Wake-riding effect-inspired opto-hydrodynamic diatombot for non-invasive trapping and removal of nano-biothreats

doi:10.21203/rs.3.rs-1967497/v1

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Wake-riding effect-inspired opto-hydrodynamic diatombot for non-invasive trapping and removal of nano-biothreats

https://doi.org/10.21203/rs.3.rs-1967497/v1

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Contamination of nano-biothreats, such as viruses, mycoplasmas, and pathogenic bacteria, is widespread in cell cultures and greatly threats many cell-based biomanufacturing and therapeutics. However, non-invasive trapping and removal of such biothreats during cell culturing is of great challenge. Here, inspired by wake-riding effect, we report a biocompatible opto-hydrodynamic diatombot (OHD) based on optical trapping navigated rotational diatom (Phaeodactylum tricornutum Bohlin) for non-invasive trapping and removal of nano-biothreats. Combining the wake-riding effect and optical trapping, this rotational OHD enables trapping of bio-targets down to sub-100 nm. Different nano-biothreats, such as adenoviruses, pathogenic bacteria, and mycoplasmas, were first demonstrated to be effectively trapped and removed by the OHD, without affecting culturing cells. The removal efficiency was greatly enhanced via reconfigurable OHD array construction. Importantly, these OHDs show remarkable antibacterial capability, and further facilitate targeted gene delivery. This OHD provides a new method for effective trapping of nano-objects in bio-microenvironment, and serves as a smart micro-robotic platform for non-invasive and active removal of nano-biothreats in cell cultures, with great promises for benefiting cell-based biomanufacturing and single-cell therapy.

Nanoscale biological threats (nano-biothreats), such as viruses, mycoplasmas, and pathogenic bacteria, are notorious and adventitious contaminants of cell cultures and bio-microenvironments. These organisms can modify both the physiology of cultured cells and the structure of recombinant biomolecules. Due to the rapid reproductive capacity, even a small number of nano-biothreats in bio-microenvironments can result in a great threat and even a disaster for different biomedical applications ranging from basic cell culturing to biomanufacturing and recombinant biotherapeutics¹. Such contamination is widespread in laboratories during cell culturing and can cause great economic losses in biomedical research^2–4. Therefore, it is of great urgence to develop efficient tools for nano-biothreat removal and anti-bacteria that can be directly used in cell cultures⁵. To further facilitate cell-based biomanufacturing and therapeutics, such tools should be biocompatible and non-invasive to bio-microenvironments and cultured cells, which otherwise would generate adverse effects for further biomedical applications such as single-cell therapy. However, the design of effective methods combating the contamination and rapid spreading of nano-biothreats in cell cultures and bio-microenvironments is always a thorny problem⁶.

Conventional nano-biothreat removal methods utilize either chemical (for example, 75% ethanol) and physical (for example, ultraviolet light) agents, which are most widely used methods for cell cultures^{7, 8}. However, the effective removal and killing of nano-biothreats requires a high dosage of these sterilization agents, which lacks selectivity and can harm other biological samples, and thus cannot be applied during cell culturing and biomedical application processes^{9, 10}. Although the use of antibiotics has increased the selectivity for bacteria killing, antibiotics increase the risk of bacteria resistance¹¹. Therefore, design of next-generation platform as nano-biothreat removal and antibacterial agents with high selectivity, efficiency as well as high biocompatibility that can actively work during cell cultures is of great importance and urgency for many biomedical applications^12–15. Different methods have been developed to meet this requirement. For example, the development of antibacterial nanomaterials has added a new dimension to nano-biothreat removal and antibacterial action^16–19. However, many of the nanomaterial-based platforms are static and the antibacterial action is passive. On the other hand, micromotors (also called microrobots) that can convert external energy into motion hold great potential for controlled navigation and active operation in bio-microenvironments^20–23. The dynamic and active feature provides a new suggestion for dynamic antibacterial and active nano-biothreat removal in bio-microenvironments. In particular, different strategies based on external energy sources such as light^24–26, magnetic^{27, 28}, and ultrasonic propulsion²⁹, have been applied for chemical-free navigation of micromotors in microenvironments. Among them, magnetic controlled micromotors are widely used for nano-biothreat removal^{30, 31}. However, all these magnetic controlled micromotors necessitate additional specific magnetic materials to respond to magnetic sources for actuation. In another scenario, a micromotor platform directly using microorganisms with self-propelling capabilities, such as sperm³², green alga³³, and rotifer³⁴, can also be used for high-efficiency nano-biothreat removal. However, these motile organisms can affect the cell growth and metabolism during culturing. In turn, the cell culture media can also affect and destroy the motility feature of the motile organisms, which makes it impossible to further work as a micromotor in cell cultures. Therefore, it is highly desirable to design an intelligent, active and biocompatible platform that can be directly used in bio-microenvironments for nano-biothreat removal. Although optical tweezers provide huge potential for trapping and manipulating of microscopic objects³⁵, and recently shows the potential for micromotor actuation^{36, 37}, the diffraction limit makes it challenging for trapping of nanoscale objects such as the nano-biothreats. In addition, the strong light intensity needed for stable trapping can induce potential harm for biological samples. On the other hand, hydrodynamic tweezers also show great potential for non-contact manipulation of micro/nano-objects via localized microfluidic flow^{38, 39}. However, neither of these two methods can achieve effective nano-biothreat removal due to their limited range of action.

