Ultrasensitive HIV viral load quantitation using designer DNA nanostructure capture probes and photonic resonator interference scattering microscopy

Abstract

Frequent, accurate, and highly sensitive HIV-1 viral load monitoring is a critical component of AIDS antiretroviral

therapy, a tool for reducing the incidence of mother-to-child HIV transmission, and a required element of routine

diagnostic testing to make people aware of their HIV status. Although enormous research and product

development effort has been applied to point-of-care viral load testing, the current paradigm of nucleic acid tests

and antigen assays continues to demonstrate fundamental limitations that derive from their inherent complexity

and lack of robustness, which in turn impact their costs and practicality for adoption in resource-limited settings.

We seek to address an important gap in the capabilities of existing technologies through a combination of three

innovations to yield an integrated, rapid, simple, ultrasensitive, highly selective, robust, and inexpensive system

for quantitative viral load measurement. First, we utilize microfluidic separation of virions from whole blood,

yielding a 10-50 µl plasma sample from 20-100 µl of whole blood in 95% virus extraction

efficiency. Second, we will achieve ultraselective recognition of intact HIV virions from the resulting serum using

designer DNA nanostructures that take the form of a macromolecular “net” whose vertices are a precise

mechanical match to the spacing and positioning of the spike gp120 protein matrix displayed on the HIV outer

surface. The DNA net vertices incorporate nucleic acid aptamer probes that have been selected for selectively

targeting the HIV gp120, resulting in multiple sites of high affinity attachment, and thus the “net” can be used as

an effective capture probe when covalently attached to a photonic crystal biosensor surface. Finally, we will

utilize a newly-invented form of biosensor microscopy called Photonic Resonator Interference Scattering

Microscopy (PRISM) in which the photonic crystal surface amplifies laser light scattering from captured intact

virions, enabling each one to be counted with high signal-to-noise ratio. Because PRISM does not require labels

or enzymatic amplification, our approach enables dynamic, real-time counting of captured virus with digital

precision and ultrasensitivity. In the proposed project, we will integrate viral separation and the photonic crystal

biosensor into a plastic cartridge and develop a rapid workflow that will be simple and rapid for compatibility with

point-of-care settings, with the goal of yielding a result in