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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study.

The aim of this study was to perform the detection of folate receptor (FR)-positive tumors with a bimodal imaging contrast agent, a perfluorocarbon (PFC)/rhodamine nanoemulsion, providing both 19F-based magnetic resonance imaging (MRI) and fluorescence imaging capabilities. The PFC/rhodamine nanoemulsion was further infused with phospholipid-anchored folate to improve the ability to target FR-expressing tumors. The preferential accumulation of the FR-targeted bimodal nanoemulsion in FR-positive tumor sites was monitored by both 19F-MRI and optical imaging. The FR-targeted PFC nanoemulsion had no significant effect on cell viability, and the size and fluorescence signal of PFC nanoemulsion were very stable. These nanoprobes were successfully delivered into FR-positive tumor xenograft models and showed significantly enhanced signal intensities of 19F-MRI and fluorescence imaging in the tumor area. The folate-PFC/rhodamine nanoemulsion has a great potential to serve as a useful optical and 19F-MRI agent for the diagnosis and targeting of FR-positive tumor.

The sintered Ca(1-x-y)Dy(x)CeyMnO3 bodies were a single phase with a perovskite structure without any impurity phases. The calculated crystallite sizes of the Ca(1-x-y)Dy(x)CeyMnO3 were in the range of 43.3 to 63.3 nm. The composition significantly affected their microstructural and thermoelectric characteristics. The doped Dy led to both an increase in the electrical conductivity as well as the absolute value of the Seebeck coefficient, resulting in an enhanced power factor. The highest power factor (5.1 x 10(-4) Wm(-1) K(-2)) was obtained for Ca(0.8)Dy(0.2)MnO3 at 800 degrees C. In this study, we systematically discussed the thermoelectric properties of the Ca(1-x-y)Dy(x)CeyMnO3, with respect to the substitution of Dy and/or Ce for Ca.

The near-infrared (NIR) fluorescence probe has better tissue penetration and lower autofluorescence. Indocyanine green (ICG) is an NIR organic dye for extensive biological application, and it has been clinically approved for human medical imaging and diagnosis. However, application of this dye is limited by its numerous disadvantageous properties in aqueous solution, including its concentration-dependent aggregation, poor aqueous stability in vitro, and low quantum yield. Its use in molecular imaging probes is limited because it loses fluorescence after binding to nonspecific plasma proteins, leading to rapid elimination from the body with a half-life of 2 – 4 min. In this study, the multifunctional perfluorocarbon (PFC)/ICG nanoemulsions were investigated with the aim of overcoming these limitations. The PFC/ICG nanoemulsions as a new type of delivery vehicle for contrast agents have both NIR optical imaging and 19 F-MR imaging moieties. These nanoemulsions exhibited less aggregation, increased fluorescence intensity, long-term stability, and physicochemical stability against external light and temperature compared to free aqueous ICG. Also, the PFC/ICG bimodal nanoemulsions allow excellent detection of lymph nodes in vivo through NIR optical imaging and 19 F-MR imaging. This result showed the suitability of the proposed nanoemulsions for non-invasive lymph node mapping as they enable long-time detection of lymph nodes. Electronic supplementary material The online version of this article (doi:10.1186/s40580-014-0006-6) contains supplementary material, which is available to authorized users.

There has been great progress in the development of functional DNA-based sensors for the detection of metal ions. However, many functional DNAs are vulnerable to hydrolysis by nucleases in human blood. In addition, the detection methods that are based on DNA often exhibit interference due to the high blood concentrations of other ions, such as K(+) and Na(+). Therefore, we selected highly Pb(2+)-specific DNA-aptamer sequences based on CD spectroscopy of 4 G-rich DNA sequences and Hg(2+)-specific T-rich DNA sequences and immobilized them on gold nanoparticles for the simultaneous detection of Pb(2+) and Hg(2+) in human serum. We used gold nanoparticles because these have a superior fluorescence-quenching efficiency over a broad range of wavelengths compared with other organic quenchers. In addition, gold nanoparticles have a stabilizing effect on the immobilized DNA, which makes it more resistant to degradation by nucleases than free DNA. As a result, even in the presence of DNase, we were able to simultaneously detect Pb(2+) and Hg(2+) in serum at concentrations as low as 128 pM and 121 pM, respectively, within 10 min. These detection limits for Pb(2+) and Hg(2+) were 39-fold and 26.4-fold lower, respectively, than the detection limits that were obtained using free DNAs. Given the multi-color-fluorescence quenching capability of the gold nanoparticles and the possibility of developing functional nucleic acids for the detection of other metal ions, this study extends the application of oligonucleotides to a point-of-care detection system for the detection of multiple harmful metal ions in body fluids.