness, and unconsciousness, making them vulnerable to crimes. To prevent drug-facilitated crimes, a GHB detection kit is developed in which PCDA-gabazine and PCDA, which strongly interact with GHB, are used as sensing materials, while PEO and PVDF are used as matrix materials that enable rapid GHB detection in a short time by increasing the penetration rate of the liquid samples. Detection kits are fabricated by electrospinning a solution containing the sensing materials and matrix materials mixed in an optimal ratio. The color of the kit distinctly changes from blue to red when a drink spiked with GHB is dropped on it (limit of detection: about 0.0096 wt%). The fabricated GHB detection kit is easy to carry and provides a visually distinguishable detection result without requiring any complicated procedure, equipment, or trained personnel. Importantly, the kit can detect GHB in colored and alcoholic beverages. Furthermore, a mobile application is developed that can aid GHB detection using the kit in diverse environments.

Influenza viruses are responsible for several pandemics and seasonal epidemics and pose a major public health threat. Even after a major outbreak, the emergence of drug-resistant influenza viruses can pose disease control problems. Here we report a novel 6E3 monoclonal antibody capable of recognizing and binding to the H275Y neuraminidase (NA) mutation, which has been associated with reduced susceptibility of influenza viruses to NA inhibitors. The 6E3 antibody had a KD of 72.74 μM for wild-type NA and 32.76 pM for H275Y NA, suggesting that it can identify drug-resistant pandemic H1N1 (pH1N1) influenza virus. Molecular modeling studies also suggest the high-affinity binding of this antibody to pH1N1 H275Y NA. This antibody was also subject to dot-blot, enzyme-linked immunosorbent assay, bare-eye detection, and lateral flow assay to demonstrate its specificity to drug-resistant pH1N1. Furthermore, it was immobilized on Au nanoplate and nanoparticles, enabling surface-enhanced Raman scattering (SERS)-based detection of the H275Y mutant pH1N1. Using 6E3 antibody-mediated SERS immunoassay, the drug-resistant influenza virus can be detected at a low concentration of 102 plaque-forming units/mL. We also detected pH1N1 in human nasopharyngeal aspirate samples, suggesting that the 6E3-mediated SERS assay has the potential for diagnostic application. We anticipate that this newly developed antibody and SERS-based immunoassay will contribute to the diagnosis of drug-resistant influenza viruses and improve treatment strategies for influenza patients.

Acanthamoeba polyphaga mimivirus (APMV) is a member of the family of giant viruses, harboring a 1,200 kbp genome within its 700 nm-diameter viral particle. The R214 gene of the APMV genome was recently shown to encode a homologue of the Rab GTPases, molecular switch proteins known to play a pivotal role in the regulation of membrane trafficking that were considered to exist only in eukaryotes. Herein, we report the first crystal structures of GDP- and GTP-bound forms of APMV Rab GTPase, both of which were determined at high resolution. An in-depth structural comparison of APMV Rab with each other and with mammalian Rab homologues led to an atomic-level elucidation of the inactive-active conformational change upon GDP/GTP exchange. APMV Rab GTPase exhibited considerable structural similarity to human Rab5, as previously predicted based on its amino acid sequence. However, it also contains unique structural features differentiating it from mammalian homologues, such as the functional substitution of a phenylalanine residue for the stabilization of the nucleotide's guanine base.

Salmonella is one of the most common foodborne pathogens, and Salmonella outbreaks are mostly associated with the intake of contaminated food or drink. Therefore, the rapid and sensitive on-site detection of Salmonella is very important. We report a naked eye detection method for Salmonella typhimurium using scanometric antibody probe. The antibody-attached glass substrate was treated with Salmonella typhimurium and the scanometric antibody probe was applied. After Ag enhancement of the probe, Salmonella typhimurium could be detected with the naked eye. The scanometric antibody probe was prepared by simply mixing Au nanoparticles, gold binding peptide-protein G, and antibody against Salmonella typhimurium. This probe can act as a signal enhancer and thus allows for an extremely simple, rapid, and efficient analysis of Salmonella typhimurium by the naked eye. We detected Salmonella typhimurium at a low concentration of 103 CFU/ml and clearly distinguished this bacterium from other foodborne pathogens. Furthermore, we successfully detected Salmonella typhimurium in milk, suggesting that this method can be useful in real-life samples. Because the scanometric antibody probe can be expanded to various types of antibodies, this naked eye detection method could be employed for the detection of various types of pathogens.

