Development of surface-enhanced Raman scattering-sensing Method by combining novel Ag@Au core/shell nanoparticle-based SERS probe with hybridization chain reaction for high-sensitive detection of hepatitis C virus nucleic acid.

The ultrasensitive detection of hepatitis C virus (HCV) nucleic acid is crucial for the early diagnosis of hepatitis C. In this study, by combining Ag@Au core/shell nanoparticle (Ag@AuNP)-based surface-enhanced Raman scattering (SERS) tag with hybridization chain reaction (HCR), a novel SERS-sensing method was developed for the ultrasensitive detection of HCV nucleic acid. This SERS-sensing system comprised two different SERS tags, which were constructed by modifying Ag@AuNP with a Raman reporter molecule of 4-ethynylbezaldehyde, two different hairpin-structured HCR sequences (H1 or H2), and a detection plate prepared by immobilizing a capture DNA sequence onto the Ag@AuNP layer surface of the detection wells. When the target nucleic acid was present, the two SERS tags were captured on the surface of the Ag@AuNP-coated detection well to generate many "hot spots" through HCR, forming a strong SERS signal and realizing the ultrasensitive detection of the target HCV nucleic acid. The limit of detection of the SERS-sensing method for HCV nucleic acid was 0.47 fM, and the linear range was from 1 to 105 fM.

A novel pH-activated AIEgen probe for dynamic lysosome tracking and high-efficiency photodynamic therapy.

A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.

Endogenous Enzyme-Activatable Spherical Nucleic Acids for Spatiotemporally Controlled Signal Amplification Molecular Imaging and Combinational Tumor Therapy.

Due to the adjustable hybridization activity, antinuclease digestion stability, and superior endocytosis, spherical nucleic acids (SNAs) have been actively developed as probes for molecular imaging and the development of noninvasive diagnosis and image-guided surgery. However, since highly expressed biomarkers in tumors are not negligible in normal tissues, an inevitable background signal and the inability to precisely release probes at the chosen region remain a challenge for SNAs. Herein, we proposed a rationally designed, endogenous enzyme-activatable functional SNA (Ep-SNA) for spatiotemporally controlled signal amplification molecular imaging and combinational tumor therapy. The self-assembled amphiphilic polymer micelles (SM-ASO), which were obtained by a simple and rapid copper-free strain-promoted azide-alkyne cycloaddition click reaction between dibenzocyclooctyne-modified antisense oligonucleotide and azide-containing aliphatic polymer polylactic acid, were introduced as the core elements of Ep-SNA. This Ep-SNA was then constructed by connecting two apurinic/apyrimidinic (AP) site-containing trailing DNA hairpins, which could occur via a hybridization chain reaction in the presence of low-abundance survivin mRNA to SM-ASO through complementary base pairing. Notably, the AP site-containing trailing DNA hairpins also empowered the SNA with the feasibility of drug delivery. Once this constructed intelligent Ep-SNA nanoprobe was specifically cleaved by the highly expressed cytoplasmic human apurinic/apyrimidinic endonuclease 1 in tumor cells, three key elements (trailing DNA hairpins, antisense oligonucleotide, and doxorubicin) could be released to enable subsequent high-sensitivity survivin mRNA imaging and combinational cancer therapy (gene silencing and chemotherapy). This strategy shows great application prospects of SNAs as a precise platform for the integration of disease diagnosis and treatment and can contribute to basic biomedical research.

A New Gene SCY3 Homologous to Scygonadin Showing Antibacterial Activity and a Potential Role in the Sperm Acrosome Reaction of Scylla paramamosain.

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain.

A novel AIEgen photosensitizer with an elevated intersystem crossing rate for tumor precise imaging and therapy.

Herein, we have designed and synthesized a quinolinyl-AIE photosensitizer (TPE-4QL+) with an alternative elevated intersystem crossing (ISC) rate, which exhibits not only highly efficient photosensitivity but also high tumor cell specificity and an excellent mitochondrial targeting ability. In vitro experiments indicate that using TPE-4QL+ as a photosensitizer can induce a series of tumor cells to die with a low dose of radiation, but with no obvious toxicity to normal cells. The in vivo studies on a mouse model bearing a subcutaneous 4T1 xenograft also show that TPE-4QL+ can be used with high efficiency as a photosensitizer in PDT.

