nihms715840.pdf

3D Printed Artificial Micro-Fish

Wei Zhu

†

Jinxing Li

†

Yew J. Leong

Isaac Rozen

Xin Qu

Renfeng Dong

Zhiguang Wu

Wei Gao

Peter H. Chung

Joseph Wang

, and

Shaochen Chen

Department of NanoEngineering, University of California, San Diego, La Jolla, CA 92093 (USA)

Keywords

functional microswimmers; 3D microprinting; functionalized nanoparticles; hydrogel;

detoxification

To maneuver within their environment, aquatic organisms employ a variety of locomotive

strategies. These diverse mechanisms offer inspiration in designing artificial

microswimmers for applications ranging from directed drug delivery to accelerated

environmental decontamination.

[

–

]

One challenge in adapting naturally-evolved designs

for “smart” microswimmer systems lies in replicating their complex biomimetic form and

function. Here, using a rapid 3D microprinting platform – microscale continuous optical

printing (

COP) – we engineered hydrogel microfish featuring biomimetic structures,

locomotive capabilities, and functionalized nanoparticles. The

COP system can print

complex 3D structures within seconds at high resolution (~1 μm) across multiple orders of

magnitude in scale. The 3D-printed microfish exhibit propulsion that is highly-efficient,

chemically-powered, and magnetically-guidable. By incorporating polydiacetylene

nanoparticles, we demonstrate the microfish’s utility in toxin-neutralization applications.

The multiple capabilities integrated within these proof-of-concept microfish highlight the

technical flexibility and broad applicability of our approach in engineering advanced

functional bio-robotics for actuation, sensing, and detoxification.

With recent advances in nanoscience and nanomanufacturing technologies, the areas of

biomimetic micro-robotics and nanomotors have seen rapid development in realizing

functionalities mimicking natural organisms with self-propulsion.

[

–

]

Significant progress

has been made over the past decade towards the design and fabrication of micro-/

nanoswimmers with various locomotion mechanisms.

[

–

]

For instance, techniques for

creating rolled-up nanostructures have led to the fabrication of tubular microjet engines and

microdrillers.

[

–

]

Template-assisted electrochemical deposition has also been used to

synthesize high-performance tubular micro-rockets and nanowire motors.

[

]

These

microswimmers feature relatively simple spherical or cylindrical geometries, indicating the

limitation of these techniques in creating complex 3D designs to mimic the sophisticated

structure and function of their biological counterparts. Moreover, most microswimmers are

Correspondence to: josephwang@ucsd.edu and chen168@eng.ucsd.edu.

†

These authors contributed equally to this work.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

HHS Public Access

Author manuscript

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composed of homogeneous inorganic materials and thus lack the capability of multiplexing

sophisticated functionalities. While laser direct-writing enables the fabrication of

microswimmers with more sophisticated 3D designs, such as helical structures used for

targeted cargo transport,

[

]

the technique offers limited scalability due to the serial nature of

its raster-scanning process, which is prohibitively time-consuming for large batch

fabrication. Molding approaches have been used to create biomimetic polydimethylsiloxane

(PDMS) microswimmers, propelled by either external magnetic fields or contractile

cardiomyocytes.

[

–

]

However, the ferromagnetic materials or contractile cells must be

post-deposited or seeded after the PDMS molding process, preventing more complex

functionalization of the microswimmers. In addition, the near-millimeter size of these

PDMS swimmers limits their applicability in micro- or nano-motion studies. The capability

to fabricate complex architectures and miniaturize the dimension is highly desired for

designing and customizing more functionalized, integrated and intelligent micromachines

for different applications as well as investigating the fundamental aspects of microscale

locomotion.

[

]

In light of these challenges, rapid 3D optical printing offers a promising

alternative for efficiently manufacturing controllable microswimmers with complex 3D

microscale structures composed of patterned heterogeneous materials as well as different

functional components.

[

–

]

In this work, we have developed a rapid 3D printing technology - microscale continuous

optical printing (

COP) - to engineer functionalized artificial microfish with diverse

biomimetic structures and locomotive capabilities. The

COP technology offers efficient

fabrication speeds, provides improved feature resolution, and requires no harsh chemicals

for fabrication. To 3D print artificial microfish with high-fidelity shapes and structures in an

economical and scalable manner, we optimized this

COP system to construct freely

swimming microfish composed of polyethylene glycol diacrylate (PEGDA)-based hydrogels

and functional nanoparticles as a proof of concept. By extending the μCOP technology to

print advanced materials that incorporate three different types of functional nanoparticles –

platinum (Pt) nanoparticles for chemically-mediated propulsion, iron oxide (Fe

)

nanoparticles for magnetic guidance, and polydiacetylene (PDA) nanoparticles for toxin

neutralization – we can create highly functionalized microfish using an approach that can be

more broadly applied to engineering complex biomimetic micromotors with diverse custom-

designed functions.

The setup of the

COP platform is schematically illustrated in Figure 1a. The key

component of the

COP system is a digital micromirror device (DMD) chip composed of

approximately two million micromirrors for continuous optical pattern generation. The

DMD chip modulates the UV light and projects the optical pattern – dictated by the custom

designed computer-aided design (CAD) model – onto the photopolymer solution. The 3D

model can be defined using commercially available CAD software and sliced into a series of

digital images. Through a computer interface coupled with in-house developed software,

these images are automatically and continuously loaded one-by-one onto the DMD chip and

then projected onto the photopolymerizable materials, which are loaded on a motorized

stage. Areas illuminated by UV light crosslink within seconds, while leaving the dark

regions uncrosslinked, forming a patterned layer in a specific polymerization plane. By

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iterating through the sequence of optical patterns in continuous synchrony with the

movement of the sample stage during the required UV exposure time, we can construct

polymer scaffolds with complex structures and submicron feature resolutions. PEGDA, a

biocompatible hydrogel that has been widely used in medical implants, drug delivery

devices, and tissue culture, was chosen as the photopolymerizable matrix material for the

fabrication of the fish body and the encapsulation of functional nanoparticles.

