Rolosense: Mechanical detection of SARS-CoV-2 using a DNA-based motor

Abstract

Assays detecting viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here we present Rolosense, a virus sensing platform that transduces the motion of synthetic DNA-based motors transporting 5-micron particles on RNA fuel chips. Motors and chips are modified with virus-binding aptamers that lead to stalling of motion. Therefore, motors perform a "mechanical test" of viral target and stall in the presence of whole virions which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 10³ copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as we demonstrated one-pot detection of influenza A and SARS-CoV-2. As a proof-of-concept, we show readout can be achieved using a smartphone camera in as little as 15 mins without any sample preparation steps. Taken together, mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost, point-of-care screening of circulating viruses.

Figure 1.. Optimizing Rolosense with GFP-labeled virus-like particles (VLPs).

a, Schematic workflow of the Rolosense assay. The presence of virus particles leads to motor stalling which reduces the net displacement or distance travelled by the motors. Readout can be performed using simple brightfield timelapse imaging of the motors. In principle, readout can be performed in as little as 15 min using a smartphone camera. b, Schematic of DNA motor and chip functionalization. The DNA motors were modified with a binary mixture of with DNA leg and aptamers that have high affinity for SARS-CoV-2 spike protein. The Rolosense chip is a gold film also comprised of two nucleic acids: the RNA/DNA chimera, which is referred to as the RNA fuel, and the same aptamer as the motor. c, Schematic of the detection of SARS-CoV-2 virus. In the presence of VLPs expressed with spike protein (spike VLPs), the motors stall on the Rolosense chip following the addition of the RNaseH enzyme as the stalling force (red arrow) is greater than the force generated by the motor (green arrow). When incubated with the bald VLPs, or VLPs lacking the spike protein, the motors respond with motion and roll on the chip in the presence of RNAseH. d, Brightfield and fluorescence imaging of DNA motors detecting GFP-labeled spike VLPs. The RNA fuel was tagged with Cy3, shown here in red. Motors were incubated with 25pM of GFP-labeled bald and spike VLPs diluted in 1xPBS. Samples with GFP-labeled spike VLPs show stalled motors and no Cy3 depletion tracks in contrast to samples GFP-labeled bald VLPs. Note that stalled motors often showed GFP signal colocalization. e, Plots showing the trajectory of motors with bald and spike VLPs. All the trajectories are aligned to the 0,0 (center) of the plots for time = 0 min. Color indicates time (0 → 30 min). f, Plot showing net displacement of over 100 motors incubated with 25 pM bald and spike VLPs. **** indicates P<0.0001. Experiments were performed in triplicate. g, Plot showing the difference in net displacement between the bald/spike VLPs normalized by the bald VLP displacement in conditions using different aptamers. Each data point indicates the pooled average for an independent experiment. Error bars show the standard deviation.

Figure 2.. Detecting SARS-CoV-2 virus in artificial saliva.

a, Fluorescence and brightfield imaging of DNA motors detecting the presence of 10⁸ copies/mL of UV-inactivated SARS-CoV-2 WA-1 spiked in artificial saliva. DNA motors were incubated for 30 min with the virus samples. Samples with SARS-CoV-2 show stalled motors and no depletion tracks in contrast to samples lacking the virus. b, Plots showing the trajectories of motors with no virus and 10⁸ copies/mL of UV-inactivated SARS-CoV-2 WA-1 strain spiked in artificial saliva. All the trajectories are aligned to the 0,0 (center) of the plots for time = 0 min. Color indicates time (0 → 30 min). c, Plots of net displacement of over 300 motors for each sample that was incubated with ranging concentrations of SARS-CoV-2 WA-1, B.1.617.2, and BA.1. UV-inactivated SARS-CoV-2 samples were spiked in artificial saliva and incubated with the motors functionalized with aptamer 3 at room temperature for 30 min. Each sample was performed in triplicate. **** and ns indicate P < 0.0001 and not statistically significant, respectively.

