Nanocavity integrated virus digital chip
The 110-nm-diameter silicon nitride (Si3N4) nanocavity array on a silicon oxide substrate is illuminated by a projected large-sized Gaussian beam (wavelength: 532 nm; diameter > 10 µm) to excite the Trapping mode in the dielectric nanocavities (Fig. 1a). The Trapping mode confines the light field tightly in the holes of the nanocavities, generating isolated hotspots with high light intensities. When a virus particle flows to the light illuminating area, it is trapped in the beam and hops between isolated hotspots. The virus also experiences an optical gradient force that pushes it towards the nanohole28. Viruses with size smaller than the nanoholes will eventually be caged inside the nanoholes, while bigger viruses will be released by the microfluidic drag force when the laser is switched off. To ensure that the viruses flowing to the laser illuminating area experience enough optical forces to pull them to the surface and nanoholes, we fabricate an optofluidic virus digital chip (VDC) with a 1-µm shallow microchannel as shown in Fig. 1b. The shallow microchannel is fabricated by spin-coating a 1-µm Polydimethylsiloxane (PDMS) onto the surface of a quartz block and ablating the pattern of microchannel using the laser engraving. The detailed fabrication process is discussed in Methods.
The nanocavities are designed to have the Trapping mode being confined inside the nanoholes (Fig. 1c), which is used to trap viruses as the light is tightly focused to generate a strong optical gradient force. On the other hand, the Futile mode is guided along the dielectric layer, leading to a much weaker optical gradient force that is incapable of trapping viruses. Figure 1d presents the simulated resonances of the TE polarized field when the hole radius and gap are 55 nm and 295.1 nm, respectively. The Q-factor plot (Supplementary Fig. 2) shows that mode 3 and mode 4 at point Γ have Q-factor up to 109. The mode 1 has a quality factor < 103 but has the largest optical gradient force (Fig. 1e) on the polystyrene nanoparticle (radius: 50 nm; Refractive index RI: 1.58) placed right above the nanohole (Supplementary Fig. S2c). This is because perfect BIC mode traps light without leakage, resulting in a relatively small optical gradient force. On the other hand, the leaking mode 1 obtains a balance between light confinement and leakage to achieve a strong optical gradient force, creating optical trapping potential wells in the nanocavities for virus trapping. Mode 2 (at point Γ) has the smallest optical attractive force towards the hole, resulting from light wave deflecting from the hole. More simulations of the resonances and optical forces of the TM modes (Supplementary Figs. 2-5). Since the nanocavities are made of dielectric material (Si3N4), it does not absorb light efficiently and therefore does not generate heat during virus trapping (Supplementary Movie 1), conserving the properties and viability of trapped viruses.
Figure 2a shows the reflection spectrum of the nanocavities with different nanohole radii and intervals g under an illumination of a 532 nm laser. The strong coupling in the parametric space produces an avoided resonance crossing with low-Q (modes 1, 3 and 5) and high-Q (modes 2, 4 and 6) modes on the left and right sides of the spectrum, respectively. Modes 1 and 6 traps light inside the holes so that they can generate large optical forces on the nanoparticle. And modes 2 and 5 disperse light in the solid Si3N4 so that the optical force is small. Figure 2b shows the enhancement factor S which is defined as S = |max Ehole|/|Ein| where |maxEhole| is the maximum normalized electric field inside the nanohole and |Ein| is the incident electric intensity. S reaches a maximum of 73, showing that the laser intensity can be enhanced by approximately 5,300 folds. Meanwhile, as light is tightly confined in a circle with a diameter Dh ≤ 110 