In nature, dolphins often cruise the boat wakes to catch a free ride, allowing them to swim and migrate with far less energy than usual. This phenomenon is called the wake-riding effect. Inspired by this wake-riding effect, in this work, by combing optical trapping and wake-riding effect, we report an opto-hydrodynamic diatombot (OHD) for trapping and removal of nano-biothreats in bio-microenvironments of cell cultures based on optical trapping-navigated triradiate Phaeodactylum tricornutum Bohlin (PTB, a widespread diatom in nature) (Fig. 1a). Nano-biothreats near the wakes of the fast-rotating OHD are easily trapped via wake-riding effect, where the hydrodynamic resistance of nano-biothreats is significantly reduced. This effect greatly increased the nanoscale trapping efficiency of optical tweezers. These OHDs are capable for active and on-demand removal of different nano-biothreats in bio-microenvironments of cell cultures, such as viruses, mycoplasmas, and pathogenic bacteria, without affecting cultured cell viability. By coating with chitosan, these OHDs show remarkable antibacterial capability after bacteria collection, without affecting the viability of cultured cells and greatly increasing the survival rate of living cells. These features further facilitate targeted gene delivery for single-cell therapy. This OHD provides a new method for effective trapping of nano-objects in bio-microenvironment, and will serve as a new micro/nano-robotic platform for non-invasive, high-efficiency, and broad-spectrum removal of nano-biothreats in cell cultures, with great promises for benefiting cell-based biomanufacturing, single-cell analysis and therapy.

Wake-riding effect-inspired OHD for nanoscale trapping. Natural PTB exhibits a triradiate morphology with three elongated diatom arms in the normal growth condition (Fig. 1b, average size of the arm: 7.9 µm in length and 1.8 µm in width, average angle 120°, details see Fig. S1 of the Supplementary Information). This natural PTB, however, is an immotile diatom and is not able for locomotion. By applying an annularly scanning optical trap (scanning radius R: 10 µm, trapping power P, scanning frequency f) on the PTB (Fig. 1c) based on a standard optical tweezers system (Aresis Tweez 250 si, operation wavelength: 1064 nm, details see Fig. S2 of the Supplementary Information), an immobile PTB can be turned into a controllably motile OHD (Movie S1). It should be noted that high-intensity laser power will inevitably affect biological activity. Therefore, to get an effective rotation and minimalize the light damage to the biological sample, we selected the parameters of P = 50 mW and f = 8 kHz to control the OHD in the experiments. A typical rotation trajectory of an OHD (see Fig. S3a of the Supplementary Information). The rotation speed of the OHD increases with laser power and scanning frequency and stabilizes at a maximum value of about 200 rpm (see Fig. S3b and S3c of the Supplementary Information). The high-speed rotation of the OHD induces highly localized flow fields and hydrodynamic vortex around the OHD arms. By moving the OHD along a predefined trajectory of laser trap, the OHD can be navigated to designated locations for targeted nano-biothreat removal as shown in Fig. 1c.