A simple scheme using a hairpin DNA aptasensor was established to detect Salmonella Typhimurium (S. Typhimurium). The hairpin DNA aptasensor was a single-stranded oligonucleotide including both a target-binding sequence and a reporting DNAzyme called horseradish peroxidase-mimicking G-quadruplex. Four hairpin DNA aptasensors were investigated to optimize the sequence with two enzymatic substrates for different signaling modes. Covalent conjugation of hemin on an aptasensor strand was preliminarily executed in colorimetric assays and then conducted with p-coumaric acid in chemiluminescence assays, to lower the background signal arisen from free hemin. A limit of detection (LOD) of this detection scheme was 100 CFU mL(-1) in colorimetric assays and was even further improved to 2 CFU mL(-1) in chemiluminescent assays. The present detection scheme is expected to have application as a simple, rapid method for detection of various types of hazardous targets.

Prostate cancer (PC) is the second leading cause of cancer death for men worldwide. The serum prostate-specific antigen level test has been widely used to screen for PC. This method, however, exhibits a high false-positive rate, leading to over-diagnosis and over-treatment of PC patients. Extracellular microRNAs (miRNAs) recently provided valuable information including the site and the status of the cancers and thus emerged as new biomarkers for several cancers. Among them, miR141 and miR375 are the most pronounced biomarkers for the diagnosis of high-risk PC. Herein, we report an attomolar detection of miR141 and miR375 released from living PC cells by using a plasmonic nanowire interstice (PNI) sensor. This sensor showed a very low detection limit of 100 aM as well as a wide dynamic range from 100 aM to 100 pM for all target miRNAs. In addition, the PNI sensor could discriminate perfectly the diverse single-base mismatches in the miRNAs. More importantly, the PNI sensor successfully detected the extracellular miR141 and miR375 released from living PC cell lines (LNCaP and PC-3), proving the diagnostic ability of the sensor for PC. We anticipate that the present PNI sensor can hold great promise for the precise diagnosis and prognosis of various cancer patients as well as PC patients.

Influenza viruses cause respiratory infection, spread through respiratory secretions, and are shed into the nasal secretion and saliva specimens. Therefore, nasal fluid and saliva are effective clinical samples for the diagnosis of influenza virus-infected patients. Although several methods have been developed to detect various types of influenza viruses, approaches for detecting mutant influenza viruses in clinical samples are rarely reported. Herein, we report for the first time a surface-enhanced Raman scattering (SERS)-based sensing platform for oseltamivir-resistant pandemic H1N1 (pH1N1) virus detection in human nasal fluid and saliva. By combining SERS-active urchin Au nanoparticles and oseltamivir hexylthiol, an excellent receptor for the pH1N1/H275Y mutant virus, we detected the pH1N1/H275Y virus specifically and sensitively in human saliva and nasal fluid samples. Considering that the current influenza virus infection testing methods do not provide information on the antiviral drug resistance of the virus, the proposed SERS-based diagnostic test for the oseltamivir-resistant virus will inform clinical decisions about the treatment of influenza virus infections, avoiding the unnecessary prescription of ineffective drugs and greatly improving therapy.

We have proposed a novel strategy for miRNA detection through enzyme-free signal amplification by self-circulation of the hybridization between the miRNAs and molecular beacon (MB) circuits. Unlike general MB-based miRNA detection based on the one-to-one (1 : 1) hybridization between MBs and miRNA, our system consists of four species of MBs (MBs A, B, C and D) (MB circuits) and is activated by a hybridization chain reaction. MBs stably coexist as hairpin structures that hardly show fluorescence signals in the absence of target miRNA. After miRNA detection, this MB circuit is able to generate fluorescence signals and amplify the fluorescence signal, contributing to improvement in detection sensitivity under iso-thermal conditions without an enzyme. Furthermore, in vitro and in vivo studies have proven that MB circuits can detect low levels of miRNA with high sensitivity, compared to when only one MB alone is used. Therefore, the MB circuits can provide a useful platform for target miRNA detection.