nm (Dh is the diameter of the hole), the optical gradient force, which is proportional to the intensity gradient (related to the maximum value in the hole), is hugely enhanced29. Meanwhile, the distribution of the Poynting vector shows that the optical scattering forces acted on a particle placed above nanohole are also pointed towards the nanohole (Fig. 2c), double ensuring the caging of viruses into the nanoholes with the optical force. To explore the trapping limit of the nanocavities, we investigate the trapping force on viruses with diameter ranging from 20 to 100 nm and RI of 1.4 (virus) and 1.58 (polystyrene nanoparticle) in Fig. 2d. Two cases are simulated (see the sketch in Fig. S10a): (1) the nanohole diameter is set to 110 nm, and (2) nanohole diameter Dh = D + 10 nm, where D is the diameter of the nanoparticle. Different intervals are chosen according to the radii of holes to match the Trapping modes in Figs. 2a and 2b. The trapping force is negligible when D < 40 nm but remains relatively large when D ≥ 40 nm. Therefore, the trapping limit of the Trapping mode nanocavities is 40 nm. The abrupt reducing of optical force when D < 40 nm is partly because that the optical gradient force decreases with D3, and because the interference of cavity resonance from the trapped nanoparticle will become prominent when both the particle size and hole size are very small. Above the surface, some weaker light spots occur and generate sub-potential wells for the 100-nm polystyrene nanoparticle, as shown in Fig. 2e. The refractive index of the nanoparticle and laser wavelength are 1.58 and 532 nm, respectively. Nanoparticles and viruses will hop from the sub-potential wells to the central deep potential well. More importantly, the change of temperature is negligible (< 0.01K) when illuminated with a laser intensity of 1 mW/µm2 (Fig. 2f), making it suitable to manipulate viruses without heating them up and altering their viability.
Manipulation of adenoviruses
With a Gaussian beam (wavelength 532 nm) being projected on the nanocavity array, the virus tends to hop from the side lobe potential well to the deeper potential well in the centre (Fig. 3a) at nanoscale (g = 295.1 nm). In the scale of a few micrometres, the virus gradually moves to the nanohole in the centre of the beam by multiple hopping, showing the principle of precise positioning by optical hopping. Simultaneously, the virus is attracted towards the nanohole by the optical gradient force. The hopping time of virus (D = 100 nm; RI = 1.4) from the side hotspot to the central hotspot is less than 10 ms (Fig. 3b). Figure 3c shows the scanning electron microscope (SEM) image of the fabricated nanocavities with a dimeter of 110 nm and an interval g of 295.1 nm, realizing the designed Trapping mode. Since we can flexibly move the laser beam, we can easily trap and position viruses in the array of nanocavities based on the virus hopping mechanism. Four prominent Movie frames are shown in Fig. 3d to illustrate the caging process. An adenovirus (yellow circle) was first spotted at t = 0. Meanwhile, the laser spot (green circle) was moved to the path of the virus. Then, the adenovirus was trapped and ready to be transported at t = 2.5 s. It started to be caged when reaching the target position at t = 5.5 s and remained caged inside the hole when the beam is moved away at t = 20.1 s. 50-nm upconversion particles, 70- and 100-nm polystyrene nanoparticles, and adenoviruses are easily patterned in various signs in the nanocavity array (Fig. 3e and Supplementary Movies 2,3). The stable caging of nanoparticles or viruses smaller than the nanohole can be verified by experimentally flushing the surface with an extremely fast flow stream (e.g., 2.5 mm/s in Fig. 21). The nanoparticles will be easily flushed away if their sizes are larger than the hole because of the perfect surface treatment (see Methods) to avoid surface adhesion.
