RESUMEN
Here, a separation-free and label-free portable aptasensor is developed for rapid and sensitive analysis of tumor-derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid-specific conformations, and a low-cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD-L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T-Hg2+-T and cytosine (C)-rich single-stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell-derived exosomes can be as low as 30 particles mL-1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology.
Asunto(s)
Exosomas , Neoplasias Pulmonares , Técnicas de Amplificación de Ácido Nucleico , Humanos , Exosomas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Biopsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Aptámeros de Nucleótidos , Biomarcadores de Tumor/genética , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentaciónRESUMEN
Simple and reliable profiling of tumor-derived exosomes (TDEs) holds significant promise for the early detection of cancer. Nonetheless, this remains challenging owing to the substantial heterogeneity and low concentration of TDEs. Herein, we devised an accurate and highly sensitive electrochemical sensing strategy for TDEs via simultaneously targeting exosomal mucin 1 (MUC1) and programmed cell death ligand 1 (PD-L1). This approach employs high-affinity aptamers as specific recognition elements, utilizes rolling circle amplification and DNA nanospheres as effective bridges and signal amplifiers, and leverages methylene blue (MB) and doxorubicin (DOX) as robust signal reporters. The crux of this separation- and label-free method is the specific response of MB and DOX to G-quadruplex structures and DNA nanospheres, respectively. Quantifying TDEs using this strategy enabled precise discrimination of lung cancer patients (n = 25) from healthy donors (n = 12), showing 100% specificity (12/12), 92% sensitivity (23/25), and an overall accuracy of 94.6% (35/37), with an area under the receiver operating characteristic curve (AUC) of 0.97. Furthermore, the assay results strongly correlated with findings from computerized tomography and pathological analyses. Our approach could facilitate the early diagnosis of lung cancer through TDEs-based liquid biopsy.
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Aptámeros de Nucleótidos , Antígeno B7-H1 , Técnicas Biosensibles , Doxorrubicina , Técnicas Electroquímicas , Exosomas , Neoplasias Pulmonares , Humanos , Técnicas Biosensibles/métodos , Exosomas/química , Técnicas Electroquímicas/métodos , Neoplasias Pulmonares/química , Aptámeros de Nucleótidos/química , Doxorrubicina/química , ADN/química , Azul de Metileno/química , Nanosferas/química , G-CuádruplexRESUMEN
This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patología , Neoplasias Pulmonares/diagnóstico , Mucina-1/genética , Técnicas Biosensibles/métodos , ADN/química , Aptámeros de Nucleótidos/química , Técnicas Electroquímicas/métodosRESUMEN
Escherichia coli is the major pathogen that causes bloodstream infections (BSI). It is critical to develop nonculture identification methods which can meet the urgent need of clinical diagnosis and treatment. In this study, we reported a homogeneous fluorescence E. coli analysis system using ß-galactosidase (ß-Gal) as the biomarker and double-stranded DNA-templated copper nanoparticles (dsDNA-Cu NPs) as the signal output. The product of the enzymatic hydrolysis reaction, p-aminophenol (PAP), could reduce Cu2+ to Cu+, triggering the alkyne-azido cycloaddition reaction (CuAAC). Subsequently, the hybrid chain reaction (HCR) was initiated, producing the dsDNA template used to generate Cu NPs in situ. The system achieved a wide linear range for ß-Gal and E. coli 1-104 mU/L and 10-2-10 colony-forming unit (CFU)/mL, and a detection limit of 0.3 mU/L and 0.003 CFU/mL, respectively. 65 samples (45 blood and 20 urine) were collected to evaluate the clinical practicality. The results demonstrated remarkable area under the curve (AUC) values of 0.95 and 0.916 from uncultured urine and blood, respectively. It had 100% specificity and 83.3% sensitivity. The whole duration of the strategy was 3.5 h, which significantly reduced the turnaround time (TAT) and facilitated early BSI diagnosis to improve patients' prognosis. Our work had the potential to be an alternative to culture-based methods in clinics.
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Técnicas Biosensibles , Sepsis , Humanos , Escherichia coli/genética , Química Clic , Cobre/química , ADN/químicaRESUMEN
Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.
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Compuestos de Cadmio , Carcinoma Hepatocelular , Neoplasias Hepáticas , Puntos Cuánticos , Biocatálisis , Biomarcadores de Tumor , Carcinoma Hepatocelular/patología , Glipicanos/metabolismo , Humanos , Neoplasias Hepáticas/patología , Telurio , alfa-FetoproteínasRESUMEN
BACKGROUND: Angiopoietin-like 4 (ANGPTL4) is a secreted glycoprotein that plays an important role in endothelial injury and the inflammatory response. Experimental models have implicated ANGPTL4 in acute respiratory distress syndrome (ARDS), but its impact on the progression of ARDS is unclear. METHODS: Paired bronchoalveolar lavage fluid (BALF) and serum samples were obtained from patients with ARDS (nâ=â56) within 24âh of diagnosis and from control subjects (nâ=â32). ANGPTL4, angiopoietin-2, interleukin (IL)-6, and TNF-α levels were measured by magnetic Luminex assay. BALF albumin (BA) and serum albumin (SA) were evaluated by enzyme-linked immunosorbent assay. RESULTS: BALF and serum ANGPTL4 concentrations were higher in patients with ARDS than in controls and were even higher in non-survivors than in survivors. The serum ANGPTL4 level was higher in indirect (extrapulmonary) ARDS than in direct (pulmonary) ARDS. Furthermore, BALF and serum ANGPTL4 levels correlated well with angiopoietin-2, IL-6, and TNF-α levels in BALF and serum. BALF ANGPTL4 was positively correlated with the BA/SA ratio (an indicator of pulmonary vascular permeability), and serum ANGPTL4 was associated with the severity of multiple organ dysfunction syndrome based on SOFA and APACHE II scores. Moreover, serum ANGPTL4 was better able to predict 28-day ARDS-related mortality (AUC 0.746, Pâ<â0.01) than the APACHE II score or PaO2/FiO2 ratio. Serum ANGPTL4 was identified as an independent risk factor for mortality in a univariate Cox regression model (Pâ<â0.001). CONCLUSION: ANGPTL4 levels were elevated in patients with ARDS and significantly correlated with disease severity and mortality. ANGPTL4 may be a novel prognostic biomarker in ARDS.