Tannic Acid-Assisted Biomineralization Strategy for Encapsulation and Intracellular Delivery of Protein Drugs.

Protein therapy has been considered to be one of the most direct and safe ways to regulate cell function and treat tumors. However, safe and effective intracellular delivery of protein drugs is still a key challenge. Herein, we developed a tannic acid-assisted biomineralization strategy for the encapsulation and intracellular delivery of protein drugs. RNase A and glucose oxidase (GOD) were choose as the protein drug model. RNase A, GOD, TA, and Mn2+ are mixed in one pot to attain RG@MT, and CaCO3 coating is subsequently carried out to construct RG@MT@C through biomineralization. Once RG@MT@C is endocytosed, the acidic environment of the lysosome will dissolve the protective layer of CaCO3 and produce plenty of CO2 to cause lysosome bursting, ensuring the lysosome escape of the RG@MT@C and thus releasing the generated TA-Mn2+, RNase A, and GOD into the cytoplasm. The released substances would activate starvation therapy, chemodynamic therapy, and protein therapy pathways to ensure a high performance of cancer therapy. Due to simple preparation, low toxicity, and controlled release in the tumor microenvironment, we expect it can realize efficient and nondestructive delivery of protein drugs and meet the needs for precise, high performance of synergistically antitumor therapy in biomedical applications.

Colorimetric nano-beacon and magnetic separation-based rapid and visual assay for gram-negative bacteria.

Food-borne diseases caused by pathogenic bacteria are one of the serious factors affecting human health. However, the most commonly used detection methods for pathogenic bacteria not only require expensive instruments, but also take a long time due to the complicated and cumbersome detection process. Therefore, the development of a fast, simple, and low-cost detection method for pathogenic bacteria is crucial for food safety and human health. In this work, based on the high binding ability of antimicrobial peptide (AMP) and polymyxin B (PMB) to bacteria, combined with magnetic separation technology, a new enzyme-free colorimetric strategy was constructed to achieve visual detection of Gram-negative bacteria in complex samples. The sensor system was divided into the following two parts: a colorimetric signal amplification nanoprobe, which was modified with AMP to enable effective binding of the colorimetric probe to the surface of bacteria, and a PMB-modified magnetic nanobead (MNB), which was used as the capture and enrichment unit of Gram-negative bacteria, as a result of which PMB could effectively distinguish Gram-negative bacteria from Gram-positive bacteria. Under optimized conditions, the detection limit of the method for Gram-negative bacteria (e.g. E. coli (G-)) was as low as 10 CFU/mL, and it was successfully applied to complex real samples. In addition, the developed colorimetric sensor offered advantages, such as fast response, less time consumption, high sensitivity, and low cost. It can be expected to become a new diagnostic tool for on-site detection of pathogenic bacteria in remote areas.

Combination of alkaline phosphatase/graphene oxide nanoconjugates and D-glucose-6-phosphate-functionalized gold nanoparticles for the rapid colorimetric assay of pathogenic bacteria.

Pathogenic bacteria are a major cause of foodborne diseases, which not only seriously threaten human safety but also cause significant losses for the national economy. Therefore, it is very important and urgent to develop a method for the detection of pathogenic bacteria with high accuracy, high sensitivity, and easy interpretation for use in food safety and medical hygiene. Herein, based upon the sensitive color changes induced by the dispersion and aggregation states of gold nanoparticles (AuNPs), a point-of-care (POCT) colorimetric assay was constructed for the rapid and sensitive visual detection of pathogenic bacteria. The POCT visual sensing system is composed of two individual elements: (1) an alkaline phosphatase/graphene oxide (GO@PEI-ALP) nanoconjugate that can release free ALP molecules in the presence of pathogenic bacteria; (2) D-glucose-6-phosphate (pGlu) and 3-aminobenzene boric acid (AMBA)-functionalized AuNPs (pGlu/AMBA-AuNPs) that are cross-linked upon the digestion of pGlu by free ALP molecules, resulting in a significant color change. Under optimized conditions, the detection limit of this sensing system for target bacteria was as low as 24 CFU mL-1 and was successfully applied to complex real samples. This proposed rapid colorimetric assay has high sensitivity, accuracy, and practicability with an intuitive signal and is expected to provide new inspiration for the detection of pathogenic bacteria.

Characterization of Purified Mulberry Leaf Glycoprotein and Its Immunoregulatory Effect on Cyclophosphamide-Treated Mice.