Since the diameter of the nanoholes is 110 nm, only viruses smaller than 110 nm can be efficiently caged in the nanoholes. Larger viruses will be flushed away by the fluidic drag force when the laser is switched off. Sorting of nanoparticles and adenoviruses can be achieved whereby larger nanoparticles and adenovirus could only be temporarily trapped by the laser spot. Once the laser spot was moved away, these larger nanoparticles and adenoviruses were released and eventually flushed away by the microflow. This size-selective mechanism is demonstrated by the nanoparticles and adenovirus, as shown in Figs. 4a and 4b, respectively. The 100-nm nanoparticle can be caged and patterned. However, the big nanoparticle conjugate will be released when the beam is moved swiftly at t = 47.4 s in Fig. 4a (Supplementary Movie 5). Similarly, adenovirus larger than 110 nm can be trapped above the hole at t = 26.0 s in Fig. 4b (Supplementary Movie 6) and released at t = 29.4 s. Some adenoviruses were not caged in the nanocavity array because of the broad size distribution of adenoviruses, measured by the Nanosizer (Fig. 4c). Based on the sorting mechanism, the nanocavity array is also capable to measure the size distribution of adenoviruses using different designs with various nanohole sizes. For instance, the 90-nm nanoholes can determine the percentage of viruses larger or smaller than 90 nm. We fabricate holes with different sizes (i.e., 90, 100, 110, and 120 nm) in different areas in one chip, then use them to trap viruses to determine how much percentage of viruses are larger than 90, 100, 110, and 120 nm. The comparison of the size distribution using the Trapping mode nanocavity array and Nanosizer is shown in Fig. 4d. The results from the nanocavity array and Nanosizer are comparable, but the nanocavity array has higher measured percentage. This is caused by the unexpected escape of adenoviruses smaller than the size of the holes due to the less trapping time and Brownian motion.
We can also concentrate massive viruses in the nanocavity array (Fig. 5a) by continuously illuminating the array with a high laser power (e.g., 60-100 mW, equivalent laser intensity ~ 1 mW/µm2). In the higher laser power, instead of a single nanohole, more nanohole (hotspots) will have stronger optical gradient forces to attract the viruses inside. The experimental results in Fig. 5b shows the caging of dozens of adenoviruses within the laser spot when the laser power is 100 mW. Instead of being concentrated within a circle, the virus will move to the nanohole in the centre of the beam by optical hopping when illuminated with a lower laser intensity. This is because the input Gaussian beam has a gradient. Thus, only the nanohole in the centre of the beam has enough optical gradient force for the caging when the laser power is low, while more nanoholes are capable of caging viruses when the laser power is high. Figure 5c illustrates the motion trajectories of adenoviruses being flushed to the laser spot by the fluidic drag force30, and subsequently, hopping to the centre of the laser spot under a laser power of 10 mW (equivalent laser intensity ~ 0.1 mW/ µm2). The SEM image of caged 87-nm nanoparticles is shown in Fig. 5d. Most nanoparticles reside at the bottom of the hole (depth 240 nm), so they appear a little bit dim. The nanoparticle can be brighter when it sticks to the side wall of the hole and resides close to the upper surface of hole. Each hole tends to trap one nanoparticle when the sizes of particle and hole are close because the trapped nanoparticle can disturb the light field and weaken the optical force from the hole, causing difficulties in trapping more nanoparticles. The probability of a virus going into the nanohole (Ptrapping) depends on the trapping time. In principle, a virus has a higher probability to be caged in the nanohole with a longer the trapping time. However, in some cases, the virus was not caged in the nanohole for a long trapping time (Supplementary Movie 7). Pcaging also depends on the beam moving velocity when the virus is transported. When the beam velocity vb > 10 µm/s, Pcaging < 3% because the virus does not have sufficient time to be caged. Meanwhile, the probability of virus being transported with the beam (Ptransporting) is > 80% when vb < 20 µm/s. When the beam moves too fast, e.g., vb > 20 µm/s, the virus is unlikely to be transported because of the inertia. Therefore, the optimal vb for the transporting of the adenovirus is between 10-20 µm/s (Fig. 5e). Pcaging for the adenovirus is > 95% when the trapping time is longer than 20 s (laser beam holding still) and the laser power is larger than 80 mW (Fig. 5f). The high caging efficiency demonstrates a high reliability that the Trapping mode nanocavity array can be used to efficiently localize single viruses in the nanoholes. This probability of virus caging is done by measuring the viruses with relatively weak intensity to guarantee that their sizes are smaller than the hole size. The intensity can be easily visualized because most of the viruses have similar scattering intensity because their sizes are close. It shows a high caging probability given certain power and flow velocity when the virus size is smaller than the hole.