To show the nano-biothreat removal capability of the OHD, numerical simulation based on three-dimensional (3D) finite-element method was carried out. In the simulation, the OHD was subjected to a focused laser beam and locomoted along a predefined circular trajectory (radius R: 10 µm, scanning frequency f: 8 kHz), resulting in a localized flow field with a maximum of 83 µm/s around the OHD arm, while the maximum relative velocity Δv_max to the OHD is 4.6 µm/s. (Fig. 1d). Meanwhile, the maximum relative flow velocity near the OHD arm linearly increases with the increase in OHD rotation speed (Fig. 1e). The situation that nano-biothreats moving along OHD is similar to dolphin riding the wake of a boat to catch a free ride, as shown in Fig. 1f. To show the wake-riding effect of nano-biothreats to the OHD, the flow field distribution of a OHD modelled micro-boat was simulated (Fig. 1g, the forward velocity of the micro-boat was set the same as the tangential velocity of the rotating OHD, 45 µm/s). Without wake-riding effect, the fluidic resistance exerted on the nano-biothreat (polystyrene particle, PS, 150 nm) is up to 50 pN with the forward velocity of 45 µm/s. This fluidic resistance is increased with the increasing in forward velocity. On the contrary, under the wake-riding effect, the calculated maximum fluidic resistance of nano-biothreat is only 5 pN, which is ten times smaller than that without wake-riding effect (Fig. 1h). Benefiting from the wake-riding effect, a small optical force exerted on the nano-biothreat can thus overcome this fluidic resistance, resulting in the trapping and collection of the nano-biothreat by the OHD.

To further show the wake-riding effect-assisted trapping of nano-biothreat by OHD, we analyzed the trapping efficiency of both sole optical trapping and wake-riding assisted-trapping for 150-nm PS nanoparticle (Fig. 2a). With a focused laser beam irradiated (power: 50 mW), microscopic objects can be trapped by optical force (F_O) via photon momentum and optical pressure change (Fig. 2bⅠ). It can be seen that the optical pressure (P_x) on both sides of PTB is equal in value and opposite in direction, resulting in zero resultant optical force. However, as the PTB deviates from the optical axis, the symmetry destruction of the optical pressure generates a spring-like optical force (F_O−PTB) that tends to draw the PTB back onto the optical axis (Fig. 2bⅡ). The maximum of F_O−PTB (F_Om−PTB = 0.89 pN) exists at the distance of 0.28 µm. The optical force (F_O−NP) exerted on nanoparticle follows a similar law to PTB, but the value is more than two orders of magnitude smaller, and the maximum value (F_Om−NP = 5.6 fN) exists at the distance of 0.6 µm. The results above indicate that the sole optical trapping has a weak confinement ability on nanoparticles. The motion of PTB and nanoparticles in liquid is restricted by the fluidic resistance (f_v), which is linearly increased with the relative velocity (Δv) of the object to the fluid (Fig. 2bⅢ). Calculation results indicate that the maximum fluidic resistance that can be overcome by F_Om−NP exists with the maximum relative velocity of Δv_max = 6 µm/s for trapping of 150-nm particle, while this Δv_max for PTB is 90 µm/s (Fig. 2bⅢ). Smaller particles move slower in liquids when the optical force and fluidic resistance are balanced during the trapping process. The relative velocity Δv_max is linearly increased with the increase in particle size (Fig. 2bⅣ). This is because the fluidic resistance is linearly increased with particle size, while F_Om−NP is quadratically increased with increasing in particle size (Fig. 2bⅣ). However, in real experiments, considering the Brownian motion, the instantaneous velocity of random motion for 150-nm particle can even be larger than the value of Δv_max, and thus the particle can escape from the trap. Therefore, for sole optical trapping, it is challenging for effective trapping of nanoscale objects.

Inspired by the wake-riding effect, in the simulation, we further considered the nanoscale trapping in a moving OHD (v_OHD = 90 µm/s, same to the calculated Δv_max for PTB motion in Fig. 2bⅢ) controlled by optical trapping (Fig. 2c). The flow field around the motion OHD in water is depicted in Fig. 2cⅠ. Effective trapping of nanoparticles in this flow field requires that the particles remain stationary relative to the moving OHD. Therefore, we concerned the relative velocity field (Δv) to OHD as reference (Fig. 2cⅡ). For nanoparticles in such a flow field, a small optical trapping force (F_O−NP) can overcome this velocity difference, allowing the nanoparticles remaining synchronized with the OHD, which is similar to wake-riding effect. Combining the data of Δv_max for different sized particles that can be synchronized to the OHD by optical force in Fig. 2bⅣ and the Δv distribution in Fig. 2cⅡ, we can obtain the effective capture range of particles with different sizes by the OHD in the flow field (Fig. 2cⅢ). It can be seen that larger particles own a wider capture range. For sole optical trapping, the random motion of nanoparticles that can be suppressed and trapped by optical force is very limited as discussed in Fig. 2b. For example, for 150-nm nanoparticle, the maximum particle velocity is only 1.5 µm/s. This velocity is gradually increased with the increase in particle size. However, due to the wake-riding effect, the nanoparticles can be confined by the OHD, and rotated together with the OHD (Fig. 2cⅣ), therefore, realizing the transition of trapping/removal velocity. This efficiency is more obvious for particles with smaller sizes.