Mulberry leaf protein is a potentially functional food component and health care agent with antioxidant and anti-inflammatory properties. However, its composition, immunoregulatory effects, and gut microbial regulatory effects are unclear. Herein, ultra-filtrated and gel-fractionated mulberry leaf protein (GUMP) was characterized. Its effects on cyclophosphamide-induced immunosuppressed mice were further investigated. The results indicated that GUMP is a glycoprotein mainly containing glucose, arabinose, and mannose with 9.23% total sugar content. Its secondary structure is mainly ß-sheet. LC-MS/MS analysis showed that GUMP closely matched with a 16.7 kDa mannose-binding lectin and a 52.7 kDa Rubisco's large subunit. GUMP intervention significantly improved serous TNF-α, IL-6, and IL-2 contents; increased serum immunoglobulins (IgA and IgG) levels; and reversed splenic damage prominently. Moreover, GUMP administration increased fecal shot-chain fatty acid concentration and up-regulated the relative abundance of Odoribacter, which was positively correlated with SCFAs and cytokine contents. Overall, GUMP alleviated immunosuppression through the integrated modulation of the gut microbiota and immune response. Therefore, GUMP could be a promising dietary supplement to help maintain gut health.

Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.

Microemulsion-Confined Assembly of Magnetic Nanoclusters for pH/H2O2 Dual-Responsive T2-T1 Switchable MRI.

In this work, a T2-T1 switchable superparamagnetic iron oxide nanoprobe with a pH/H2O2 dual response was obtained using a microemulsion method. This novel method for the controllable assembly of small iron clusters followed by their independent modification was reported, which could not be prepared by common synthetic methods. The size of the assembled nanoprobe was uniform and controllable, with a stable T2 magnetic resonance imaging (MRI) signal under a single condition. When the nanoprobe was exposed to the tumor environment, the higher H+ and H2O2 concentrations at the tumor site could dissociate the nanoprobe and redisperse into small iron clusters. When this occurred, the T2 MRI signal was converted into a T1 MRI signal, achieving specific detection of tumors by a pH/H2O2 dual-response T2-T1 MRI.

Microemulsion-Confined Biomineralization of PEGylated Ultrasmall Fe3O4 Nanocrystals for T2-T1 Switchable MRI of Tumors.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are a novel T1 contrast agent with good biocompatibility and switchable imaging signal that have not been widely applied for magnetic resonance imaging (MRI) because it is difficult to induce their relatively close ideal agglomeration. Here, by combining the microemulsion method with the biomineralization principle, a pH-responsive T2-T1 switchable MRI nanoprobe was constructed via the microemulsion-confined biomineralization of PEGylated USPIONs (PEG-USPIONs). The size of the formed CaCO3-coated PEG-USPION conjugates (PEG-USPIONs@CaCO3 nanoprobe) was uniform and controllable, and the preparation method was simple. The PEG-USPIONs inside the nanoconjugates agglomerate more tightly, and the T1-MRI signal of the nanoprobe is converted to the T2-MRI signal. When exposed to the acidic environment of the tumor tissue or internal organelles, the CaCO3-coating of the nanoprobes is dissolved, and free PEG-USPIONs are released, thus realizing the T1-weighted imaging of the tumors. The suitability of the PEG-USPIONs@CaCO3 nanoprobe for tumor MRI detection was successfully demonstrated using a mouse model bearing a subcutaneous 4T1 xenograft.

Synergistically enhanced multienzyme catalytic nanoconjugates for efficient cancer therapy.

Tumors are complex and highly variable, making it difficult for a single treatment strategy to be significantly effective for cancer therapy. Herein, we report a robust cascade biomimetic nanoplatform that integrates chemiluminescence-induced photodynamic therapy (CL-PDT), Fenton reaction-based chemodynamic therapy (CDT), and glucose oxidase (GOD)-mediated starvation therapy to synergistically enhance cancer treatment. For the nanoplatform of CPPO@porphyrin-MOF@Cancer cell membrane-GOD (C1@M@C2G), the ferric ion-linked porphyrin-MOF can trigger a Fenton reaction to reach CDT, the carried CPPO as an energy donor is used to excite a photo-sensitive porphyrin-MOF in situ to generate singlet oxygen (1O2) for PDT, GOD catalyzes glucose into H2O2 and gluconic acid to realize starvation therapy, and the cancer cell membrane wrapped onto the nanoparticle plays a key role in homologous targeting, which is conducive to achieving better therapeutic effects. Significantly, the porphyrin-MOF with catalase-like activity can generate O2 to effectively relieve tumor hypoxia, thereby enhancing the catalytic effect of GOD and the efficacy of PDT. Additionally, the produced H2O2 and gluconic acid can further improve the CPPO-H2O2-triggered CL-PDT and promote the low pH-dependence Fenton reaction-based CDT, respectively. Both in vitro and in vivo studies showed that the constructed nanoplatform displays an excellent cooperative anti-tumor performance, so we firmly believe that this simple nanoplatform broadens the pathway to fight against cancer through effective cascade catalysis.

