Design and Control of a Novel Corkscrew Clawbot for Cell Manipulation

Abstract

Magnetic microrobots are untethered microscale objects that, due to their small size, have the potential for biomedical applications such as targeted drug delivery and cellular patterning. In this paper, we present a new magnetic microrobot design resembling a corkscrew microrobot with claws. This novel magnetic corkscrew clawbot is capable of performing single cell manipulation. Closed loop control was also implemented to demonstrate the precise actuation capabilities of the clawbot in a fluidic environment on a glass slide. Cell viability was assessed via trypan blue viability assay on Chinese Hamster Ovary (CHO) cells. In addition, CHO cells were chosen as the representative cell line for the cell manipulation and patterning studies.

I. Introduction

Magnetic actuation is one of the most common actuation mechanisms for microrobots since it is non-invasive, has strong penetration and precise control which make it suitable for targeted drug delivery, microscale manipulation and in vivo applications[1]. Magnetic actuation is especially well suited for interventions inside the body since it is less invasive compared to other modes of actuation such as light and chemical [2]. Also, imaging techniques such as magnetic resonance imaging (MRI) can be utilized for tissue ablation, needle biopsy, and microrobot tracking and control of devices put into the body for navigation via the vascular system[3], [4], [5], [6]. The biocompatibility of these magnetic microrobots make them ideal for cellular manipulation [7], [8].

With advancements in fabrication methods such as glancing angle deposition [9], soft-lithography, and two-photon polymerization techniques [10], different shapes of microrobots have been synthesized based on specific applications. For example,helical-shaped spherical-shaped microrobots were made to obtain uniform rolling motion [11]. Another microrobot that was synthesized is a helical shaped microrobot that can be propelled with the help of a bacterial flagella [12] and penetrate through biological tissue [13]. A cylindrical microrobot of rolling motion was also proposed in [14], [15], [16]. Although many magnetic microrobots have been used for cell manipulation [17], [18], typically their design lacks specific features that optimize their functionality for manipulation specifically.

Cell manipulation is important for various fields like tissue engineering and synthetic biology [19]. The use of microrobots offers a distinct advantage for cellular manipulation due to their flexibility, adaptability, biodegradability, and the possibility of intracellular delivery [17], [20]. Cell patterning involves manipulating cells into predefined specific configurations on a surface. Microrobots facilitate cell patterning by positioning single cells or clusters of cells via remote control. By controlling the spatial organization of cells, it is possible to study single-cell behavior, cell-cell communications, tissue development, and disease mechanisms [21], [22]. To achieve precise cell manipulation, advanced control and navigation techniques are needed to ensure accurate localization and delivery methods. For example, closed-loop control algorithms allows for the automatic guidance of microrobots to desired locations [6]. A good control algorithm requires an accurate model of the microrobot motion which depends on individual microrobot design and micro-scale forces like surface tension, drag forces, and viscosity.

In this paper, we present a new microrobot design that can be controlled by a customized closed loop system to perform cell manipulation and cell delivery under a magnetic field. The design consists mainly of a cylindrical component with a helical cut sweep to provide better maneuverability and stability of motion. It also consists of ‘claws’ that can be used as grippers. The following sections discuss the design and fabrication, control of the microrobot and cellular manipulation using the corkscrew clawbot.

II. Methods

A. Design and Fabrication

The microrobot measures 70 microns in length, consisting of a cylindrical body at 40 microns and claws of 30 microns at the head with a 30-micron diameter. A helix sweep cut, 2.25 microns thick, is threaded on the cylinder’s surface to enhance maneuverability. The use of the claws can be helpful to perform tasks such as single cell manipulation, clot removal and drug delivery. The entire design mimics microdrillers with the claws at the front and the cylindrical part with helix cut providing the stability and motion. The microrobots are fabricated on a coverslip substrate using the Microlight 3D printer with OrmoBio photoresist, utilizing a 20x Objective in arrays through the two photon lithography technique. Prints undergo development by removing unpolymerized resin with OrmoDev developer for 10 minutes and then treated with an IPA solution for 2 minutes. Subsequently, a 100 nm - thick Ni layer is deposited perpendicular to the microrobot’s lateral surface using the Ebeam evaporation technique, resulting in microrobots with a half-coated layer. The Ni layer provides the microrobot with magnetic properties for actuation.

B. Magnetic Actuation

The actuation of the microrobot is primarily achieved using the magnetic fields generated by a customized Helmholtz coil system that generates a uniform magnetic field at the center of the system. The electromagnetic coil system generates the magnetic field by passing current in a sinusoidal fashion.