To further show the nanoscale trapping and removal capability of the rotating OHD, simulations on multiple 150-nm PS nanoparticle trapping and collection via both annularly scanning optical tweezers (OT) and OHD were both performed. In the simulation, the scanning frequency of both methods was kept the same of 8 kHz. As shown in Fig. 2d, effective collection is achieved after two circles of OHD rotation, while no obvious collection is achieved even after three circles for OT (details see Movie S2). This collection performance was also demonstrated experimentally. Effective collection and removal of Escherichia coli (E. coli) was realized at an operation time of t = 13.5 s for OHD, while 80% of the bacteria were still randomly distributed in the microchannel for OT (see Fig. S4 of the Supplementary Information). Repeated experiments show that about 100% removal rate can be achieved after 14 s for OHD, with a much higher efficiency than that of OT in the same time (see Fig. S5a of the Supplementary Information). Due to the synergic effect of optical force and hydrodynamic force, this removal capability also depends on the power of the trapping laser. As shown in Fig. 2e, the simulated removal rate of OHD increases with the increase in optical intensity, and it is much higher than that for OT under the same optical intensity. In the experiments, we find that a removal rate of about 100% can be achieved with an optical power higher than 40 mW for OHD, while OT is only about 11% under the power of 40 mW (see Fig. S5b of the Supplementary Information). Both the simulation and experimental results show the wake-riding effect inspired OHD exhibits a remarkable removal capability due to the synergic opto-hydrodynamic force, and thus can be used for nano-biothreat trapping and removal.

Non-invasive trapping and removal of nano-biothreats. As three most widely existed nano-biothreats in cell cultures, the contamination of viruses, pathogenic bacteria, and mycoplasmas are great threats for many cell-based biomanufacturing and therapeutics. To show the non-invasive nano-biothreat trapping and removal capability of our OHD, demonstration of nano-biothreat trapping and removal in a microfluidic channel was carried out (Fig. 3a). Randomly distributed nano-biothreats are first trapped and collected around the OHD arms during OHD rotation (200 rpm). The collected nano-biothreats can be swept to designated location via navigating the OHD. By turning off the trapping laser, the localized flow fields around the OHD arms disappear, and the collected nano-biothreats can be released at designated location. Subsequently, the annularly scanning optical trap is switched to a central trap with lower power (5 mW), and the OHD can be navigated to other positions for repeated use in subsequent experiments.

As some examples, Fig. 3b-3d show the trapping and removal of adenoviruses (90–100 nm in diameter)⁴⁰, pathogenic bacteria (rod-shaped Gram-negative E. coli), and mycoplasmas by using our OHD (P = 50 mW and f = 8 kHz) in a disc-shaped microfluidic channel (diameter: 100 µm, depth: 50 µm, fabrication details see Methods). As shown in Fig. 3b, about 20 randomly distributed adenoviruses were completely trapped by the OHD at t = 20 s. By turning off the laser power, the collected adenoviruses were released from the OHD and completely removed from the channel at t = 25 s, and the OHD was then moved to another position. Details of the trapping and removal of adenoviruses are shown in Movie S3. To show the trapping and removal capacity of the OHD for different sized nano-biothreats, PS particles with different sizes were used as the nano-biothreat models for demonstration. Experimental results show that our OHD is capable for the effective trapping and removal of PS particles with sizes from 100 nm to 2 µm (see Fig. S6-S8 of the Supplementary Information). In addition to the effective removal of immotile abiotic particles that imitate nano-biothreats, importantly, our OHD can also be used for effective removal of motile nano-biothreats, for example pathogenic bacteria. Pathogenic bacteria are a common contamination during cell cultures. For example, contamination of a small number of pathogenic E. coli can result in the death of both HeLa cells and human promyelocytic leukemia cell line HL-60 within 12 h (see Fig. S9 of the Supplementary Information), due to the rapid bacterial reproduction in the cell culture medium. Effective removal of rod-shaped E. coli and spherical S. aureus were realized respectively (see Fig. 3c and Fig. S10 of the Supplementary Information, details see Movie S4).