Figure 3.. Motors demonstrate a specific response to SARS-CoV-2 viruses.

a, Schematic of motors modified with SARS-CoV-2 aptamer stalling when incubated with SARS-CoV-2 virus particles which is in contrast to motors incubated with other viruses. b, Plot showing the net displacement for over 100 motors incubated with 10⁷ copies/mL of UV-inactivated HCoV OC43, HCoV 229E, influenza A, SARS-CoV-2 WA-1, SARS-CoV-2 B.1.617.2, and SARS-CoV-2 BA.1 spiked in artificial saliva. The motors were functionalized with aptamer 3 and incubated for 30 min with each sample. All measurements were performed in triplicate. ns, *, **, and **** indicate not statistically significant, P=0.018, P=0.0015, and P<0.0001, respectively.

Figure 4.. Multiplexed detection of SARS-CoV-2 and influenza A viruses.

a, Schematic showing multiplexed detection of IAV (influenza A virus) and SARS-CoV-2. Two types of motors specific to SARS-CoV-2 (blue, 6μm polystyrene) and IAV (grey, 5 μm silica) were encoded based on the size and composition of the microparticles and used to simultaneously detect these two respiratory viruses. The two types of motors were mixed together and incubated for 30 min with the virus sample. b, Fluorescence and brightfield imaging of DNA motors with no virus, 10⁷ copies/mL of UV-inactivated SARS-CoV-2 WA-1, and 10¹⁰ copies/mL of IAV. Representative images showing the two different DNA motors are shown and each type of motor can be identified based on the brightfield particle size and contrast. Samples with SARS-CoV-2 show stalled 6 μm motors, while the IAV samples showed only stalled 5 μm particles. Samples lacking any virus showed motion of both types of motors. (c) Plots showing the net displacement for over 300 motors incubated with 10⁷ copies/mL of UV-inactivated SARS-CoV-2 WA-1 and 10¹⁰ copies/mL of IAV spiked in 1xPBS supplemented with 1.5mM Mg⁺². Experiments were run in triplicate. ns and **** indicate not statistically significant and P<0.0001, respectively.

Figure 5.. Detecting SARS-CoV-2 WA-1 and B.1.617.2 using smartphone readout.

a, Set-up of cellphone microscope (Cellscope) which is 3D printed and includes an LED flashlight along with a smartphone holder and simple optics. The representative microscopy image shows an image of DNA motors that have been analyzed using our custom particle tracking analysis software. Moving particles show a color trail that indicates position-time (0→30 min). Scale bar is 100 pixels, and the diameter of the motors is 5 μm. b, Plots showing net displacement of motors incubated with different concentrations of UV-inactivated SARS-CoV-2 Washington WA-1 and B.1.617.2 samples spiked in artificial saliva. The net displacement of the motors was calculated from 15-min videos acquired using a cellphone camera. The motors were functionalized with aptamer 3 and experiments were run in triplicate. ** and **** indicate P=0.0015 and P<0.0001, respectively.

Figure 6.. Detecting SARS-CoV-2 virus in breath condensate.

a, (Top) Schematic of breath condensate sample collection and incubation of DNA motors with spiked-in virus particles. i) Fluorescence and brightfield imaging of aptamer 3 modified DNA motors without virus and with 10⁷ copies/mL of SARS-CoV-2 B.1.617.2. ii) Fluorescence and brightfield imaging of aptamer 4 modified DNA motors without virus and with 10⁷ copies/mL of SARS-CoV-2 BA.1. Samples without virus show long depletion tracks in the Cy3-RNA channel but no tracks are observed following sample incubation with 10⁷ copies/mL of SARS-CoV-2 B.1.617.2 and BA.1. b, Plots of net displacement of over 300 motors with no virus and different concentrations of UV-inactivated SARS-CoV-2 B.1.617.2, and BA.1. UV-inactivated SARS-CoV-2 samples were spiked in breath condensate and incubated with the motors functionalized with aptamer 3 (B.1.617.2) and aptamer 4 (BA.1) at room temperature. Experiments were done in triplicate. **** indicates P < 0.0001.