The components can be described as follows,

$${\mathit{B}}_{\mathit{x}}=-B\left[\cos \left(\gamma \right)\cos \left(\alpha \right)\cos \left(\omega t\right)+\sin \left(\alpha \right)\sin \left(\omega t\right)\right]$$ (1)

$${\mathit{B}}_{\mathit{y}}=-B[\cos (\gamma )\sin (\alpha )\cos (\omega t)-(\cos \left(\alpha \right)\sin \left(\omega t\right)\left)\right]$$ (2)

$${\mathit{B}}_{\mathit{z}}=B\sin \left(\gamma \right)\cos \left(\omega t\right)$$ (3)

where γ is the polar angle from the z axis, α is the azimuthal angle from the x axis, B is the magnetic field magnitude, and ω is the frequency of the field. The default γ is 90°, and changing α incrementally allows us to steer the microrobots when actuating under magnetic control.

The magnetic moment of an object under a magnetic field action is related to the torque T, given by

$$\mathit{T}=\mathit{M}\times \mathit{B},$$ (4)

where M is the Magnetic moment, and B is the Magnetic Field see Fig. 3(b).

Fig. 3.

a) Overview of the experimental setup b) Illustration of rolling motion of microrobot in deionized (DI) water on glass slide under magnetic field

C. Control System

To control the actuation of the microrobot a comprehensive custom-built control system primarily consisting of Arduino Mega 2560 for computational abilities, controlled power supply, and H-Bridge PWM Drivers for varying and controlling the direction of the current flow in the coil system are used. The Helmholtz coil system consists of 6 electromagnet coils wired with 260 turns of enamel coated copper wire to produce a magnetic field of 5 mT at the center of the workspace as in Fig. 3(a).

1). Open loop Control System:

The microrobots can be controlled with the help of a customized joystick to roll and steer on the surface of the glass slide. The open-loop control is primarily governed by moving the joystick in the desired direction to send a corresponding signal to the control system that varies the current flow in the coils. Subsequently, the direction of the magnetic field changes, resulting in the movement of the microrobot in the desired direction. The velocity of the microrobots is primarily due to the rotating magnetic field’s frequency.

2). Closed loop Control:

A closed-loop control is applied to demonstrate the ability of the microrobot to move along the specific path from point A to point B. The user defines the trajectory by drawing it on the screen. The algorithm provides a logical technique to guide the micro-robots along the trajectories defined by the user. It starts by analyzing a trajectory array that the camera extracted, which contains nodes with their corresponding coordinates in the Cartesian (x, y) plane. The algorithm iteratively computes the difference between the current node’s position and the desired position, then moves on to the next node if the error is less than a predefined threshold. When the inaccuracy exceeds the threshold, the algorithm calculates the magnetic field vector required to direct the micro-robot to the intended position, taking into account characteristics of the equations mentioned in 1,2,3.The algorithm computes the frequency at which the microrobot moves along the trajectory. By discretizing trajectories and generating appropriate magnetic field vectors, this algorithm offers a fundamental framework for achieving precise navigation of rolling micro-robots in 2D environment.

D. Cell Culture and Viability

Chinese Hamster Ovary (CHO) cells were used to demonstrate cell manipulation using the microrobots. Cells were maintained in Dulbecco’s Modified Essential Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, BenchStable, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Corning Life Sciences, USA) and 1% penicillin-streptomycin(Thermo Fisher Scientific, USA) at 37 °C and with 5% CO₂ in a humidified cell culture incubator. For the experiments, cells were used after their third passage, and cultured when they reached 90% confluency. In vitro, cytotoxicity was determined via trypan blue exclusion assay to directly identify live/dead cells in the cell suspension after microrobot treatment. CHO cells were cultured under standard cell culture conditions with the claw-shaped microrobots in a 35 mm cell culture dish (Thermo Fisher Scientific, USA) at a cell density of 3 × 10⁵ overnight. After incubation, the media was collected, centrifuged, and resuspended in Phosphate Buffer Saline (PBS) (Gibco, BenchStable, USA). The cell suspension was mixed with 0.4% trypan blue (Thermo Fisher Scientific, USA) in a 1:1 ratio, and live/dead cells were counted using a cell counter (Nexcelom Cellometer Vision Trio Cell Profiler, USA). Cell morphology after staining was observed under an optical microscope (ZOE Fluorescent Cell Imager, USA).