In addition to the effective trapping and removal capability, the non-invasiveness and biocompatibility of the OHD is also very important for further cell-based biomedical applications. To show the non-invasiveness of the OHD during nano-biothreat removal, we further carried experiments on the removal of mycoplasmas in the channel containing both mycoplasmas and cultured mammalian cells (HL-60). Mycoplasma is one of the most common contaminants during cell culture, and can result in the destroy of healthy cells (see Fig. S11 of the Supplementary Information). Because of the small size and deformability, it is difficult to remove mycoplasma efficiently by traditional filtration methods. By using our OHD, it is capable for highly efficient and selective removal of the mycoplasmas. As shown in Fig. 3d, about 19 mycoplasmas were collected and removed at t = 32.6 s. Importantly, during the mycoplasmas collection and removal, the HL-60 cell can be avoided from the affection of the OHD, and it was kept intact during the mycoplasma removal process (details see Movie S5). This indicates that the OHD exhibits a non-invasive feature for nano-biothreat removal. The effective removal time of a single OHD for different nano-biothreats is different (Fig. 3e). In addition, the removal efficiency of OHD for different nano-biothreats is also related to its rotation speed. As shown in Fig. 3f, when the rotation speed reaches 200 rpm, the removal rate can reach the best of 100%. The collection and removal capacity is different for different nano-biothreats. As shown in Fig. 3g, for the nano-biothreats we used, the saturation removal numbers of adenoviruses, E. coli, S. aureus, and mycoplasmas were about 39, 40, 45, and 71 with a single OHD, respectively.

To further show the non-invasiveness and biocompatibility feature of the OHD, we tested the biocompatibility of OHD to two different mammalian cell lines (adherent HeLa cells and suspending HL-60 cells). After co-culturing OHD with the cells for 24 hours, both cell lines show no obvious decrease in cell viability (Fig. 3h and 3i, green fluorescence for living cells, blue fluorescence for nuclei). The cell viability is not affected by OHD, which is similar to that for normal cell culturing (Fig. 3j). These results indicate that the PTB-based OHD is highly biocompatible to bio-microenvironments and mammalian cells.

OHD array for efficiency-enhanced removal. Although single OHD is capable for non-invasive nano-biothreat trapping and removal, the efficiency is limited by individual operation. As the number of nano-biothreat increases, the collection and removal capacity can be saturated, and the removal rate of the OHD is gradually decreased when exceeding the saturation number (see Fig. S12 of the Supplementary Information). Therefore, the formation of OHD arrays with highly reconfigurable and controllable capabilities is important for efficiency enhanced multi-task execution and manipulation, with higher speed and larger collection volume. Fortunately, our OHD can be extended into OHD arrays with high reconfigurability and controllability, and multiple OHDs can operate sequentially or simultaneously. By extending the single optical trap into trap arrays, multiple traps with designated patterns can be formed. Multiple PTB cells can then be turned into OHD arrays. The rotation of each OHD element in the array can be controlled similar to that for a single OHD. As some examples, Fig. 4a shows the formed OHD arrays with the pattern of ‘DIATOMBOT’. These OHD arrays with designated patterns will provide more choices for cooperative robotic operation and on-demand task execution with higher efficiency.

These OHD arrays can work independently and collaboratively for nano-biothreat removal with higher efficiency than that for a single OHD. As shown in Fig. 4b, for a single OHD, the trapping of 25 E. coli was completed at 14 s. The completion collection time was reduced to 7.3 s and 6.3 s for a two-OHD and three-OHD array, respectively (Fig. 4c and d) (details see Movie S6). The efficiency for a three-OHD array is more than twice of that for a single OHD. A comparison of the collection and removal for a single OHD and OHD arrays is also shown in Fig. 4g. These OHD arrays can be directly used in cell cultures for high-efficiency nano-biothreat removal without affecting the cultured cells. As shown in Fig. 4e, for a single OHD, removal of 14 E. coli in cultured HL-60 cells was completed within 15 s. For a three-OHD array, the removal efficiency was more than twice of that for a single OHD. Removal of similar number of E. coli with three-OHD array was completed within only 7 s (Fig. 4f). During the removal, the viability of the cultured cells was not affected. Due to the collection capacity limit of a single OHD, the total removal capacity for different OHD arrays is also different, and this capacity is increased with the increase in OHD number in the array (Fig. 4h and i). For bacteria number that exceeds the removal capacity of a single OHD, although a single OHD cannot completely removal the bacteria, OHD arrays can get a perfect removal efficiency (Fig. 4h and see Fig. S13 of the Supplementary Information). For a given number of E. coli, the removal time is decreased with the increase in the number of OHD in the array (Fig. 4i). These results indicate that we can build OHD arrays with more OHDs to further get a higher removal efficiency and capacity.

