Cascade Signal Amplification Strategy for Subattomolar Protein Detection by Rolling Circle Amplification and Quantum Dots Tagging

Abstract

A cascade signal amplification strategy was proposed for detection of protein target at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized quantum dots (QDs), multiplex binding of the biotin-strepavidin system, and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of QDs, which presented per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100 microL sample with a linear calibration range from 1 aM to 1 pM and was amenable to quantification of protein target in complex biological matrixes. The proposed cascade signal amplification strategy would become a powerful tool for proteomics research and clinical diagnostics.

Signal Amplification for Highly Sensitive Immunosensing

“…The developed immuno-PCR methods have been used for detection of biomarkers in clinic medicine. Moreover, these molecular biological technologies can conveniently be combined with the advantages of nanomaterials for development of two dimensional signal amplification strategies, leading to the occurrence of extremely high sensitivity of immunoassay, which can detect several molecules of protein marker with general electrochemical detection [58].…”

“…Since Ward et al firstly described an adaptation of the RCA for the detection of protein, termed ''immuno-RCA'' in 2000 [67], RCA has been explored as important strategies for signal enhancement in immunoassay due to the sensitivity, simplicity, and versatility of the RCA technique [68]. For example, Cheng et al [58] introduced the biotin-streptavidin system to bind primers to antibody and combined the RCA technique with oligonucleotide functionalized quantum dots (QDs), and anodic stripping voltammetric test, realizing detection of protein target at ultralow concentration (Fig. 16).…”

Aptameric Molecular Switch for Cascade Signal Amplification

“…Thus, the development of assay signal amplification paths has become important, and several amplification techniques have been developed, including catalytic nanoparticles (1,2 ), PCR (3,4 ), rolling-circle amplification (5,6 ), enzyme-triggered strand displacement amplification (7)(8)(9)(10), entropydriven signal amplification (11,12 ), and cascade signal amplification (13,14 ). The PCR is currently the most widely used thermal-cycling technique for DNA amplification.…”

A Highly Efficient Electroaptasensor for VEGF Detection by Employing Peroxidase-Like Activity Mesoporous Pt Nanospheres

“…with DNA amplifcation-assisted strategies such as immune-PCR [10], rolling circle amplifcation (RCA) [11,12], hybridization chain reaction (HCR) [13], Isothermal Amplification Reactions [14], and Chemiluminescence [15,16], surface plasmon resonance [17], field-effect transistor [18], and ELISA based sensors have been developed and recognized as a powerful tool to improve the sensitivity for protein detection [19]. Nevertheless, these methods still require the help of expensive reagents and instruments, complex experimental procedures, skilled labor, and many side reactions are easy to take place, which may limit their applications in resource-constrained countries.…”