RESUMEN
An electrochemical aptasensing method is described for the determination of the biomarker CA125. It combines aptamer recognition and target-triggered strand displacement amplification. Flower like gold nanostructures were electrodeposited on a screen-printed carbon electrode to increase the sensor surface, to assemble more toehold-containing hairpin probe 1 (Hp1), and to improve the accessibility for DNA strands. Under the optimal conditions, this assay has a linear response in the 0.05 to 50 ngâ¢mL-1 CA125 concentration range, with a low detection limit of 5.0 pgâ¢mL-1. This method is specific and stable. It was successfully applied to the detection of CA125 in spiked biological samples, with recoveries between 82.5% and 104.1%. Graphical abstract.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Antígeno Ca-125/análisis , Técnicas Electroquímicas/métodos , Proteínas de la Membrana/análisis , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/genética , Antígeno Ca-125/sangre , Antígeno Ca-125/orina , Carbono/química , ADN/química , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Técnicas Electroquímicas/instrumentación , Electrodos , Oro/química , Humanos , Límite de Detección , Proteínas de la Membrana/sangre , Proteínas de la Membrana/orina , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , Saliva/químicaRESUMEN
An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5'-thiol group (5'-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL-1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis. Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , ADN/química , Molécula de Adhesión Celular Epitelial/análisis , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico/métodos , Electroquímica , Molécula de Adhesión Celular Epitelial/metabolismo , Estudios de Factibilidad , Oro/química , HumanosRESUMEN
A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) based on deoxyribonuclease I-aided target recycling signal amplification was proposed. In this paper, in the absence of MUC1, the MUC1 aptamer is absorbed on the surface of graphene oxide (GO) via π-stacking interactions. This results in quenching of the fluorescent label and no fluorescence signal is observed. Upon adding MUC1, the probe sequences could be specifically recognized by MUC1, leading to an increase in the fluorescence intensity. The detection limit is as low as 10 pg mL-1, and a linear range from 50 pg mL-1 to 100 ng mL-1. The assay is specific and sensitive, and successfully applied to the determination of MUC1 in spiked human serum, urine and saliva. Importantly, the proposed aptasensing strategy has great potential in detecting various protein and even cancer cells.