Use of Nanotrap particles for the capture and enrichment of Zika, chikungunya and dengue viruses in urine.

Shih-Chao Lin,Anurag Patnaik,Amy K Smith,Benjamin Lepene,Victoria Callahan,Brian D Carey,Ji-Hyun Lee,Kylene Kehn-Hall,Nicole Bracci,Aarthi Narayanan,Pierre Roques

doi:10.1371/journal.pone.0227058

Abstract

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.

Highlights

Nanotrap1 (NT) particles are cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with various chemical affinity baits that aid in the collection and binding of target analytes that may range from peptides, proteins, nucleic acids and whole pathogens
Our study addresses the ability of NT particles to serve as a pre-diagnostic processing platform to enhance RT-qPCR detection of various arboviruses causing febrile illness
One possible explanation for these results is that the constitutes of the envelope structures between alphavirus (CHIKV) and flavivirus (ZIKV) are fundamentally different; distinct viral glycoproteins may have different affinities for the NT particles conferring differentiated enrichment

Summary

Introduction

Nanotrap (NT) particles are cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with various chemical affinity baits that aid in the collection and binding of target analytes that may range from peptides, proteins, nucleic acids and whole pathogens. Selectivity of NT particles is maintained through the use of chemically fixed affinity baits that consist of reactive dyes such as reactive red, cibacron blue or acrylic acid affinity bait residues [3]. These residues are covalently coupled to the NT core and provide broad spectrum retention of analytes. The microspheres containing sulphonic acid shells aid in decreasing non-specific absorption of albumin, enhancing the affinity of NT particles to their targeted analyte [4]

Methods

Results

Conclusion