Photoactivatable Red Chemiluminescent AIEgen Probe for In Vitro/Vivo Imaging Assay of Hydrazine.

Here, we have developed a novel photoactivatable red chemiluminescent AIEgen probe (ACL), based on the combination of the red-emission AIEgen fluorophore (TPEDC) that shows excellent singlet oxygen (1O2)-generation ability and the precursor of Schaap's dioxetane (the linker connected to adamantane is the C═C bond) that can be modified to target various analytes, for in vitro and in vivo measurement of hydrazine. Prior to applying for sensing detection, the C═C bond connected to adamantane in ACL was first converted into dioxetane by irradiation to form the activated chemiluminescent AIEgen probe (ACLD). Then, the self-immolative reaction was triggered upon the deprotection of the acylated phenolic hydroxyl group in ACLD in the presence of hydrazine, resulting in the release of the high energy held in the 1,2-dioxetanes, and then, the chemiexcitation was triggered, thereby producing red chemiluminescence through the intramolecular chemiluminescence resonance energy transfer from Schaap's dioxetane to TPEDC. This chemiluminescent AIEgen probe was evaluated in a clean buffer environment as well as using living cells and mouse models.

PEGylated AIEgen molecular probe for hypoxia-mediated tumor imaging and photodynamic therapy.

We report a novel PEGylated aggregation-induced emission luminogen (AIEgen) molecular probe for hypoxia-mediated tumor imaging and photodynamic therapy by linking a PEG chain to the AIE-active photosensitizer via a hypoxia-sensitive azo group.

A CaO2 @Tannic Acid-FeIII Nanoconjugate for Enhanced Chemodynamic Tumor Therapy.

Chemodynamic therapy (CDT) is an effective tumor treatment strategy in which FeII reacts with hydrogen peroxide (H2 O2 ) in tumor cells to produce highly toxic hydroxyl radical (. OH) through the Fenton reaction. However, the content of endogenous H2 O2 in cells is limited, and the reaction between FeIII and H2 O2 is inefficient, greatly limiting the efficiency of the Fenton reaction and reducing the effectiveness of tumor treatment. Therefore, in this work, we designed and synthesized a new type of nano-system (CaO2 @TA-FeIII ) for the enhanced CDT of tumors, in which the polyphenolic compound- tannic acid (TA) and FeIII formed a TA-Fe nano-coating on the surface of calcium peroxide (CaO2 ) nanospherical aggregates. When the CaO2 @TA-FeIII nanoconjugates reach the tumor site, the CaO2 contained in the nanoconjugates produces H2 O2 after disintegration in tumor cells, and the carried TA rapidly reduces FeIII to FeII , solving the two major shortcomings in CDT of (1) insufficient content of H2 O2 in cancer cells, and (2) low catalytic efficiency of the Fenton reaction. Additionally, the . OH produced in the Fenton reaction induces oxidative stress for the tumor cells, promoting the occurrence of the "calcium overload" process, and thereby accelerating the death of tumor cells. Experimental results in vitro and in vivo showed that CaO2 @TA-FeIII nanoconjugates can effectively kill cancer cells and display an excellent tumor therapeutic effect. We believe that the CaO2 @TA-FeIII nanoconjugates are a promising new nano-platform for highly effective tumor treatment.

Biomineralization of Aggregation-Induced Emission-Active Photosensitizers for pH-Mediated Tumor Imaging and Photodynamic Therapy.

