Dual-emissive europium doped UiO-66-based ratiometric light-up biosensor for highly sensitive detection of histidinemia biomarker.

BACKGROUND: Lanthanide metal-organic frameworks (Ln-MOFs) are a kind of emerging crystalline porous materials with high fluorescence and easy-to-tunable properties, making them ideal for sensing applications. However, current Ln-MOFs based fluorescent probes are primarily single-emissive or fluorescence-quenched, which greatly limited the detection performances such as sensitivity, accuracy and repeatability, thereby hindering their applications in efficient target monitoring and related disease diagnosis. To address these issues, the reasonable design of Ln-MOFs equipped with dual fluorescence emissions and light-up mode is urgently needed for a high-performance biosensor. RESULTS: A dual-emissive europium doped UiO-66 (Eu@UiO-66-NH2-PMA)-based ratiometric fluorescent biosensing platform was constructed for highly sensitive and selective detection of the histidinemia biomarker-histidine (His). Eu@UiO-66-NH2-PMA (pyromellitic acid abbreviated as PMA) was synthesized utilizing a post-synthetic modification method via coordination interactions between the free -COOH of UiO-66-NH2-PMA and Eu3+, which exhibited characteristic peaks of broad ligand emission and sharp Eu3+ emissions simultaneously. Considering that Cu2+ had the excellent fluorescence quenching ability toward Eu3+ and superior affinity with His, it was deliberately introduced into the Eu@UiO-66-NH2-PMA, acting as active sites for target His responsiveness. The Eu@UiO-66-NH2-PMA/Cu2+/His ternary competition system demonstrated a low detection limit of 74 nM, excellent selectivity and good anti-interference capability that allowed for sensitive analysis of His levels in milk and human serum samples. SIGNIFICANCE: Attributing to the superior luminescent properties, good stability and self-calibration capability of Eu@UiO-66-NH2-PMA, the developed ratiometric light-up sensing platform enabled sensitive, selective and credible analysis of His in complex practical samples, which might provide an available tool for food nutrition guideline and diagnostic applications of His related diseases.

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein.

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78% to 96%. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Polydopamine@ZIFs with enhanced electrochemiluminescence quenching performance for mycotoxin detection.

Quench-type electrochemiluminescence (ECL) immunosensors are appealing for detecting small molecule contaminants in signal-on mode, for which efficient ECL quenchers are highly desirable. Here, the classical quencher of polydopamine (PDA) was transformed into a unique structure by introducing zeolite imidazole frameworks (ZIFs). Besides the inherent energy transfer quenching effect on ECL, the resulting PDA@ZIFs exhibits a high scavenging property against electrogenerated coreactant-radicals and inhibits the formation of excited luminophore. A quench-type ECL immunosensor for ochratoxin A (OTA) was developed using the PDA@ZIFs as a quencher and the g-C3N4 as a luminophore. The immunosensor showed a good response towards the OTA with a linear range of 10.0 fg/mL-1.0 ng/mL and a detection limit of 4.8 fg/mL. Acceptable recoveries of 85.7 to 109.2 % were achieved for the detection of OTA in spiked foods. This work offers valuable insight for improving the performance of quench-typed ECL biosensors.

SERS detection of foodborne pathogens in beverage with Au nanostars.

The aim of this study is to develop a simple but rapid method for the determination of foodborne pathogens in complex matrices (beverages) by surface enhanced Raman spectroscopy (SERS) combined with Au nanostar solid-phase substrates. The star-shaped singlet Au nanostructure was formed on the surface of a stainless steel sheet by chemical replacement reaction. Rhodamine 6G verified the sensitivity and reproducibility of this substrate, and the relative standard deviations of the SERS intensity at 1312 cm-1, 1364 cm-1, and 1510 cm-1 displacements were 3.40%, 5.64%, and 3.48%, respectively. By detecting four pathogens in beverage samples on Au nanostar substrates, the utility of the SERS assay was demonstrated, while the combination of principal component analysis (PCA) and hierarchical cluster analysis (HCA) further enabled the isolation and identification of pathogens. The results of spiked beverages were validated in conventional culture identification and Vitek 2 Compact biochemical identification system experiments. Thus, this research demonstrated that Au nanostar substrates can be effectively utilized for the recognition of pathogenic bacteria and have immense promise to advance the progress of quick detection of foodborne pathogens and food safety.

