Ultrasensitive aptamer-based protein detection via a dual amplified biocatalytic strategy

Abstract

We present an ultrasensitive aptasensor for the electronic monitoring of proteins through a dual amplified strategy in this paper. The target protein thrombin is sandwiched between an electrode surface confined aptamer and an aptamer-enzyme-carbon nanotube bioconjugate. The analytical signal amplification is achieved by coupling the signal amplification nature of multiple enzymes with the biocatalytic signal enhancement of redox-recycling. Our novel dramatic signal amplification strategy, with a detection limit of 8.3fM, shows about 4 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal single enzyme-based assay. This makes our approach an attractive alternative to other common PCR-based signal amplification in ultralow level of protein detection.

Fig. 1

Different amplification schemes for thrombin detection based on enzyme labels. (A): (a) Single enzyme labeled detection of thrombin; (b) Cyclic voltammogram for 1 μg mL^-1 thrombin in the presence of 1 mM p-APP substrate. (B): (a) TBA 2-ALP-SWCNT-based detection of thrombin; (b) Cyclic voltammogram for 10 ng mL^-1 thrombin in the presence of 1 mM p-APP substrate. (C): (a) TBA 2-ALP-SWCNT/DI bi-enzyme-based redox-recycling amplified detection of thrombin; (b) Cyclic voltammogram for 1 ng mL^-1 thrombin in the presence of 1 mM p-APP, 2 μM DI and 4 mM NADH. EC measurements were performed in Tris-HCl buffer (100 mM Tris-HCl, 0.2 g L^-1 MgCl₂, pH=9.0) at a scan rate of 10 mV s^-1 from -0.25 V to 0.15 V.

Fig. 2

(A): Cyclic voltammograms for (a) 0 ng mL^-1 of thrombin; (b) 100 ng mL^-1 of mouse-IgG; (c) 100 ng mL^-1 of lysozyme; (d) 100 pg mL^-1 of thrombin in the sample. (B): Zoom in current responses for (A) (a) 0 ng mL^-1 of thrombin; (b) 100 ng mL^-1 of mouse-IgG; (c) 100 ng mL^-1 of lysozyme in the sample. (C): Cyclic voltammograms for increasing concentrations of thrombin (a) 0; (b) 0.001; (c) 0.1; (d) 10 ng mL^-1. (D): The resulting calibration plot for thrombin over the 0.001 to 1000 ng mL^-1 range. The error bars represent the standard deviations of three parallel samples at each target concentration. Other conditions, as in Fig. 1Cb.

Scheme 1

Illustration of the dual amplified protocol for ultrasensitive thrombin detection. A) Electrochemical polymerization of p-ABA on a GCE. B) EDC-NHS coupling of TBA 1 probe molecule and ethanolamine blocking of active sites. C) Specific recognition of the thrombin target protein. D) Binding of TBA 2-ALP-SWCNT bioconjugate. E) EC measurement of current response in the presence of p-APP, NADH and DI.