ABSTRACT
There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.
Subject(s)
Tuberculosis , Humans , Enzyme-Linked Immunosorbent Assay , Tuberculosis/diagnosis , Antibodies , Biological Transport , BiomarkersABSTRACT
Rapid and accurate quantification of low-abundance protein biomarkers in biofluids can transform the diagnosis of a range of pathologies, including infectious diseases. Here, we harness ultrabright plasmonic fluors as "digital nanolabels" and demonstrate the detection and quantification of subfemtomolar concentrations of human IL-6 and SARS-CoV-2 alpha and variant proteins in clinical nasopharyngeal swab and saliva samples from COVID-19 patients. The resulting digital plasmonic fluor-linked immunosorbent assay (digital p-FLISA) enables detection of SARS-CoV-2 nucleocapsid protein, both in solution and in live virions. Digital p-FLISA outperforms the "gold standard" enzyme-linked immunosorbent assay (ELISA), having a nearly 7000-fold lower limit-of-detection, and outperforms a commercial antigen test, having over 5000-fold improvement in analytical sensitivity. Detection and quantification of very low concentrations of target proteins holds potential for early detection of pathological conditions, treatment monitoring, and personalized medicine.
Subject(s)
COVID-19 , Humans , Enzyme-Linked Immunosorbent Assay , COVID-19/diagnosis , Fluoroimmunoassay , SARS-CoV-2 , Biomarkers , Sensitivity and SpecificityABSTRACT
BACKGROUND: TP73 antisense RNA 1 (TP73-AS1) is a long noncoding RNA which has been shown to be involved in the progression of multiple malignant tumors. Previous studies have demonstrated the oncogenic role of TP73-AS1 in breast cancer. However, its molecular mechanism remains largely unknown in breast tumorigenesis. METHODS: Expression of TP63-AS1, miRNA-125a-3p (miR-125a) and metadherin (MTDH) was detected by real-time quantitative PCR and western blotting. The malignancy was evaluated by cell counting kit 8 (CCK-8), transwell assays, flow cytometry and western blotting. The target binding was confirmed by dual luciferase reporter assay. Xenograft tumor model was performed to detect tumor growth in vivo. RESULTS: Expression of TP73-AS1 was higher in breast cancer tissues and cell lines. Biologically, its knockdown could promote cell apoptosis rate, and inhibit proliferative capacity, migration and invasion ability in HCC-70 and MB231 cells, accompanied with higher cleaved caspase 3 level and lower Ki67, N-cadherin and Vimentin level. Moreover, TP73-AS1 downregulation restrained the tumor growth of HCC-70 cells in vivo. Mechanically, TP73-AS1 functioned as a molecular "sponge" for miR-125a to modulate MTDH, a downstream target of miR-125a. Intriguingly, both miR-125a overexpression and MTDH silencing exerted a tumor-suppressive effect in the malignant progression of HCC-70 and MB231 cells, which was counteracted by TP73-AS1 upregulation and miR-125a downregulation, respectively. CONCLUSION: Knockdown of TP73-AS1 inhibited cell proliferation, migration and invasion, but facilitated apoptosis in breast cancer cells in vitro through targeting miR-125a and upregulating MTDH, suggesting a novel TP73-AS1/miR-125a/MTDH pathway in the malignant progression of breast cancer.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Membrane Proteins/metabolism , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA-Binding Proteins/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Movement , Cell Proliferation , Disease Progression , Female , Humans , In Vitro Techniques , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Prognosis , RNA-Binding Proteins/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Graphitic carbon nanocages (GCNCs) are unique graphene-based nanomaterials that can be used for cancer photothermal therapy (PTT). However, low toxicity GCNC-based organic/inorganic hybrid biomaterials for microwave irradiation assisted PTT have not yet been reported. In the present study, chitosan (CS)-coated GCNCs (CS-GCNCs) loaded with 5-fluorouracil (5Fu) were used for cancer therapy when activated by 808-nm laser and microwave co-irradiation. The cytotoxicity of GCNCs was significantly reduced after coating with CS. For example, fewer cell-cycle defects were caused by CS-GCNCs in comparison with non-coated GCNCs. The release rate of 5Fu from CS-GCNCs was significantly slower than that of 5Fu from GCNCs, providing sustained release. The release rate could be accelerated by 808-nm laser and microwave co-irradiation. The 5Fu in CS-GCNCs retained high cancer cell killing bioactivity by enhancing the caspase-3 expression level. The cancer cell killing and tumor inhibition efficiencies of the 5Fu-loaded nanomaterials increased significantly under 808-nm laser and microwave co-irradiation. The strong cell killing and tumor ablation activities were due to the synergy of the enhanced GCNC thermal effect caused by laser and microwave co-irradiation and the chemotherapy of 5Fu. Our research opens a door for the development of drug-loaded GCNC-based nano-biomaterials for chemo-photothermal synergistic therapy with the assistance of microwave irradiation. STATEMENT OF SIGNIFICANCE: Graphitic carbon nanocages (GCNCs) are graphene-based nanomaterials that can be used for both drug loading and cancer photothermal therapy (PTT). Herein, we showed that chitosan (CS)-GCNCs hybrid biomaterials had very low cytotoxicity, high ability for loading drug, and exhibited sustained drug release. In particular, although low-power microwaves alone are unable to trigger cancer cell damage by GCNCs, the cell killing and mouse tumor inhibition efficiencies were significantly improved by near-infrared (NIR) laser and microwave co-irradiation compared with laser-triggered PTT alone. This combined use of laser and microwave co-irradiation promises essential therapeutic modality and opens a new avenue for PTT.