The use of Nanotrap particles for biodefense and emerging infectious disease diagnostics

Abstract

Detection of early infectious disease may be challenging due to the low copy number of organisms present. To overcome this limitation and rapidly measure low concentrations of the pathogen, we developed a novel technology: Nanotrap particles, which are designed to capture, concentrate, and protect biomarkers from complex biofluids. Nanotrap particles are thermoresponsive hydrogels that are capable of antigen capture through the coupling of affinity baits to the particles. Here, we describe recent findings demonstrating that Nanotrap particles are able to capture live infectious virus, viral RNA, and viral proteins. Capture is possible even in complex mixtures such as serum and allows the concentration and protection of these analytes, providing increased performance of downstream assays. The Nanotrap particles are a versatile sample preparation technology that has far reaching implications for biomarker discovery and diagnostic assays.

Keywords: Rift Valley fever virus; Venezuelan equine encephalitis virus; diagnostics; influenza.

Figure 1

Functionalized Nanotrap particles with core and core–shell architecture. (a) Nanotrap particles are capable of sequestering low‐molecular‐weight biomolecules out of complex solutions, although some high‐abundance, high‐molecular‐weight species may nonspecifically bind to the hydrogel matrix. Note how the low‐molecular‐weight biomolecules are pulled away from carrier proteins (such as albumin) upon binding to the Nanotrap particles. (b) Addition of a cross‐linked shell to the particle architecture increases the sieving performance of the particles, effectively preventing high‐molecular‐weight molecules from binding. (c) Common affinity baits incorporated onto the particle matrix.

Figure 2

Virus particle capture. (a) Nanotrap particles can capture human coronavirus (HCoV), influenza A, and adenovirus. One hundred microliters of viral supernatants were incubated with 75 µL of each Nanotrap particle for 30 min at room temperature. Nanotrap particles were washed four times with water and viral RNA or DNA extracted and amplified with viral specific primers using either RT‐PCR or PCR. PCR products were separated on 2% agarose gels and visualized with ethidium bromide. NT = no Nanotrap particle control. (b) A proposed model of Nanotrap particle binding to viruses. Virus particles can be separated from biofluid samples via interaction between the affinity bait(s) incorporated into the particle matrix and specific ligands present on the virus particle.

Figure 3

Particles with modified core–shell architecture. The thermoresponsive nature of the Nanotrap particles enables them to expand or shrink in response to changes in pH and temperature, although the species allowed to enter the interior of the particles are limited by the pore sizes of the hydrogel particles. (a) Larger virus particles may not be able to enter the interior of the Nanotrap particles. (b) Inclusion of a non‐cross‐linked shell onto the particle matrix gives the Nanotrap environmentally responsive ‘arms’ that can capture larger virus particles.

Figure 4

RNA protection. The Nanotrap particles can sequester viral RNA from complex biofluids while protecting them from degradation by RNase.

Figure 5

Nanotrap particles are capable of capturing and enriching RVFV NP. (a) Nanotrap particle screening for RVFV NP capture. One hundred microliters of purified RVFV His‐NP (0.02 mg mL⁻¹) was incubated with 75 µL of seven different Nanotrap particles (NT45, 46, 53, 55, 69, 71, 76). Samples were incubated at room temperature for 30 mins, washed four times with water, and separated by SDS‐PAGE. Western blot analysis was performed with anti‐His antibody. NP input is 0.02 mg mL⁻¹ as a positive control and had no Nanotrap particles added. (b) NT45 is capable of capturing RVFV NP from serum. RVFV His‐NP (20 µg mL⁻¹) was spiked into 1 mL of 100% serum and incubated with 150 µL of NT45. Samples were incubated at room temperature for 30 mins, washed four times with water, and separated by SDS‐PAGE. Western blot analysis was performed with anti‐His antibody. NP (100 µg mL⁻¹) was included as a positive control.