Constructing a 3D interconnected network of Ag nanostructures for high-performance SERS detection of food coloring agents.

The design and preparation of various effective three-dimensional (3D) silver nanostructures is a frontier area of research in the field of surface-enhanced Raman scattering (SERS). This paper demonstrates a simple and novel method for the preparation of a substrate, whose surface was covered by a 3D interconnected network of Ag nanostructures, and the resulting network structure surface is free of organic contaminants. The EDS measurements confirm the metallic nature of the formed 3D Ag nanonetwork substrate. Additionally, the influence of experimental parameters on the morphology of the 3D Ag nanonetwork was also investigated, such as reaction time, hydrofluoric acid concentration, silver nitrate concentration and sodium citrate concentration. The 3D Ag nanonetwork has good uniformity. Importantly, the 3D Ag nanonetwork substrate was used to accurately and reliably detect amaranth (AR) and sunset yellow (SY) in beverages, with the lowest detection limit of 3 and 0.1 µg L-1, respectively. Therefore, this substrate is expected to be a promising candidate for SERS detection and offers attractive potential for a wider range of applications.

Production of high affinity monoclonal antibody and development of indirect competitive chemiluminescence enzyme immunoassay for gentamicin residue in animal tissues.

To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC50) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.

Boronate carbon nanoparticles featuring efficient FRET for activatable two-photon fluorescence imaging of sialic acid surface-abundant tumor cells.

Two-photon carbon-based nanoprobes hold great potential for biomedical applications as a result of their advantages of low fluorescence background, deep tissue imaging penetration and enhanced spatial resolution. However, the development of an activatable two-photon fluorescence carbon-based nanoprobe that simultaneously has the ability to target desired organs or cells is highly desired but remained a largely unsolved challenge. Herein, we developed boronate affinity BCNP@MnO2 nanocomposites, constructed by one step in situ growth of MnO2 nanosheets on the surface of aminophenylboronic acid-functionalized CNPs (BCNPs) via a redox reaction, which can feature efficient fluorescence energy transfer quenching to the BCNPs, allowing for tumor-specific affinity recognition and two-photon fluorescence activation imaging. By utilizing the inherent two-photon optical properties and sialic acid (SA) specific targeting ability of the BCNPs, good biocompatibility of the nanocomposites as well as highly sensitive and selective responses of MnO2 nanosheets towards GSH, the developed nanocomposites have demonstrated specific two-photon fluorescence activation imaging in target cancer cells and nude mouse tissues. Therefore, our proposed novel strategy could be used for monitoring GSH-triggered two-photon fluorescence activation events in SA-overexpressed cancer cells and has promising applications in both biological exploration and clinical diagnosis.

DNAzyme cascade circuits in highly integrated DNA nanomachines for sensitive microRNAs imaging in living cells.

DNA molecular probes have emerged as powerful tools for fluorescence imaging of microRNAs (miRNAs) in living cells and thus elucidating functions and dynamics of miRNAs. In particular, the highly integrated DNA probes that can be able to address the robustness, sensitivity and consistency issues in a single assay system were highly desired but remained largely unsolved challenge. Herein, we reported for the first time that the development of the novel DNA nanomachines that split-DNAzyme motif was highly integrated in a single DNA triangular prism (DTP) reactor and can undergo target-activated DNAzyme catalytic cascade circuits, allowing amplified sensing and imaging of tumor-related microRNA-21 (miR-21) in living cells. The DNA nanomachines have shown dynamic responses for target miR-21 with excellent sensitivity and selectivity and demonstrated the potential for living cell imaging of miR-21. With the advantages of facile modular design and assembly, high biostability, low cytotoxicity and excellent cellular internalization, the highly integrated DNA nanomachines enabled accurate and effective monitoring of miR-21 expression levels in living cells. Therefore, our developed strategy may afford a reliable and robust nanoplatform for tumor diagnosis and for related biological research.

Tumor-selective catalytic nanosystem for activatable theranostics.

A novel tumor-selective catalytic nanosystem that enables efficient chemodynamic therapy (CDT) and activatable fluorescence imaging in H2O2-rich tumor microenvironments has been developed.

Tumor-Targeted Graphitic Carbon Nitride Nanoassembly for Activatable Two-Photon Fluorescence Imaging.

