Amperometric Immunosensor Based On Carbon Nanotubes Magnetic Nanocomposites

In this article, we synthesized some magnetic nanocomposites, then, the nanocomposites were used to immobilize antibody to prepare a series of biomagnetic nanoprobes for detection of tumor maker. The nanoprobes can realize the separation and enrichment of the target antigen selectively, and then the obtained immunomagnetic beads were immobilized on the surface of screen-printed electrodes (SPCE) to realize detection. After determination, the electrode surface can be reusable by removing the magnetic field, which constructed renewable, non-mediated amperometric immunosensor for the detection of tumor maker. The main works are as follows:The synthesis and characterization of iron oxide nanoparticles (Fe₃O₄ NPs), core-shell structured Fe₃O₄ @Au nanoparticles (GMPs). Oleic-coated Fe₃O₄ NPs were initially prepared by co-precipitation method and subsequently coated with gold nanoparticles (Au NPs) under oleylamine reduction of HAuCl4. The characterization results showed that the size and morphology of Fe₃O₄ NPs and GMPs are relatively homogeneous, and the preparation process is simple, save time with a mild reaction condition.A magnetic amperometric immunosensor for carcinoembryonic antigen (CEA) was fabricated. Firstly, multi-walled carbon nanotubes (MCNTs)-Thionine(Thi)-Nafion composite was prepared and dropped on the surface of SPCE to prepare the basic electrode (SPCE│MCNTs-Thi-Nafion). Secondly, horseradish peroxidase (HRP) labeled CEA antibody (HRP-anti-CEA) and bovine serum albumin (BSA) were assembled on GMPs to form the nanoprobes, which were introduced on the surface of the basic electrode by permanent magnet to prepare immunosensor (SPCE│MCNTs-Thi-Nafion/HRP-anti-CEA/GMPs/BSA). Through one-step immunoassay format, the immunosensor was incubated with CEA solution, the decreased current was proportional to the CEA concentration from 0.1 to 5.0 and 5.0 to 80 ng/mL with a detection limit of 0.03 ng/mL at signal/noise ratio of 3. This method reduced the cost and simplified the preparation process of the immunosensor. The immunosensor can simultaneously realize separation, enrichment and determination, with high sensitivity and good stability, which would be valuable for clinical immunoassay for CEA in human serum. Further, we constructed a kind of amperometric immunosensor, based on magnetic and electroactive nanoprobes, which can realize the incubation of multiple samples simultaneously and using SPCE as the detection electrode. First, Au NPs-Fe₃O₄ /hemin/MCNTs-COO^- nanocomposites were prepared using self-assemble method, using alpha fetoprotein (AFP) as a model analyte, AFP antibody (anti-AFP) was absorbed on the surface of Au NPs, BSA was then used to block sites against non-specific binding, and finally formed anti-AFP/AuNPs-Fe₃O₄/hemin/MCNTs-COO^- named AFHM nanoprobes. Then, AFHM nanoprobes incubated with differenet concentration of AFP simultaneously, the formed antigen-antibody complex was immobilized onto the SPCE would result in a decreased electrochemical signal of hemin for quantitative determination of AFP. The results showed that the nanoprobe-based electrochemical immunosensor was sensitive to AFP detection at a concentration of 0.1 to 200 ng/mL with a detection limit of 0.04 ng/mL. The electroactive nanoprobes can be massively prepared, easily immobilized on the SPCE for target detection and rapidly renewed with a magnet. Compared with our previous work, the proposed immunosensor had a higher antibody labeled density, shorter incubation time, without any modification process of the electrode, and minimizing background interference. The immunosensor can be prepared massively, nontoxic, reusable, which can be used on site and line detection.