III. Results and Discussion

A. Microrobot Motion

The microrobot exhibits rolling motion along its longitudinal axis when subjected to a rotating magnetic field. A magnetic object subjected to an externally rotating magnetic field would be rotated due to the tendency of alignment between its internal magnetization and the field. This rolling motion of the microrobot is generated by the torque acting along its major axis. Although the primary mode of motion is rolling, instances of tumbling motion, i.e. rotation about its minor axis, are also observed. We believe that the tumbling motion of the microrobot indicates that the magnetic moment may not be entirely along the short axis of the microrobot and that hydrodynamic and/or electrostatic forces may influence the type of rotational motion as well. Our analysis reveals that the microrobot’s speed increases incrementally with an increase in the applied frequency, see Fig. 4. However, the decrease in the speed at some frequencies could be due to the hydrodynamic and electrostatic forces that can be predominant at the surface of the glass slide.

Fig. 4.

Average speed vs rolling frequency of the microrobots

B. Cell Transport

In this study, we used CHO cells to demonstrate single-cell manipulation via the claw-shaped microrobots. The supporting video shows a single CHO cell being transported by a single microrobot in its ’claws’ through rolling motion. As seen in the video, the microrobot approaches and picks up a single cell and moves to the user defined position. Then it releases the cell at the desired position by employing a small amount of fluid pressure. Following the successful delivery of a single cell, a cell pattern was created using the clawbots where the cells were arranged in a straight line. As shown in Fig. 5 and the supporting video, in total, 5 CHO cells were arranged to comprise a straight line in 150 seconds. The results indicate the versatility and controllability of the claw-shaped microrobots for single-cell delivery and patterning applications.

Fig. 5.

a,b,c) Illustration of single cell transport d,e,f) Frames showing the cell patterning into a line

C. Closed Loop Control

To demonstrate the ability of the microrobot to follow the above-mentioned algorithm a ”UD” shape and rectangle-shaped figures were drawn on the screen (see Fig. 6). For the rectangle-shaped path it took 10 seconds to complete the trajectory. The differences in the original path and the trajectory followed by the microrobot are minimal, while for the UD shaped path, it took 30 seconds with a maximum difference of about 30μm which is an acceptable limit since it is within the dimensions of the microrobot.

Fig. 6.

Overview of the closed-loop control implementation. a,b) Frames of the UD-shaped path and trajectory c) Comparison of the UD Shaped trajectory d,e) Frames of the rectangle-shaped path and trajectory c) Comparison of the rectangle trajectory

D. Cell Viability

In this study, we demonstrated the biocompatibility of our microrobots by determining the quantitative cell viability via trypan blue viability assay in CHO cells. Briefly, the dye can integrate into the cell membrane, and detect live (unstained) and dead (stained) cells in the cell population. In addition to the quantitative results, we were also able to visualize the cell morphology of the treated and untreated cells. Fig. 7 shows the microscopic images of CHO cells (10x magnification) after microrobot treatment along with the control group without any microrobots. Cell viability was 89% after 24 hours of incubation, and 90% for the control group. The assay indicated that the microrobots did not have any noticeable impact on the viability of the cells (see Fig 7c). In addition, the cell morphology and growth rate were similar indicating that these microrobots don’t have toxic effects on the cells, and are suitable for further biomedical applications.

Fig. 7.

Images of the microrobot-treated and untreated CHO cells immediately after (a, d) and 24 hours after treatment (b, e). Cell viability was determined as 89% (c) for treated, and 90% for untreated control CHO cells (f). Dead cells are stained and shown in red circles. CV represents the cell viability.

IV. Conclusion

In this work, we present a new microrobot design that is capable of performing cell transport and making cell patterns suitable for potential biomedical applications like tissue engineering and gene therapy. For single-cell delivery, various magnetically actuated microrobot designs have been reported. Peanut-shaped microrobots were designed to create a “HIT” pattern with NIH/3T3 cells [23], cup-like shaped microrobots were used to target and transport different types of cells such as neuron cells, mouse embryonic stem cells, and yeast cells [24]. However, our microrobots present a distinct advantage over these shapes due to the presence of the ‘claws’ which can function as a gripper. We also discuss closed-loop control of our corkscrew clawbots for precise navigation in 2D fluidic environments. In addition, we also investigated the magnetic actuation capabilities of the microrobot in deionized (DI) water, analyzing its speed response across varying frequencies under the influence of a magnetic field. These findings emphasize the versatility and effectiveness of our microrobot design for a range of biomedical applications. Further studies will explore the microrobot’s utility in clot removal and in vivo applications such as targeted drug delivery. Additionally, we will also analyze the motion of the microrobot in various biological fluids as well as implement the closed-loop system in a 3D environment.

Fig. 1.

a) Schematic showing printing of the microrobots on the coverslip using a two-photon lithography technique b) Overview of the printed microrobots on the substrate after development c) Illustration of the Ni layer coating on the microrobot d) SEM Image of the Ni-coated microrobot

Fig. 2.

Nonlinear Closed loop Trajectory Discretization Algorithm for Microrobots