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Simple, rapid and selective methods for simultaneous determination of ultralow amount of multiple protein biomarkers in complicated biological samples are in great demand for early-stage diagnostics and biomedical research. Herein, we present a novel polydopamine nanoparticle (PDANP)-based multicolor fluorescent proximity immunoassay for ultrasensitive and multiplexed analysis of proteins in a single solu- tion using isothermal quadratic amplification (IQA). The assay system consists of DNA tethered-antibody pairs for different protein targets, different fluorophore-labeled DNA hairpin probes, DNA polymerase, T7 exonuclease and PDANPs. The DNA tethered- antibodies and fluorophore-labeled hairpin probes can coexist stably, and fluorophore- labeled DNA hairpin probes are adsorbed onto the PDANPs, leading to the significant quenching of the fluorescence of all fluorophores. When target proteins enter into the system, each pair of DNA tethered-antibodies simultaneously binds to a specific pro- tein target and forms the proximate complexes via proximity-dependent DNA hybridi- zation. The formed proximate complexes can then unfold the hairpin structures of the respective probes, leading to DNA polymerase/T7 exonuclease-catalyzed recycling of both proximate complexes and DNA strands to achieve IQA. This IQA results in the di- gestion of many fluorophore-labeled hairpin probes, causing a substantial increase in the fluorescence of corresponding fluorophores for detection of target proteins. As a proof of concept, we demonstrate that a three-color proximity immunoassay can si- multaneously detect prostate-specific antigen (PSA), carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) with ultralow attomolar detection limits, high specificity and 6-log dynamic range. Furthermore, the application of this assay for direct and simulta- neous determination of multiple proteins in clinical sera has also been demonstrated. Our newly proposed assay can be expected to provide a generic method platform for multiplexed analysis of a variety of low-abundance proteins.

We develop a novel peptide-based four-color fluorescent polydopamine nanoprobe for multiplexed sensing and imaging of tumor-related proteases in living cells. This nanoprobe responds rapidly and selectively, enabling simultaneous and high-contrast imaging of multiple proteases in living cells. Furthermore, it could detect the changes of protease expression in living cells.

The extraordinary fluorescence quenching capability of graphene oxide (GO) was coupled to the specific recognition capability of aptamers to design a four-color fluorescent nanoprobe for multiplexed detection and imaging of tumor-associated proteins in living cells. Specifically, alpha-fetoprotein (AFP), vascular endothelial growth factor-165 (VEGF165), carcinoembryonic antigen (CEA), and human epidermal growth factor receptor 2 (HER2) were detected. Due to strong π interaction, the fluorescence of labeled aptamers is quenched by GO. Four fluorophore-labeled aptamers that bind the tumor-associated proteins were adsorbed on GO to form the four-color nanoprobe with quenched fluorescence. The nanoprobes were internalized into cells via endocytosis, where the aptamer/GO nanoprobes bind the intracellular tumor-associated proteins. The aptamer-protein complexes thus formed detach from GO, and fluorescence recovers. Each analyte has its typical color (AFP: blue; VEGF165: green; CEA: yellow; HER2: red). As a result, simultaneous detection and imaging of multiple tumor-associated proteins in living cells were achieved. This nanoprobe has a fast response and is highly specific and biocompatible. The linear ranges for AFP, VEGF165, CEA, and HER2 are 0.8 nM–160 nM, 0.5 nM–100 nM, 1.0 nM–200 nM, and 1.2 nM–240 nM, respectively. Detection limits were 0.45 nM for AFP, 0.30 nM for VEGF165, 0.62 nM for CEA, and 0.96 nM for HER2. The probe allows for a fast distinction between tumor cells and normal cells via imaging.

Primary pyridylcarboxamides are prevalent parent structures in bioactive molecules and have the apparent advantages over N-protected derivatives as synthetic building blocks. However, no practical methods have been developed for direct synthesis of this compound class from unfunctionalized pyridines. We herein present a general, safe, concise, acid-free, and highly selective method for the C2-carba­moylation of pyridines with unprotected formamide and N-methyl formamide through the cleavage of two C–H bonds.

