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Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)3(2+) probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)3(2+)-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead-based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)3(2+)). The complex consisting of the analyte, the capture probe, the detector probe and the magnetic bead is brought into contact with the electrode by using a magnetic field. The Ru(bpy)3(2+) reacts with TPrA in solution to generate the ECL signal. The full protocol, including the synthesis and labeling of the bioactive Ru(bpy)3(2+), requires 5-6 d to complete. ECL immunoassays or nucleic acid tests only require 1.5-2 h, including the sample preparation time.

Immunotherapy assays using immunoadjuvants and tumor antigens could greatly increase the survival rates of patients with malignant tumors. As effective carriers, metal-organic frameworks (MOFs) have been widely utilized in cancer therapy due to their remarkable histocompatibility and low toxicity. Herein, we constructed a multimodal imaging-guided synergistic cancer photoimmunotherapy by employing a specific MOF (MIL101-NH 2 ) as the core carrier; the MOF was dual-dressed with photoacoustic and fluorescent signal donors (indocyanine green, ICG) and immune adjuvants (cytosine-phosphate-guanine sequence, CpG) and named ICG-CpG@MOF. This nanocarrier could passively target the tumor site through the EPR effect and achieve multimodal imaging (fluorescence, photoacoustic, photothermal and magnetic resonance imaging) of the tumor. Synergistic cancer photoimmunotherapy was achieved via simultaneous photodynamic and photothermal methods with 808 nm laser irradiation. ICG-CpG@MOF achieved the GSH-controlled release of immunoadjuvant into the tumor microenvironment. Furthermore, the released tumor-associated antigen along with CpG could induce the transformation of tumor cells from cold to hot by activating the immune system, which significantly enhanced tumor cytotoxicity and achieved high cure rates with minimal side-effects. This strategy utilizing multimodal imaging and synergistic cancer photoimmunotherapy provides a promising approach for the diagnosis and treatment of cancer.

MicroRNAs (miRNAs) participate in important processes of life course. Because of their characters of small sizes, vulnerable degradabilities, and sequences similarities, the existing detection technologies mostly contain enzymatic amplification reactions for acquisition of high sensitivities and specificities. However, specific reaction conditions and time-dependent enzyme activities are caused by the accession of enzymes. Herein, we designed a target-triggered enzyme-free amplification platform that is realized by circulatory interactions of two hairpin probes and the integrated electrochemiluminescence (ECL) signal giving-out component. Benefiting from outstanding performances of the enzyme-free amplification system and ECL, this strategy is provided with a simplified reaction process, high sensitivity, and operation under isothermal conditions. Through detection of the miRNA standard substance, the sensitivity of this platform reached 10 fmol, and a splendid specificity was achieved. We also analyzed three tumor cell lines (human lung adenocarcinoma, breast adenocarcinoma, and hepatocellular liver carcinoma cell lines) through this platform. The sensitivities of 10(3) cells, 10(4) cells, and 10(4) cells were, respectively, achieved. Furthermore, clinical tumor samples were tested, and 21 of 30 experimental samples gave out positive signals. Thus, this platform possesses potentials to be an innovation in miRNA detection methodology.

Exosomes serve as significant information carriers that regulate important physiological and pathological processes. Herein, functionalized DNA is engineered to be a hinge that anchors quantum dots (QDs) onto the surface of exosomes, realizing a moderate and biocompatible labeling strategy. The QDs‐labeled exosomes (exosome–DNA–QDs complex) can be swiftly engulfed by tumor cells, indicating that exosome–DNA–QDs can be applied as a specific agent for tumor labeling. Furthermore, the engineered artificial vesicles of M1 macrophages (M1mv) are constructed via a pneumatic liposome extruder. The results reveal that the individual M1mv can kill tumor cells and realize desirable biological treatment. To reinforce the antitumor efficacy of M1mv and the specificity of drug release, a target‐triggered drug delivery system is constructed to realize a specific microRNA‐responded delivery system for visual therapy of tumors. These strategies facilitate moderate labeling and functionalization of exosomes/vesicles and construct artificial drug‐delivery vesicles that simultaneously possess biological treatment and chemotherapy functions, and thus have the potential to serve as a new paradigm for tumor labeling and therapy.

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Infectious diseases, especially pathogenic bacterial infections, pose a growing threat to public health worldwide. As pathogenic bacteria usually exist in complex experimental matrixes at very low concentrations, developing a technology for rapid and biocompatible sample enrichment is essential for sensitive diagnosis. In this study, an Fe3O4/Vancomycin/PEG magnetic nanocarrier was constructed for efficient sample enrichment and in situ nucleic acid preparation of pathogenic bacteria for subsequent gene sensing. We attached Vancomycin, a well-known broad-spectrum antibiotic, to the surface of Fe3O4 nanoparticles as a universal molecular probe to target bacterial cells. Polyethylene glycol (PEG) was introduced to enhance the nanocarrier's water solubility and biocompatibility. Results show that the proposed nanocarrier achieved a 90% capture efficiency even if at a Listeria monocytogenes concentration of 1×10(2) cfu/mL. Contributing to the good water solubility achieved by the employment of modified PEG, highly efficient enrichment (enrichment factor 10 times higher than PEG-free nanocarrier) can be completed in 30 min. Moreover, PEG would also develop the nanoparticles' biocompatibility by passivating the positively charged unreacted amines on the magnetic nanoparticles, thus helping to release the negatively charged bacterial genome from the nanocarrier/bacteria complexes when an in situ nucleic acids extraction step was executed. The outstanding bacterial capture capability and biocompatibility of this nanocarrier enabled the implementation of a highly sensitive gene-sensing strategy of pathogens. By employing an electrochemiluminescence-based gene-sensing assay, L. monocytogenes can be rapidly detected with a limit of detection of 10 cfu/mL, which shows great potential for clinical applications.

Tuberculosis (TB) causes a global burden with its high rates of infection and death, especially the irrepressible threats of latent infection and drug resistance. Therefore, it is important to construct efficient theranostics for the prevention and control of TB. Herein, we created a targeted theranostic strategy for TB with a rifampicin-loaded aggregation-induced emission (AIE) carrier and performed testing in laboratory animals. The AIE carrier was constructed to localize in the granulomas and emit fluorescent signals at the early stage of infection, enabling the early diagnosis of TB. Subsequently, reactive oxygen species (ROS) were generated to eradicate infection, and the loaded rifampicin (RIF) was released for the synergistic treatment of persistent bacteria. Furthermore, targeted TB therapy was performed with the light-controlled release of ROS and accurate delivery of RIF, which realizes an antiinfection effect, providing an especially important treatment for drug-resistant TB. Thus, targeted theranostics for TB in laboratory animals possess the potential to become granulomas-tracking and anti-infection strategies for the diagnosis and treatment of TB.