Unique physicochemical characteristics of graphitic carbon nitride (g-CN) nanosheets suit them to be a useful tool for two-photon fluorescence bioimaging. Current g-CN nanosheets based imaging probes typically use the "always-on" design strategies, which may suffer from increased fluorescence background and limited contrast. To advance corresponding applications, g-CN nanosheets based activatable two-photon fluorescence probes remain to be explored. For the first time, we developed an activatable two-photon fluorescence probe, constructed from a nanoassembly of g-CN nanosheets and hyaluronic acid (HA)-gold nanoparticles (HA-AuNPs), for detection and imaging of hyaluronidase (HAase) in cancer cells. The deliberately introduced HA in our design not only functions as the buffering layer for stabilizing AuNPs and inducing corresponding self-assembly on g-CN nanosheets but also as a pilot for targeting HA receptors overexpressed on cancer cell surfaces. Our results show that the developed nanoassembly enables specific detection and activatable imaging of HAase in cancer cells and deep tissues, with superb signal-to-background ratio and high sensitivity. This nanoassembly can afford a promising platform for highly specific and sensitive imaging of HAase and for related cancer diagnosis.

Enhancement of the Intrinsic Peroxidase-Like Activity of Graphitic Carbon Nitride Nanosheets by ssDNAs and Its Application for Detection of Exosomes.

The present work investigates the capability of single-stranded DNA (ssDNA) in enhancing the intrinsic peroxidase-like activity of the g-C3N4 nanosheets (NSs). We found that ssDNA adsorbed on g-C3N4 NSs could improve the catalytic activity of the nanosheets. The maximum reaction rate of the H2O2-mediated TMB oxidation catalyzed by the ssDNA-NSs hybrid was at least 4 times faster than that obtained with unmodified NSs. The activity enhancement could be attributed to the strong interaction between TMB and ssDNA mediated by electrostatic attraction and aromatic stacking and by both the length and base composition of the ssDNA. The high catalytic activity of the ssDNA-NSs hybrid permitted sensitive colorimetric detection of exosomes if the aptamer against CD63, a surface marker of exosome, was employed in hybrid construction. The sensor recognized the differential expression of CD63 between the exosomes produced by a breast cancer cell line (MCF-7) and a control cell line (MCF-10A). Moreover, a similar trend was detected in the circulating exosomes isolated from the sera samples collected from breast cancer patients and healthy controls. Our work sheds lights on the possibility of using ssDNA to enhance the peroxidase-like activity of nanomaterials and demonstrates the high potential of the ssDNA-NSs hybrid in clinical diagnosis using liquid biopsy.

Graphitic Carbon Nitride Nanosheets-Based Ratiometric Fluorescent Probe for Highly Sensitive Detection of H2O2 and Glucose.

Graphitic carbon nitride (g-C3N4) nanosheets, an emerging graphene-like carbon-based nanomaterial with high fluorescence and large specific surface areas, hold great potential for biosensor applications. Current g-C3N4 nanosheets based fluorescent biosensors majorly rely on single fluorescent intensity reading through fluorescence quenching interactions between the nanosheets and metal ions. Here we report for the first time the development of a novel g-C3N4 nanosheets-based ratiometric fluorescence sensing strategy for highly sensitive detection of H2O2 and glucose. With o-phenylenediamine (OPD) oxidized by H2O2 in the presence of horseradish peroxidase (HRP), the oxidization product can assemble on the g-C3N4 nanosheets through hydrogen bonding and π-π stacking, which effectively quenches the fluorescence of g-C3N4 while delivering a new emission peak. The ratiometric signal variations enable robust and sensitive detection of H2O2. On the basis of the glucose converting into H2O2 through the catalysis of glucose oxidase, the g-C3N4-based ratiometric fluorescence sensing platform is also exploited for glucose assay. The developed strategy is demonstrated to give a detection limit of 50 nM for H2O2 and 0.4 µM for glucose, at the same time, it has been successfully used for glucose levels detection in human serum. This strategy may provide a cost-efficient, robust, and high-throughput platform for detecting various species involving H2O2-generation reactions for biomedical applications.

Melanin-Like Nanoquencher on Graphitic Carbon Nitride Nanosheets for Tyrosinase Activity and Inhibitor Assay.

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.