A novel microfluidic chip and antibody-aptamer based multianalysis method for simultaneous determination of several tumor markers with polymerization nicking reactions for homogenous signal amplification

摘要

To detect trace level of tumor markers (TMs) plays a crucial role in the early diagnosis of cancer. However, the fabrication of assays to simultaneously detect trace levels of TMs was still challengeable. The study was aimed to develop a novel microchip electrophoresis (MC) and antibody-aptamer based hybrid detection strategy for simultaneous determination of prostate specific antigen (PSA), carcino-embryonic antigen (CEA) and carbohydrate antigen 125 (CA125) in human serum. The assay comprises the generation of magnetic aptamer capture probes and anti-TMs labeled encoded signal tags, then employment of nicking enzyme for signal amplification. Firstly, the aptamers of TMs were co-immobilized on surface of Fe3O4@AuNPs (AuMPs) as capture probes. Simultaneously, the antibodies of TMs labeled with different double stranded DNA (dsDNA) as nicked fragment-induced strand was prepared as encoded signal tags. Secondly, the TMs, capture probes and encoded signal tags were simultaneously incubated to form sandwich complex. After magnetic separation, the complex was collected by adding the nicking enzyme. As a result, the dsDNA on the complex can initiate nicking enzyme cleavage reaction to produce many single stranded DNA (ssDNA) products with different length corresponding to different targets. Finally, the ssDNA products were injected into MC for separation and determination of TMs, respectively. Under optimized conditions, the assay can simultaneously detect three TMs with detection limits of 0.1, 0.15, 0.12 pg mL(-1) for PSA, CEA and CA125, respectively (S/N = 3). Moreover, the magnetic aptamer probes exhibited good stability and can be reused for 20 times with the recovery above 80% after heat treatments. The multianalysis method exhibits distinct advantages such as high-throughput, -sensitivity, easy-to-operate and offers a powerful tool for early diagnosis of cancers.