Amplified QCM-D biosensor for protein based on aptamer-functionalized gold nanoparticles

Abstract

A highly sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) biosensor for protein was developed using aptamer-functionalized gold nanoparticles (Apt-GNPs) for amplification. Human α-thrombin, an important physiological protease found in blood, was chosen as the target protein. Captured by immobilized aptamers, thrombin was determined on-line using Apt-GNPs to enhance both frequency and dissipation signals. The fabricated sandwich of aptamer/thrombin/Apt-GNPs on chip surface was confirmed by atomic force microscopy (AFM). Compared to direct assay, the detection limit for thrombin was down to 0.1 nM, yielding about 2 orders of magnitude improvement in sensitivity. This aptamer-based QCM-D biosensor also showed good selectivity and repeatability in complex matrix. For the first time, the dual-signal enhancement of Apt-GNPs on QCM-D sensing was demonstrated, and such design could provide a promising detection strategy for proteins with two binding sites.

Introduction

Aptamers are ligand-binding oligonucleic acids selected in vitro through systematic evolution of ligands by exponential enrichment (SELEX) (Ellington and Szostak, 1990, Tuerk and Gold, 1990). They have been applied in biochemical analysis as alternative recognition elements due to their relative low price, high binding affinity and good stability (Tombelli et al., 2005b). Up to now, many aptamer-based biosensors for proteins have been reported by using electrochemical, fluorescent, colorimetric methods, etc. (Cho et al., 2009, Liu et al., 2009). Most of these methods suffer from time-consuming steps for labeling and off-line analysis. Hence, a fabrication strategy for biosensor that avoids labor-intensive labeling steps and allows real-time, on-line measurements is of great interest for simple and rapid protein analysis.

Alternatively, quartz crystal microbalance (QCM) is a useful analytical tool for label-free and real-time analysis of biological molecules (Sauerbrey, 1959). Aptamer-based QCM biosensors have been constructed for proteins (Hianik et al., 2005, Liss et al., 2002, Min et al., 2008, Tombelli et al., 2005a, Yao et al., 2009). However, method with higher sensitivity is still desirable especially for protein biomarkers. And signal amplification would be one of the major approaches for designing and developing highly sensitive QCM biosensors. Gold nanoparticles (GNPs) are frequently used for signal amplification in protein analysis because GNPs have unique electrical and optical properties and also are easy to functionalize (Zhang et al., 2007a). Recent work on signal amplified colorimetric (Pavlov et al., 2004), electrochemical (Deng et al., 2008, Ding et al., 2010) and surface plasmon resonance (SPR) (Wang et al., 2009) assays based on aptamer-functionalized gold nanoparticles (Apt-GNPs) demonstrated that Apt-GNPs could be used for effective signal enhancement.

QCM with dissipation monitoring (QCM-D) technique could provide information on the viscoelastic properties of the mass bound by simultaneous measurements of the frequency and dissipation factor (Rodahl et al., 1997). QCM-D system can be used to characterize the formation of thin films from proteins, polymers and cells on surfaces (Höök and Kasemo, 2007). There have been relatively few efforts made to adapt QCM-D to biosensor development (Grieshaber et al., 2008, Poitras and Tufenkji, 2009). To the best of our knowledge, the amplification of Apt-GNPs with QCM-D detection has not been investigated.

In this work, we reported a highly sensitive QCM-D biosensor for protein detection (human α-thrombin as the target) using Apt-GNPs to enhance both frequency and dissipation signals. According to the fact that two aptamers (15-mer and 29-mer thrombin binding aptamer, TBA15 and TBA29 in abbreviation respectively) recognize different sites on thrombin (Bock et al., 1992, Tasset et al., 1997), a sensing system in sandwich manner was constructed. TBA15 was firstly immobilized on a QCM-D chip to capture thrombin, then TBA29-functionalized gold nanoparticles (TBA29-GNPs) were introduced to obtain a TBA15/thrombin/TBA29-GNPs sandwich structure. The entire process was monitored by QCM-D and the expected sandwich structure was characterized by atomic force microscopy (AFM). The capability of Apt-GNPs in amplifying both frequency and dissipation signals of QCM-D was presented. Meanwhile, the sensitivity, selectivity and repeatability performances of such designed QCM-D biosensor were evaluated in detail. The thrombin standard spiked fetal calf serum sample was determined to explore the potential of this method in practical application.