Putrescine and cadaverine are important volatile indicators for the evaluation of food spoilage. In this study, a metal-organic framework (MOF)-coated surface-enhanced Raman scattering (SERS) paper platform for the detection of putrescine and cadaverine is developed. Au@ zeolite imidazolate framework-8 (ZIF-8) SERS paper is fabricated by the coating of ZIF-8 layer on a Au nanoparticle-impregnated paper that is prepared by dry plasma reduction. The Au@ZIF-8 SERS paper is characterized by scanning electron microscope, energy-dispersive X-ray spectroscopy, X-ray diffraction, and N2 sorption isotherm. The ZIF-8 layer enables the accumulation of gaseous molecules and also provides enhancement of SERS signals. The fluorescence, SERS, and simulation results prove the improved detection ability of the Au@ZIF-8 platform for the volatile molecules. For the selective detection of putrescine and cadaverine, the Au@ZIF-8 SERS paper is functionalized with 4-mercatobenzaldehyde (4-MBA). The 4-MBA molecule acts as a Raman reporter and also a specific receptor for the volatile amine molecules. Using the intensity ratiometric detection of 4-MBA-functionalized Au@ZIF-8 SERS paper, putrescine and cadaverine are quantitatively detected with detection limits of 76.99 and 115.88 parts per billion, respectively. Furthermore, the detection of volatile amine molecules released from spoiled salmon, chicken, beef, and pork samples is demonstrated. It is anticipated that the MOF-coated SERS paper platforms will be applicable not only in food safety but other applications including disease diagnosis and environmental monitoring.

To develop a reliable system for identifying multiple S haplotypes controlling self-incompatibility (SI) in radish (Raphanus sativus L.), the genomic organization of the S locus region was identified from radish draft genome sequences. An initial attempt to find the S receptor kinase (SRK) gene, the female determinant of SI, failed due to presence of 15 homologous genes. Using synteny between the radish and Chinese cabbage genomes, the putative S locus region was identified in the radish R7 linkage group. One scaffold anchored to this R7 region contained the S-locus glycoprotein (SLG) gene, which is one of the S locus genes. Using the high homology between the SLG and S domain of SRK, the full-length radish SRK gene containing the largest 6861-bp intron1 was assembled by connecting two scaffolds harboring the S receptor and kinase domains, respectively. A scaffold containing the full-length S-locus cysteine-rich protein (SCR)/S locus protein 11 (SP11) gene, the male-determinant of SI, was identified using information reported previously. Finally, 53,785, 42,804, and 10,165 bp sequences containing the S locus genes and their flanking sequences were obtained. Unlike the various orientations of the SRK or SCR/SP11 genes, the position of SLL2 located at the border region of the S locus was conserved among haplotypes. Sequencing of the SLL2 gene from 31 inbred lines showing differential SI responses revealed 26 polymorphic alleles. Four additional SLL2 alleles were identified from analysis of diverse breeding lines. Based on the polymorphic SLL2 sequences, a new S haplotyping system was developed for efficient marker-assisted selection of the S haplotypes in radish.

We developed a nanoprobe for the label-free detection of antibodies associated with infectious diseases, through a method based on the antibody catalyzed water oxidation pathway (ACWOP). First, methoxy poly(ethylene glycol) (mPEG)–chlorin e6 was chemically conjugated to mPEG and chlorin e6 by esterification, to enable both singlet oxygen generation and loading of coumarin boronate. Subsequently, the nanoprecipitation method was used for spontaneous nanoprobe self-assembly from coumarin boronate and mPEG–chlorin e6, which resulted in a mean nanoprobe size of ∼200 nm. The potential of the nanoprobe as an antibody detection agent was evaluated by using human immunoglobulin antibodies as a model antibody. We confirmed that the nanoprobe generated singlet oxygen (1O2) via the photodynamic chemical effect of the chlorin e6 in mPEG–chlorin e6 and that water (H2O) was subsequently oxidized to hydrogen peroxide (H2O2) in the presence of the singlet oxygen through the ACWOP. The generated H2O2 was detected via the fluorescence of coumarin boronate in the nanoprobe. Specifically, the fluorescence intensity increased linearly with increasing antibody amounts, thus demonstrating the potential of the system as an alternative method for label-free antibody detection.