A Novel Signal-Amplified Immunoassay for Myoglobin Using Magnetic Core-Shell Fe₃O₄@Au- Multi Walled Carbon Nanotubes Composites as Labels Based on One Piezoelectric Sensor

Abstract

An ultrasensitive and amplified piezoelectric immunosensor (PEI) with highly sensitivity has been proposed for the detection of ultra-trace of Myoglobin (Mb) in human serum based on precipitation of an insoluble product on the functionalized gold electrode. The core-shell Fe₃O₄@Au (with 85 nm as particle size)-multi-walled carbon nanotubes nanostructures labeling with Myoglobin first antibody (Mb Ab1) was firstly synthesized as capture probe(Fe₃O₄@Au-Mb Ab1-MWCNTs). It was modified on the surface of quartz crystal gold electrode. After a sandwich-type immunoreactions, the immunocomplex formed between Fe₃O₄@Au-Mb Ab1-MWCNTs/Mb and the HRP linked secondary antibody (HRP-Mb Ab2), can lead to the decrease of frequency (Δf) of the piezoelectric crystal electrode. Moreover, HRP enzyme labeled on the immunocomplex can catalyze 3- Amino-9-ethylcarbazole (AEC) in the presence of H₂O₂, resulting in an insoluble product on the electrode surface. The above two matters, immunocomplex and AEC's oxidation product, can add more weight on the detector and achieve a great amplification of Δf. Based on the change of Δf, ultrasensitive Mb detection was achieved. Under optimal conditions, Δf depended linearly on the Mb concentration with a range from 0.001 to 5 ng/mL, with a detection limit of 0.3 pg/mL. Comparing with the conventional ELISA, the immunoassay provides a more stable, specific protocol for Mb, which can greatly lower the sensitivity for detection of ultra-trace level of Mb in serum. It can be employed to quantify other biomarker if changed the detection antibody.

Keywords:

• Magnetic core-shell Fe₃O₄@-multi-walled carbon nanotubes composites
• Myoglobin
• Piezoelectric immunosensor
• 3- Amino-9-ethylcarbazole

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30901367), the Natural Science Foundation of Zhejiang province and Ningbo (LY12C20004, Y3110479, 2011R09025-06, 2011A610018, 2011A610006, 2011B82014, 2011C23126, 2011C5003, 2011C31037, Y13C200010), the K.C. Wong Magna Fund in Ningbo University.