A novel gold nanoparticle-based four-color fluorescence proximity immunoassay is developed for one-step, multiplexed detection of protein biomarkers using ribonuclease H signal amplification. This assay could detect proteins with detection limits down to pg mL−1 and a 5-log dynamic range. Furthermore, it is suitable for rapid, direct quantification of multiple proteins in biological samples.

e14650 Background: Gene fusions, resulting from chromosomal breakage and rearrangement, play a critical role during oncogenesis. Recently, a growing number of gene fusions are being considered as important therapeutic targets in oncology, for instance, Anaplastic lymphoma receptor tyrosine kinase (ALK) fusion, which confer exquisite sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, available methods are inadequate for unbiased identification of potentially targetable fusions. Hence, we developed a novel fusion gene analyzing method named AcornFusion by DNA-seq data. Methods: The method is purely based on the mapping results of soft-clipped reads and the distribution of the positions where the candidate breakpoints locate, which is significantly different from the traditional methods with assembly of short reads and efficient way to do the detection. 309 patients with advanced non-small cell lung cancer (NSCLC) were enrolled. Formalin-fixed, paraffin embedded (FFPE) samples were used to DNA extracting and analyzed by AcornFusion method. Immunohistochemistry (IHC) and Reverse transcriptase polymerase chain reaction (RT-PCR) platform were also applied. Results: Of 309 pts, 17 patients were identified as fusion-positive cases by AcornFusion. 15 patients were EML4-ALK fusion positive and 1 patient was HIP-ALK fusion positive, which were all confirmed by IHC. 1 patient was ROS1 rearrangement positive, which was validated by Reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: With this advantage of high consistency with IHC and RT-PCR, AcornFusion can be a useful tool for investigating gene fusion events in clinical settings.

Primary pyridylcarboxamides are prevalent parent structures in bioactive molecules and have the apparent advantages over N-protected derivatives as synthetic building blocks. However, no practical methods have been developed for direct synthesis of this compound class from unfunctionalized pyridines. We herein present a general, safe, concise, acid-free, and highly selective method for the C2-carba­moylation of pyridines with unprotected formamide and N-methyl formamide through the cleavage of two C–H bonds.

The expedient and efficient copper powder catalyzed direct ring-opening arylation of benzazoles with aryl iodides in polyethylene glycol that proceeds in the absence of an added ligand has been developed. The protocol provides facile access to 2-(arylthio)anilines and 2-phenoxyanilines in high yields with a wide tolerance of functional groups. Transmission electron microscopy confirmed that the active catalyst results from the in situ generation of copper nanoparticles under standard reaction conditions, which is an alternative avenue to develop a highly effective metallic copper catalyst. Moreover, the catalytic system can be recycled up to six times.

This reaction yields aniline derivatives via ring opening of benzothiazoles or benzoxazoles, while thiazole and N-methyl-benzylimidazole do not react under these reaction conditions.

We report a facile methodology for one-step synthesis of the porous palladium nanospheres (Pd-NSS) using polyallylamine hydrochloride (PAH) as the complex-forming agent and stabilizing agent under mild reaction conditions (35 °C). The size, integrity, nature and composition of the Pd-NSS are investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption–adsorption experiments, energy-dispersive X-ray analysis (EDX), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), zeta potential, ultraviolet-visible (UV-vis) spectroscopy and Fourier transform infrared spectroscopy (FT-IR), etc. These spectral studies confirm that the PAH functionalized Pd-NSS are three-dimensionally interconnected porous nanostructures with primary nanoparticles as building blocks. Further experiential investigations show that the particle size of the Pd-NSS can be readily controlled by altering the ratio of PdCl2 to PAH during synthesis, and the Pd-NSS possess high catalytic activity and good stability for the Mizoroki–Heck reaction.