Neutral DNA-avidin nanoparticles as ultrasensitive reporters in immuno-PCR

Abstract

We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific "stickiness" and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.

Figure 1.

Schematic of the immuno-nanoparticle PCR assay. A) Assembly of DNA-avidin core-shell nanoparticles. DNA plasmids carrying the synthetic PCR template are sequentially assembled with avidin and biotin-polyethylene glycol (PEG). B) Workflow of immuno-nanoparticle PCR. Target protein molecules are captured by a capture antibody and detected with nanoparticles via a DTT-cleavable-biotin-linked detection antibody. The captured nanoparticles are disassembled by heat to expose the PCR template for PCR amplification (not to scale).

Figure 2.

Nucleotide sequence of the annealed dsDNA template and restriction enzyme recognition sequences. The cohesive end annealing to the compatible SacI site on the digested plasmid pBC is shown in red. Once ligated to the plasmid, the recognition sequence for SacI is abolished by a thymine-to-cytosine nucleotide change (shown in red bold). The cohesive end which anneals to the compatible, conserved XbaI site on the digested plasmid pBC is shown in blue. Shown in green is an extra SacI recognition sequence included in the dsDNA template to be used for the introduction of additional template repeats. The 79-bp PCR reporter sequence is shown in black and underlined.

Figure 3.

qPCR standard curves of plasmid DNA constructs containing 1 to 7 repeats of target template. The plasmid with no template showed C_t over 35.

Figure 4.

qPCR standard curves of DNA-avidin nanoparticles (n=1). Particle 1 (solid black) and Particle 4 (solid red)

Figure 5.

Nanoparticle tracking analysis of DNA-avidin nanoparticles. Three distinct fields of view were observed for calculating size and concentration of both Particle 1 and Particle 4. Curves shown in shades of red correspond to Particle 1 stock solution diluted 100-fold with water. The average size and undiluted concentration of Particle 1 were found to be 109 ± 3.8 nm and 6.7×10¹⁰ particles/ml respectively. Curves shown in shades of green correspond to Particle 4 stock solution diluted 100-fold with water. The average size and undiluted concentration of Particle 4 were found to be 95 ± 3.7 nm and 7.2×10¹⁰ particles/ml respectively.

Figure 6.

Quantification of hCG spiked in PBS +1% BSA using DNA-avidin nanoparticle (with four repeats of template)-based iPCR (n=3, error bars ±1 SD; non-template control gave no C_t). The dashed red line is the detection threshold of the assay, which is defined as the average -delta C_t value of the no-hCG control plus 3 times the standard deviation of the no-hCG control. A standard approach was used to estimate the Limit of Detection as the lowest analyte concentration that gave a signal clearly distinguishable from the detection threshold. The Limit of Detection was estimated at 25 pg/ml hCG (660 fM).