Non-invasive antibacterial capability for enhanced gene delivery. Despite the non-invasive and high-efficiency nano-biothreat collection and removal capability of OHD in bio-microenvironments, further non-invasive and efficient killing of contaminated nano-biothreats in the bio-microenvironments are very important to ensure further single cell analyses. Importantly, in addition to the nano-biothreat removal, our OHD is also capable for non-invasive bacterial killing and antibacterial treatment. As shown in Fig. 5a, the OHD is modified with a chitosan (Chi) layer as a micro-robotic strategy for antibacterial treatment in cell culturing microenvironments. Chitosan has garnered increasing interest in the field of antibacterial as a renewable material due to its unique properties such as high biocompatibility, ease of decomposition, and low toxicity⁴¹. Our antibacterial strategy relies on the combination of the efficient nano-biothreat removal capability of OHD and the strong bactericidal activity of chitosan layer. Taking E. coli as an example, for naked OHD, it can realize E. coli removal. Nevertheless, it does not exhibit antibacterial property, and there still exists infection risk for the cultured cells. In this case, the final fluorescence of the OHD with collected live E. coli stained with bacterial viability kit (details see Methods) is yellow. However, by coating a layer of chitosan, the collected E. coli can be killed by the OHD, demonstrating the good antibacterial effect of OHD (see Fig. S14 of the Supplementary Information). Therefore, there is no infection risk for cultured cells. Since the OHD is fluorescent red and the dead E. coli also has red fluorescence, the measured fluorescence intensity also reflects the enhanced antibacterial effect of the chitosan-coated OHD (chi-OHD, Fig. 5b).

In order to show the antibacterial ability of the chi-OHD, we carried out a series of different controlled experiments. To show the bacterial viability after different treatments, a commercial viability kit based on two dyes (DMAO: 9-Octadecen-1-amine,N,N-dimethyl-,(9Z)- and EthD-3: Ethidium Homodimer 3) was used for E. coli staining. The DMAO dye (green) was used to label live bacteria, while the EthD-3 dye (red) could only penetrate damaged bacteria and was used to label dead E. coli. As shown in Fig. 5c, after collection of E. coli for 10 min, the bare OHD fluoresces yellow, which is the combination of red (for PTB) and green (for live E. coli) fluorescence. This phenomenon indicates the bare OHD cannot result in E. coli killing. For the treatment with chitosan only, the fluorescence of about 40% E. coli is red, while the others are still green after 10 min treatment, indicating only about 40% of the bacteria are killed by the chitosan solution. However, for chi-OHD, all the fluorescence is red, indicating all the bacteria are killed after 10 min. The high antibacterial efficiency obtained by the chi-OHD reflects the key role of the combination of chitosan and the effective rotation of OHD for antibacterial activity. The OHD rotation increases chitosan-bacteria interaction, and thus results in a high-efficiency antibacterial performance. Figure 5h and 5ishow the comparison of the antibacterial efficiency for naked OHD, chitosan solution, and chi-OHD. This antibacterial performance is dependent on the concentration of the chitosan solution for OHD coating. When the chitosan concentration reaches 0.2 mg/ml, the antibacterial efficiency of the OHD reaches 98% (Fig. 5j).

This antibacterial capability is of great importance for cell culturing and further single cell-based therapy. As shown in Fig. 5d, for HL-60 cells contaminated with E. coli, without any treatment, once the living HL-60 cells (green) are infected by active bacteria, the cell is dead after 60 min (red). However, by using chi-OHD for bacteria removal and antibacterial treatment, the HL-60 cell is not infected by bacteria even the microenvironment is contaminated with E. coli, and the cell viability is not affected after 60 min (green fluorescence, Fig. 5e). Although the chi-OHD can kill bacteria, chi-OHD is highly biocompatible and non-invasive to the cultured cells (see Fig. S15 of the Supplementary Information). This non-invasive antibacterial feature further facilitates the study of enhanced drug delivery and single cell-based therapy. To show this capability, Cy3-labeled small interfering RNA mimics (siRNAs) that fluoresce red were loaded into mesoporous silica particles (details see Methods) and added to the cultured HL-60 cells contaminated with E. coli. The bacteria contamination and infection results in cell death (red fluorescence), and siRNA cannot be delivered into the HL-60 cells (Fig. 5f). However, with the treatment of chi-OHD, the contaminated E. coli are completely removed and killed. The cell viability is thus not affected by the contaminated E. coli even after 2 h, which is similar to the cells without any contamination (Fig. 5k). Since the cell viability is not affected by the contaminated E. coli, the siRNA is thus delivered into the cell in 90 min (Fig. 5g). These results indicate that the non-invasive antibacterial capability of chi-OHD can be directly used to remove and kill the contaminated nano-biothreats in cell cultures, and further for enhanced drug delivery and subsequent single-cell analysis.

