Quantitative Detection of Single Molecules Using Enhancement of Dye/DNA Conjugate-Labeled Nanoparticles

Abstract

An ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules is developed on the basis of counting single magnetic nanobeads (MNBs) with combined amplification of DNA and dye/DNA conjugate. Highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidin-coated magnetic nanobeads (SA-MNBs) and magnetic separation. In this method, human IgG (Ag) is captured on the silanized glass substrate surface, followed by immunoreaction with biotinylated mouse antihuman antibody (BT-Ab). Then, SA-MNBs are attached to the BT-Ab through the biotin/streptavidin interaction at a ratio of 1:1. Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The resultant Ag-BT-Ab-SA-MNBs/BT-dsDNA/SYBR Green I is achieved after a fluorescent DNA probe, SYBR Green I, is added to the substrate and bound to the oligonucleotide at high ratios. Finally, epifluorescence microscopy coupled with a high-sensitivity electron multiplying charge-coupled device is employed for human IgG fluorescence imaging and detection. The number of fluorescent spots corresponding to single protein molecules on the images is counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/SYBR Green I immuotargeted on the glass slides is correlated with the concentration of human IgG target antigen in the range 3.0-50 fM.

How Nanoparticles Transform Single Molecule Measurements into Quantitative Sensors

“…However, there are a multitude of examples where the nanoparticle serves as a platform to which multiple labels are attached. An excellent example is the work of Xue et al,10a which developed a fluorescent assay where magnetic nanoparticles acted as a bridge to accommodate many fluorescent molecules onto each nanoparticle. The presence of human immunoglobulin G caused the attachment of streptavidin‐coated magnetic beads on a glass substrate modified with biotinylated mouse antihuman antibodies.…”

“…An additional benefit for using magnetic beads is that they allow for the removal of unbound magnetic beads from the surface through the application of a magnet, which helps to reduce background noise. Likewise, Zhang et al10b developed an ultrasensitive electrochemical assay that can count single molecules based on a strategy of DNA amplification in conjunction with magnetic nanoparticles labeled with alkaline phosphatase. The enzyme label converts a non‐electrochemically active substrate into its product, which can then be oxidized electrochemically, resulting in a detectable electric signal.…”

“…It has been estimated that the scattering signal produced by a single plasmonic gold nanoparticle is equal to that by 5 million fluorescein molecules or more than 10 5 QDs . Similarly, in single molecule assays, nanoparticles have been used as platforms that possess many fluorescent10a or electrochemical labels10b to give massive amplification of signals. Additionally, nanoparticles may be used to detect single entities through a blocked flux of ions, which leads to an amplified change in electric conductivity …”

“…Nanoparticles are becoming the dominant material construct for quantitative single molecule sensors because they can confine light for optical sensing and serve as platforms upon which reporters can be placed, in both cases addressing the challenge of getting significant signals above noise. Nanoparticles also provide possible solutions to the challenge of reducing response time by magnetic nanoparticles being dispersed through a sample to scavenge rare analytes before being rapidly drawn to the sensing surface back for measurement.…”

Digital immunoassay of a prostate-specific antigen using gold nanorods and magnetic nanoparticles

“…Fluorophore reporters include organic dyes, 9,11,12 quantum dots (Qds), 10,13,14 and uorescent nanoparticles. [15][16][17] However, uorescent organic dyes are limited by low absorption cross sections, 18 photobleaching 19 and photoquenching. 20 Qds demonstrate the drawbacks of photoblinking and biological toxicity.…”