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. 2012 Aug 28;6(8):6776-85.
doi: 10.1021/nn3015008. Epub 2012 Jul 24.

Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system

Michael A Nash  1 , John N Waitumbi, Allan S Hoffman, Paul Yager, Patrick S Stayton
Affiliations

Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system

Michael A Nash et al. ACS Nano. .

Abstract

There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.

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Figures

Figure 1
Figure 1
Targeted bioconjugate gold nanoparticle design. Gold nanoparticles were modified with a diblock copolymer produced via two-step RAFT polymerization. The polymer's semi-telechelic carboxyl group was conjugated to lysine groups on streptavidin enabling linkage to a biotinylated affinity protein. This universal bioconjugate design allowed facile multiplexed detection by simply mixing different biotinylated antibodies with the sample before addition of the universal streptavidin-gold detection reagent.
Figure 2
Figure 2
Depiction of the magnetic enrichment lateral flow immunoassay. A biotinylated antibody is added to a plasma sample containing the target biomarker(s). An equal volume of buffer containing streptavidin-pNIPAm-gold nanoparticles, pNIPAm magnetic nanoparticles, and free pNIPAm polymer is added. Upon heating, the mixed AuNP/mNP aggregates are separated by a magnet. After discarding the supernatant, the captured aggregates are redissolved into a smaller volume of cool buffer, resulting in particle disaggregation and 50-fold enrichment. The enriched mixture is then applied directly to an immunochromatographic assay membrane with functionalized test and control line antibody regions.
Figure 3
Figure 3
Recombinant PfHRP2 immunoassay. (a) Flow strip images, (b) green channel pixel intensity line scans offset along the y-axis for clarity, and (c) the corresponding sigmoidal curve generated by a typical magnetic enrichment immunoassay performed on recombinant PfHRP2 biomarkers in spiked human plasma. The starting sample of 500 μL was magnetically enriched 50-fold prior to analyte visualization by immunochromatography.
Figure 4
Figure 4
Comparison of magnetic enrichment and commercial assay. (a) Flow strip images from a 50-fold magnetic enrichment immunoassay (top row), and from the unmodified commercial assay performed with no enrichment (bottom row). (b) Green channel pixel intensity line scans for the magnetically enriched samples offset along the y-axis for clarity. (c) The integrated green channel pixel intensity at the test line plotted as mean ± standard deviation (n=3) for the two assay systems.
Figure 5
Figure 5
Effect of increasing sample volume on assay signal and noise. The PfHRP2 antigen was magnetically enriched from 100 or 500 μL volumes down to 10 μL, representing 10 or 50-fold volumetric enrichment, respectively. For the 100 μL processed volume, the magnetic enrichment immunoassay resulted in a false negative test result (a, top right), while for the 500 μL of processed sample (a, top left) a strong true positive signal at the test line was obtained. The assay noise remained low at each volume processed (a, bottom left and right). (b) Line scans from the positive sample, offset along the y-axis for clarity. (c) The integrated green channel pixel intensity at the test line (mean ± standard deviation, n=3) for the positive (“signal”) and negative (“noise”) samples.
Figure 6
Figure 6
Multiplexed magneto-enrichment immunoassay. After binding of the biotinylated anti-PfHRP2 and anti-aldolase IgG antibodies to the mixed antigens in human plasma, the universal streptavidin gold reagent was added, followed by magnetic enrichment and immunochromatographic readout. The top panel shows the respective concentrations of biomarkers in the original 500 μL sample.
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References

    1. Giljohann DA, Mirkin CA. Drivers of Biodiagnostic Development. Nature. 2009;462:461–464. - PMC - PubMed
    1. Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral Flow (Immuno) Assay: Its Strengths, Weaknesses, Opportunities and Threats. A Literature Survey. Anal Bioanal Chem. 2009;393:569–582. - PubMed
    1. Moody A. Rapid Diagnostic Tests for Malaria Parasites. Clin Microbiol Rev. 2002;15:66–78. - PMC - PubMed
    1. Beadle C, Long GW, Weiss WR, McElroy PD, Maret SM, Oloo AJ, Hoffman SL. Diagnosis of Malaria by Detection of Plasmodium-Falciparum Hrp-2 Antigen with a Rapid Dipstick Antigen-Capture Assay. Lancet. 1994;343:564–568. - PubMed
    1. Bell D, Peeling RW. Evaluation of Rapid Diagnostic Tests: Malaria. Nat Rev Microbiol. 2006:S34–S40. - PubMed

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