In summary, inspired by wake-riding effect, we created a non-invasive and active opto-hydrodynamic diatombot (OHD) based on a natural diatom for nano-biothreat removal, which can be directly used in cell cultures for enhanced gene delivery. The synergic optical trapping and hydrodynamic force of OHD results in high-efficiency trapping and removal of different nano-biothreats, including adenoviruses, pathogenic bacteria and mycoplasmas, in cellular environments without affecting culturing cells. This removal capability was further enhanced by reconfigurable OHD array formation. Efficient and non-invasive antibacterial performance was demonstrated in cellular environment with chitosan coated OHD, which further facilitated single-cell gene delivery.

This OHD provides a new method for effective trapping of nano-objects in bio-microenvironment, and serves as a smart micro-robotic platform for non-invasive and active nano-biothreat removal and antibacterial treatment in cellular environments, offers a seamless interface among optical, microrobotic, and biological worlds. Due to the non-invasiveness and high biocompatibility, this OHD can be directly used in cell cultures for the removal of nano-biothreat contamination, without affecting the cultured cells. This feature ensures subsequent cell-based biomanufacturing and therapeutics after nano-biothreats removal and antibacterial treatment. OHD arrays ensure the capability of larger-scale removal of nano-biothreats via the cooperation of microrobot individuals. This OHD provides a biocompatible and smart platform for non-invasive, high-efficiency, and broad-spectrum removal of nano-biothreats, especially the rapid spreading drug-resistant bacteria, in bio-microenvironments and cell cultures, with great promises for benefiting cell-based biomanufacturing and enhanced single-cell therapy.

Experimental setup of optical tweezers: A scanning optical tweezers system (Aresis, Tweez 250Si, SI Appendix, section S3) was built on an inverted optical microscope (Nikon Eclipse Ti-U) using a continuous wave solid-state laser with 1064-nm operation wavelength (maximum output power: 5 W). The laser beam was refocused into the sample chamber after passing through a 60× water immersion inverted objective (numerical aperture: 1.0) for optical manipulation. Multiple trap sequences could be constructed in a loop manner by a computer interfaced AOD system, and the high switching rate (maximum 100 kHz) ensured stable capture and precise manipulation of multiple targets at the same time. Real-time image was recorded by a computer interfaced high-speed CCD camera. Details of the setup can be seen in Fig. S2 in the Supplementary Information.

PTB preparation and OHD construction

PTB diatom solution was purchased from Zhuhai Xinrui Trading Co., Ltd, China. PTB cells were obtained through a 47 mm × 5 µm mixed cellulose ester filter, and the PTB suspension was then prepared by resuspending PTB cells into 1 mL phosphate buffered saline (PBS) buffer, with a final concentration of about 2 ×10⁴ ~ 4 × 10⁴ cell/mL. This PTB solution was then transferred to the sample slide on the scanning optical tweezers system. Next, a central optical trap was applied to trap the PTB, and an annularly scanning optical trap controlled by the AOD system was applied to the PTB for rotation and OHD construction.

Microfluidic channel fabrication: Silicon elastomer and curing agent were mixed at a weight ratio of 10:1 for channel fabrication. The solution was placed in a vacuum pump to remove air bubbles that were generated during the mixing process. 50-µm thick SU8-3005 photoresist was coated on a clean Si/SiO₂ wafer and exposed to standard lithography setup to prepare the master mold of the microfluidic channel. The bubble free polydimethylsiloxane (PDMS) was casted over the SU8 photoresist master mold and cured at 70℃ for 3 h. After the curing process, the PDMS was peeled off from the mold and bonded to the glass slide by using O₂ plasma bonding.

