8 research outputs found
Effect of the Protein Corona on Antibody–Antigen Binding in Nanoparticle Sandwich Immunoassays
We
investigated the effect of the protein corona on the function
of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich
immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1)
were conjugated to gold NPs, and another anti-NS1 Ab was immobilized
onto the nitrocellulose membrane. Sandwich immunoassay formation was
influenced by whether the strip was run in corona forming conditions,
i.e., in human serum. Strips run in buffer or pure solutions of bovine
serum albumin exhibited false positives, but those run in human serum
did not. Serum pretreatment of the nitrocellulose also eliminated
false positives. Corona formation around the NP-Ab in serum was faster
than the immunoassay time scale. Langmuir binding analysis determined
how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by
corona formation conditions, quantified as an effective dissociation
constant, <i>K</i><sub>D</sub><sup>eff</sup>. Results show
that corona formation mediates the specificity and sensitivity of
the antibody–antigen interaction of Zika biomarkers in immunoassays,
and plays a critical but beneficial role
Selective melting of NRs and NBs to switch blood clotting off and on.
<p>NR-HS-TBA and NB-HS-antidote were mixed at a ratio so that the TBA:antidote was 1:1. a) NR-HS-TBA + NB-HS-anti mixture before (black) and after 800 nm irradiation (red), after 800 nm and then 1100 nm irradiation (blue) b) fluorescence of released supernatant before (black) and after 800 nm irradiation (red), and after 800 nm+1100 nm irradiation (blue). c) Normalized <i>t<sub>plasma</sub></i> for samples before irradiation (defined as 1.0, and used to compare the statistical parameters of all samples) of mixture of NR-HS-TBA + NB-HS-antidote with excitation at 800 nm (<i>t<sub>plasma</sub></i> increases to 1.73), and 800 nm+1100 nm (<i>t<sub>plasma</sub></i> restored to 0.88), demonstrating restoration of clotting time. 1100 nm irradiation alone of the mixture NR-HS-TBA + NB-HS-anti, showing no significant increase in clotting time. Irradiation at 800 nm of NR-HS (without TBA) + NB-HS-anti showing no increase in clotting time. To test the effect of the presence of the nanoparticles, HS-NR-TBA+NB-HS-anti were not exposed to any irradiation in blood. Significant differences (p≤0.05) from baseline <i>t<sub>plasma</sub></i> are indicated with an * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>).</p
TBA and antidote affect coagulation in whole human blood.
<p>a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (<i>t<sub>plasma</sub></i>) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti] = 0 to 1000 nM (anti/TBA = 0 to 2.0).</p
Release from NR- and NB-coronas and their comparison to covalently loaded NRs.
<p>a) Absorption spectrum of NB-HS-TBA before (black) and after (red) 1100 nm irradiation, where [NB-HS-anti] = 0.3 nM, and released [anti] = 129±5 nM (430±17 anti released/NB). Inset: fluorescence spectrum of released TBA before (black) and after (red) 1100 nm irradiation. b) Absorption spectrum of NR-HS-TBA before (black) and after (red) 800nm irradiation where [NR-HS-TBA] = 2.9 nM, and released [TBA] = 663±23 nM (223±8 DNA released/NR). Inset: fluorescence spectrum of released TBA before (black) and after (red) 800 nm irradiation, c) Effect of the released TBA in blood. Comparing normalized <i>t<sub>plasma</sub></i> from released TBA from the coronas [NR-HS-TBA] = 2.9 nM (red), where released [TBA] = 663±23 nM in a clotting test. Supernatant of NR-HS-TBA with exposed to no irradiation and added to blood is defined as <i>t<sub>plasma</sub></i> = 1.0 (gray dotted line). A significant difference (p≤0.05) from baseline <i>t<sub>plasma</sub></i> is indicated with a * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>), d) <i>t<sub>plasma</sub></i> (normalized) calibration curve of free TBA in a thrombin test (stars). Released TBA from NR-HS-TBA (red circle) and extrapolated equivalent concentration (red dashed line). e) <i>t<sub>plasma</sub></i> (normalized) calibration curve of free thiolated TBA (blue X’s). Released thiolated TBA from NR-thiol-TBA (1460±108 nM, blue square) and extrapolated equivalent concentration (blue dashed line).</p
Gold nanoparticles synthesized and loaded for triggered release.
<p>Absorption spectra of a) NRs, NR-HSA-TBA coronas (LSPR max = 777 nm), b) NB, NB-HSA-antidote (LSPR max = 1093 nm). c) TEM image of NRs, scale bar = 20 nm, d) TEM image of NBs, scale bar = 100 nm, e) <i>D<sub>H</sub></i> (DLS) of NRs, NR-HS-TBA, NBs, NB-HS-antidote, indicating that a corona contains multiple not a single NR or NB, but multiple ones. f) Zeta potential of NRs, NR-HS-TBA = −9.8 mV, NBs, NB-HS-antidote = −10.1 mV g) Quantified DNA payloads of NR-HS-TBA (674±74 TBA/NR), NB-HS-antidote (1307±255 antidote/NB). h) mixture of NR-CTAB + NB-CTAB before (black) and after (red) 800 nm irradiation. i) NR-CTAB + NB-CTAB before (black) and after (red) 1100 nm irradiation.</p
Optimizing the Properties of the Protein Corona Surrounding Nanoparticles for Tuning Payload Release
We manipulate the passive release rates of DNA payloads on protein coronas formed around nanoparticles (NPs) by varying the corona composition. The coronas are prepared using a mixture of hard and soft corona proteins. We form coronas around gold nanorods (NRs), nanobones (NBs), and carbon nanotubes (CNTs) from human serum (HS) and find that tuning the amount of human serum albumin (HSA) in the NR-coronas (NR-HS-DNA) changes the payload release profile. The effect of buffer strength, HS concentration, and concentration of the cetyltrimethylammonium bromide (CTAB) passivating the NP surfaces on passive release is explored. We find that corona properties play an important role in passive release, and concentrations of CTAB, HS, and phosphate buffer used in corona formation can tune payload release profiles. These advances in understanding protein corona properties bring us closer toward developing a set of basic design rules that enable their manipulation and optimization for particular biological applications
Surface-Enhanced Raman Spectroscopy-Based Sandwich Immunoassays for Multiplexed Detection of Zika and Dengue Viral Biomarkers
Zika and dengue are
mosquito-borne diseases that present similar nonspecific symptoms
but possess dramatically different outcomes. The first line of defense
in epidemic outbreaks are rapid point-of-care diagnostics. Because
many outbreaks occur in areas that are resource poor, assays that
are easy to use, inexpensive, and require no power have become invaluable
in patient treatment, quarantining, and surveillance. Paper-based
sandwich immunoassays such as lateral flow assays (LFAs) are attractive
as point-of-care solutions as they have the potential for wider deployability
than lab-based assays such as PCR. However, their low sensitivity
imposes limitations on their ability to detect low biomarker levels
and early diagnosis. Here, we exploit the high sensitivity of surface-enhanced
Raman spectroscopy (SERS) in a multiplexed assay that can distinguish
between Zika and dengue nonstructural protein 1 (NS1) biomarkers.
SERS-encoded gold nanostars were conjugated to specific antibodies
for both diseases and used in a dipstick immunoassay, which exhibited
15-fold and 7-fold lower detection limits for Zika NS1 and dengue
NS1, respectively. This platform combines the simplicity of a LFA
with the high sensitivity of SERS and could not only improve Zika
diagnosis but also detect diseases sooner after infection when biomarker
levels are low
A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays
<p>Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.</p