No abstract

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.

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.

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.