Configurable Digital Virus Counter on Robust Universal DNA Chips

Abstract

Here, we demonstrate real-time multiplexed virus detection by applying DNA-directed antibody immobilization technique to a single-particle interferometric reflectance imaging sensor (SP-IRIS). In this technique, the biosensor chip surface spotted with different DNA sequences is converted to a multiplexed antibody array by flowing antibody-DNA conjugates and allowing specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant Vesicular Stomatitis Viruses (rVSVs) genetically engineered to express surface glycoproteins of Ebola, Marburg, and Lassa viruses in real-time in a disposable microfluidic cartridge. We also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-DNA conjugates with the virus sample in solution phase prior to flowing in the microfluidic cartridge, eliminating the antibody immobilization step. This homogenous approach achieved detection of the model Ebola virus, rVSV-EBOV, at a concentration of 100 PFU/ml in 1 hour. Finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. A concentration of 104 PFU/ml was detectable under 10 minutes for the rVSV-Ebola virus. Utilizing DNA microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. We believe these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.

Configurable Digital Virus Counter on Robust Universal DNA Chips

“…This would make the transportation and storage of the test components easier, especially when a large number of tests needs to be delivered to clinical labs and distant locations where cold storage is limited. 41 Passive Microfluidic Cartridge Test Using the Homogeneous Approach. The active flow cartridge-based approach is not practical for field testing because of the complexity of the sample flow process that uses a syringe pump and tubing.…”

“…Although the homogeneous approach adds an extra step of adding the antibody–DNA conjugates to the sample, it eliminates the need for refrigeration because the two components of the assay, DNA chips and lyophilized conjugate pellets, are stable at room temperature for extended periods of time. This would make the transportation and storage of the test components easier, especially when a large number of tests needs to be delivered to clinical labs and distant locations where cold storage is limited …”

The Role of Surface Chemistry in the Efficacy of Protein and DNA Microarrays for Label-Free Detection: An Overview

“…Finally, the substrate is incubated with the particles sample, to achieve capture, and is then analyzed with the method of choice. Sometimes the incubation and capture are performed simultaneously and detected in real time [ 88 ], as represented in Figure 5 for homogeneous virus detection on DNA microarrays.…”

“…The binding of DNA-directed antibodies to the nanoparticles and of the nanoparticles to the substrate can also take place by self-assembly, where all the incubation steps are performed simultaneously [ 91 ]. The antibodies can also be incubated with the particles prior to capture, changing the order of the binding reaction with respect to the standard methods [ 88 ]. Both these approaches demonstrate high efficiency with shorter incubation times, however, they provide less control of non-specific binding.…”

“…(I) Direct antibody spotting (II) DNA-directed antibody immobilization (III) Homogenous virus tagging in solution with antibody-DNA conjugates. Figure adapted with permission from [ 88 ]. Copyright (2021) American Chemical Society.…”