Fabrication and Application of Levan-PVA Hydrogel for Effective Influenza Virus Capture.

To prevent the transmission of pathogenic microorganisms such as the influenza virus, efficient pathogen-capturing materials are required. Here, we report a new pathogen-capturing and recovery material using levan polysaccharide. We fabricated hydrogels by blending levan and poly(vinyl alcohol) (PVA) and by using glutaraldehyde as a cross-linking agent. Fabricated levan-PVA hydrogels have a high water solubility and water adsorption ability. SEM observations showed that levan-PVA hydrogels have a 3D porous structure. We confirmed by RT-PCR analysis that the influenza virus capture efficiency of levan-PVA hydrogels is higher than that of commercial cotton swabs. Moreover, we confirmed that levan-PVA hydrogels on gauze as a filter material effectively captured bioaerosol samples. Therefore, levan-PVA hydrogels are expected to serve as simple and efficient pathogen capture and recovery materials.

High-performance nanogap electrode-based impedimetric sensor for direct DNA assays.

The rapid and sensitive detection of pathogen DNA (Deoxyribonucleic acid) would be essential for diagnosis and appropriate antibiotic treatment time. Herein, we report a novel direct DNA detectable impedimetric sensor. Direct assay of the amplified target DNA (mecA gene from methicillin-resistant Staphylococcus aureus (MRSA)) was performed using the PCR (polymerase chain reaction) product without any purification. Even though there are lots of PCR reagents and excess salts in sample PCR product, the nanogap electrode-based impedimetric sensor was able to detect DNA amplification fast in 5th PCR cycle which had 260 fM mecA gene in sample originally. The 70 nm gap electrode sensor yielded over 20% signal increase at the 5th PCR cycle and the impedance change grew up to about 60% at 25th in case of sample with 260 fM mecA gene template originally. The increased concentration of target DNA template led to the rise in impedance change such as 60% up at 5th and 120% up at 25th cycle with 260 pM, respectively. It is very outstanding result as compared with the traditional PCR agarose gel. Besides, it is 7-fold superior sensitivity to the microgap electrode. Furthermore, genomic DNA sample extracted from MRSA was detected rapidly. The nanogap electrode-based impedimetric sensor could be a good candidate for a rapid, sensitive, and low-cost electrical biosensor for DNA characterization in diagnostics and disease monitoring.

A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex.

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.

Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation.

Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for measuring alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to the hydrolysis of its phosphate groups by ALP. In addition, gold nanoparticles help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that the AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity.

A novel copper-chelating strategy for fluorescent proteins to image dynamic copper fluctuations on live cell surfaces.

Copper is indispensable in most aerobic organisms although it is toxic if unregulated as illustrated in many neurodegenerative diseases. To elucidate the mechanisms underlying copper release from cells, a membrane-targeting reporter which can compete with extracellular copper-binding molecules is highly desirable. However, engineering a reporter protein to provide both high sensitivity and selectivity for copper(ii) has been challenging, likely due to a lack of proper copper(ii)-chelating strategies within proteins. Here, we report a new genetically encoded fluorescent copper(ii) reporter by employing a copper-binding tripeptide derived from human serum albumin (HSA), which is one of the major copper-binding proteins in extracellular environments. Optimized insertion of the tripeptide into the green fluorescent protein leads to rapid fluorescence quenching (up to >85% change) upon copper-binding, while other metal ions have no effect. Furthermore, the high binding affinity of the reporter enables reliable copper detection even in the presence of competing biomolecules such as HSA and amyloid beta peptides. We also demonstrate that our reporter proteins can be used to visualize dynamic copper fluctuations on living HeLa cell surfaces.

Splitting and self-assembling of far-red fluorescent protein with an engineered beta strand peptide: application for alpha-synuclein imaging in mammalian cells.

We introduce the strategic development of self-assembling peptide/protein fragments based on the far-red fluorescent protein mPlum. The first beta strand (mPlum 1, 18 amino acids) of mPlum was engineered to spontaneously bind with the rest of the protein (mPlum 2-11, next 10 beta strands) and to form a native chromophore. The target beta strand mPlum 1 was separated from mPlum 2-11 and linked via a flexible peptide linker, resulting in fluorescently inactive circularly permuted mPlum protein (CpmPlum). In vitro evolution of this CpmPlum to a fluorescently active form and the subsequent splitting of the engineered mPlum 1 peptide afforded self-assembling mPlum fragments. Recombinantly expressed and synthetically prepared beta strand peptides were successfully assembled with the remaining mPlum protein in vitro and in cells. This developed pair of peptide/protein fragments was effectively used for peptide tag detection of alpha-synuclein in mammalian cells. Sequential expression of self-assembling mPlum fragments offered an entirely genetically encoded sensing system of naturally unfolded alpha-synuclein.