Sensitive Detection of Proteins Using Assembled Cascade Fluorescent DNA Nanotags Based on Rolling Circle Amplification

Abstract

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.

Signal Amplification for Highly Sensitive Immunosensing

“…11 Schematic diagrams of a preparation of M-DNAzyme/ AuNP, b sensor preparation and CL immunosensing procedure, and c multiplex CL imaging immunoassay of four tumor markers using an immunosensor array Fig. 12 Schematic illustration of amplifying synthesis of MnTMPyPdsDNA enzyme mimic as label for CL biosensing Reprinted with permission from [69] to its peroxidase-mimicking activity. The oxidation product of HQ, a polymer with abundant electron accepting groups, could efficiently inhibit the photocurrent of CdS QDs through photoinduced electron transfer and steric hindrances effect, resulting in a highly sensitive PEC immunosensor.…”

“…A long DNA production containing hundreds of copies complementary to the circular DNA sequence can be detected via variety of signal-reporting labels. For example, using a highly fluorescent SYBR Green I to intercalate the RCA product, Xue et al [69] proposed a cascade fluorescence signal amplification strategy based on RCA-aided assembly of fluorescent DNA nanotags as fluorescent labels for detection of protein target at ultralow concentration (Fig. 17).…”

“…17). Huang et al [69] adopted a protein detection technique using surface plasmon resonance (SPR) and RCA. When RCA amplification products characterized by oligonucleotide Fig.…”

DNA for Non-nucleic Acid Sensing

“…Furthermore, the detection particle carries a nucleotide sequence of versatile structure and can be designed to contain an enzymatic primer. The possibility of combining the barcode approach with an enzymatic amplification, essentially RCA type, is increasingly used (Ou et al 2009;Xue et al 2012c;Yan et al 2012;Zhao et al 2013a;Cao et al 2011). It would probably be desirable to develop this approach using the LAMP method, but the main advantage of the barcode strategy is the simultaneous detection of multiple targets.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Isothermal RCA has also been very popular as a signal amplification method for cell and protein arrays [66,68,72,73]. The strategy for RCA detection of cells or proteins is very similar.…”