As an efficient, noninvasive, and high spatiotemporal resolved approach, photodynamic therapy (PDT) has high therapeutic potential for cancer treatment, whereas its development still faces a number of challenges, such as the lack of efficient and stable photosensitizers (PSs) and the inadequate ability of PSs to accumulate at tumor sites and target responses. Herein, a pH-responsive calcium carbonate (CaCO3)-mineralized AIEgen nanoprobe was prepared by using bovine serum albumin as the skeleton and loaded with a mitochondria-specific aggregation-induced emission (AIE)-active PS of 1-methyl-4-(4-(1,2,2-triphenylvinyl)styryl)quinolinium iodide (TPE-Qu+), which exhibits superior singlet oxygen (1O2)-generation ability and meanwhile possesses a bright near-infrared fluorescence emission. The biomineralized nanoparticles have small sizes (100 ± 10 nm) with good water dispersion and stability. With an increase in acidity (pH = 7.4-5.0), the internal TPE-Qu+ molecules are released gradually and accumulated in the mitochondria due to their hydrophobicity and electropositivity and then generate fluorescence emission and PDT under an external light source. Tumor inhibition and low acute toxicity were further successfully confirmed by the intracellular uptake test and 4T1-tumor-bearing mouse model.

Self-Illuminated, Oxygen-Supplemented Photodynamic Therapy via a Multienzyme-Mimicking Nanoconjugate.

Current photodynamic therapy (PDT) faces several intrinsic limitations, including insufficient oxygen supply and limited penetration of external light sources. Herein, we report a nanoconjugate, which, in response to the elevated hydrogen peroxide levels associated with tumor tissues, can supplement the oxygen needed for PDT and provide local self-illumination. Consisting of a MnFe2O4 core, a metal-organic framework shell loaded with the chemiluminescence reagent luminol, and a hyaluronic acid surface coating, the nanoconjugate is highly effective for suppressing cancer tissues in vivo via PDT in the absence of externally delivered light.

A Novel DNAzyme Signal Amplification-based Colorimetric Method for RNase H Assays.

A simple visual strategy was developed for the RNase H colorimetric measurement using DNAzyme-mediated signal amplification. When RNase H was presented, the RNA strand of the duplex formed by the G-rich DNA sequence (G-Rich) and its complementary RNA sequence (cp-RNA) was digested, releasing G-Rich to form HRP-mimicking DNAzymes of the G-quadruplex/hemin complexes in the presence of hemin. These DNAzymes catalyze the oxidation reaction of the substrate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to produce green-color products of ABTS•-, allowing for the detection of RNase H. A horseradish peroxidase (HRP)-mimicking DNAzyme of the G-quadruplex/hemin complex was used to mediate the signal amplification in the sensing strategy, resulting in high selectivity and sensitivity. This proposed colorimetric method shows a low detection limit of 0.04 U/mL, with a detection range of 0.1 to 3 U/mL. Moreover, this colorimetric method has been successfully used for RNase H assays in complicated biosamples, such as cell lysates. These results indicate that our colorimetric method not only detects RNase H in an ideal system, but also in real samples.

Catalytic Hairpin Self-Assembly-Based SERS Sensor Array for the Simultaneous Measurement of Multiple Cancer-Associated miRNAs.

The abnormal expression of some miRNAs is often closely related to the development of tumors. Available detection methods or biosensors that can simultaneously quantify multiple miRNAs in a single sample have rarely been reported. Herein, a novel catalytic hairpin self-assembly (CHA)-based surface-enhanced Raman scattering (SERS) sensor array was developed to simultaneously measure multiple miRNAs associated with cancer in one sample. The sensor array with four different sensing units was constructed by immobilizing one of four different hairpin-structured DNA sequence 1 (hp1) onto one of four Au/Ag alloy nanoparticle (AuAgNP)-coated detection wells. When target miRNA is present, the SERS tags, which were prepared by modifying AuAgNPs with a Raman reporter molecule of 4-mercaptobenzonitrile (MPBN) and the related hairpin-structured DNA sequence 2 (hp2), were captured onto the corresponding sensor unit through a repeated specific CHA reaction. This generated many "hot spots" because of interactions between the SERS tags and the AuAgNP layer-coated surface of the sensor, which ultimately produced a strong SERS signal that allowed the detection of target miRNAs with the detection limit of 0.15 pM. Using this SERS sensor array, multiple cancer-associated miRNAs (miR-1246, miR-221, miR-133a, and miR-21) were successfully determined in buffer, serum, and cellular RNA extracts.