Nano-biothreat preparation

Adenoviruses were purchased from Guangzhou VectorBuilder Co., Ltd., China, and were suspended in a carbonate buffer (pH = 9.3). Adenoviruse suspension with a final concentration of about 2 ×10³ ~ 6 × 10³ per microliter was prepared. The bacteria (E. coli and S. aureus) were grown 3–4 hours in lysogeny broth in a shaker with a rotation speed of 180 rpm at 37℃. The bacteria were washed and diluted with PBS buffer to obtain a suitable concentration of about 2 ×10⁴ ~ 5 × 10⁴ bacteria per microliter. Mycoplasma-contaminated cell culture suspension (1 mL, containing HL-60 cells) mixed with PTB suspension. After preparation, the suspension and PTB mixture were injected into the microfluidic chamber by a microsyringe for following experiments. 4',6-Diamidino-2'-phenylindole (DAPI) was used to stain the cells contaminated with mycoplasma.

Cell culturing and viability test

Mammalian cell lines (HeLa cells and HL-60 cells) and PTB cells were co-cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4500 mg L^− 1, 10% fetal bovine serum (FBS) and 1% Penicillin/streptomycin (Pen/strep), and then mixed overnight in a 37°C incubator contained 5% CO₂. Mammalian cell viability was tested using dual-fluorescent calcein-AM/propidium iodide (PI) (purchased from Jiangsu KeyGEN BioTECH Corp., LTD, China). In the experiments, 2-µL AM and PI dyes were added to co-cultured cells for 25 min. For living cells, Calcein-AM can react with esterases, and then strong green fluorescence was emitted for live cells. PI cannot pass living cell membrane, but can reach the nucleus through the disordered region of the dead cell membrane. Therefore, the DNA double helix structure in the cell was bound with PI, so that red fluorescence was emitted for dead cells.

Chi-OHD preparation: Chitosan was dissolved in 2% glacial acetic acid to prepare chitosan solution. In the experiments, 1 mg/mL chitosan solution was mixed with OHD, and then the chi-OHD suspension was placed on a shaker with a rotation speed of 200 rpm to shake at 37°C for 3 ~ 4 h. Finally, the chi-OHD suspension was centrifuged for 10 min at a rotation speed of 1500 rpm, and the supernatant was removed to get the chi-OHD precipitation. The antibacterial ability of the chi-OHD was tested by analyzing the activity of E. coli cells mixed with the chi-OHD precipitation. Living E. coli were collected by chi-OHD through a standard optical tweezers system. Then the mixture was reacted for 10 min, and the E. coli were killed. Bacterial viability kit (DMAO: 9-Octadecen-1-amine,N,N-dimethyl-,(9Z)-; and EthD-3: Ethidium Homodimer 3 dyes) (purchased from Yeasen Biotech Co. Ltd. Shanghai, China) was used to test the viability of E. coli collected by chi-OHD and OHD. 2 µL of DMAO and EthD-3 dye were added to 100 µL of the mixture and incubated with E. coli for 20 min for viability characterization.

Preparation of siRNA-loaded mesoporous silica particles: Cy3-labeled small interfering RNA mimics (siRNAs, sequence: 5’-UUCUCCGAACGUGUCACGUTT) were obtained from GenePharma Co. Ltd. (Shanghai, China). Mesoporous silica Particles (MSNs) with diameter of 1 µm were obtained from Wuhan Huake Microtechnology Co., Ltd, Shanghai, China. For the loading of SiRNAs, 500 µg MSN was dissolved in 500 µL diethyl pyrocarbonate (DEPC), with a weight concentration of 10 µg/µL. Then, 50 µL siRNAs were added to 200 µL MSN solution and stirred for 24 h. Thereafter, the siRNA-loaded MSNs were centrifuged and washed twice with DEPC solution and then freeze-dried for 6 h.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (61975065 and 11904132), Guangdong Basic and Applied Basic Research Foundation (2019B151502035), Science and Technology Program of Guangzhou (202102010088).

Author information

Authors and Affiliations

Institute of Nanophotonics, Jinan University, 511443, Guangzhou, China

Jianyun Xiong, Yang Shi, Ting Pan, Dengyun Lu, Ziyi He, Danning Wang, Xing Li, Guoshuai Zhu, Baojun Li, and Hongbao Xin

Contributions

J. X. and H. X. conceived the idea, designed the experiments. Y. S. carried out the simulation. J. X., T. P., and D. L. performed the experiments. Z. H. review and revised original draft. D. W., X. L. and G. Z. discussion the manuscript. H. X. and B. L. supervised the study.

Corresponding authors

Correspondence to [email protected], [email protected]

Ethics declarations

Conflict of interest

The authors declare no completing interests.

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Wake-riding effect-inspired opto-hydrodynamic diatombot for non-invasive trapping and removal of nano-biothreats

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