Optimizing the swimming performance of microswimmers requires precise control over

their shape and size. Our

COP platform is capable of building a wide array of complex 3D

geometries, allowing for efficient iteration through various microswimmer designs. Here we

chose to print microfish of multiple biomimetic forms to demonstrate the potential for using

this approach to systematically refine the microswimmer design to optimize its swimming

performance. With this digitized and continuous optical printing technique, we are able to

fabricate a large array of 3D microfish within mere seconds. In order to characterize the 3D

morphology of the printed microfish, a 3D observation digital microscope (Keyence

VHX1000) is used to capture the images of the microfish array from bottom to top at a step-

size of 1 μm and to subsequently construct a 3D microscopy image that represents the height

profile (Figure 1b). The thickness of the microfish is approximately 30 μm and the length of

the microfish is 120 μm. These parameters can be readily varied during the 3D fabrication

process by changing the digital masks and layer settings.

The digitized nature of the

COP technique enables us to modulate the parameters as well as

the designs of the microfish conveniently (Figure 2). Figure 2a illustrates that large arrays of

microfish can be printed with excellent uniformity and precision. Multiple sizes (Figure 2b)

and shapes (Figure 2c–h) of microfish can be produced on the same substrate, with the size

of the microfish tunable from micrometer to nearly centimeter scale. Furthermore, the

PEGDA material comprising the microfish bodies can be substituted for other

photopolymerizable polymers as appropriate for different functions and applications.

Importantly, the flexibility of this technique enables us to pattern or embed different

materials at specific locations for custom-designed functions. For example, we are able to

fabricate a microfish with magnetic Fe

nanoparticles at the head and catalytic Pt

nanoparticles at the tail (Figure 3). With these two types of functional nanoparticles, the

swimming direction and speed of the microfish can be controlled readily.

The fabrication process of the chemically-powered and magnetically-guided microfish is

illustrated step by step in Figure 3a. First of all, the hydrogel body of the fish is fabricated

with photopolymerizable solutions such as PEGDA in this particular case. Secondly, the

solution is replaced with PEGDA containing catalytic Pt nanoparticles and only the tail of

the microfish is polymerized to encapsulate the Pt nanoparticles, which enables efficient

self-propulsion via decomposition of the peroxide fuel. Thirdly, the solution is replaced with

PEGDA containing magnetic Fe

nanoparticles and only the fish head is polymerized to

encapsulate the magnetic Fe

nanoparticles, which guide the motion of the microfish.

Energy-dispersive X-ray (EDX) spectroscopy images show the successful functionalization

of the microswimmers with the magnetic control and catalytic patch segments at different

regions (Figure 3b), demonstrating the capability of the

COP system to localize different

functional nanoparticles at specific positions with high accuracy. Other nanoparticles can be

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further patterned on the microfish to achieve greater multiplexing and more complex

functionalities.

Pt nanoparticle encapsulation allows for efficient propulsion of the microfish, as illustrated

in Figure 4. Hydrogen peroxide was used as the fuel source. The track lines of microfish

motion, shown in Figure 4a and 4b and corresponding to Supplementary Movie 1 and 2,

illustrate the thrust created by bubbles generated during peroxide decomposition at the tail

ends of the fish, demonstrating the efficacy of the site-specific catalytic nanoparticle

loading. The catalytically-generated oxygen bubbles expelled from microfish tail propel the

microfish while fast and high-frequency bubble ejection leads to continuous motion of the

microfish. Fundamentally, the propulsion force increases with the rate of the catalytic

reaction that leads to faster bubble generation.

[

]

Therefore, increased peroxide

concentration and loading of the Pt catalyst both lead to faster oxygen bubble generation for

a more efficient propulsion. The microfish are able to achieve a speed of 780 μm s

−1

in a 15

% peroxide solution. The Stokes’s drag force is highly related to the geometry of the

microstructures.

[

]

Different fish geometries result in different speeds under identical fuel

concentrations, owing to different Stokes’ drag at such small scales. In Figure 4c, Fish 1 and

Fish 2 are both loaded with the same concentration of Pt nanoparticles, while Fish 1 has a

shape of common fish and Fish 2 has shape of manta ray. The average speed of Fish 1 is 1.5

times higher than that of Fish 2, indicating that streamline geometry of microfish 1 can

reduce drag forces for faster locomotion compared to the micro manta rays (Figure 4c). In

general, our results indicate that the shape of the microfish can be optimized to achieve

higher speeds. The speed can also be readily tuned by tailoring the loaded concentration of

the catalytic Pt nanoparticles. In Figure 4c, Fish 1 and Fish 3 have the same shape of

common fish, yet Fish 1 is loaded with twice the amount of Pt nanoparticles than that of

Fish 3, yielding a higher speed under the same fuel concentrations.

The encapsulation of Fe

nanoparticles also allows for magnetic guidance. Such

encapsulation of magnetic nanoparticles within the fish head provides a net magnetization,

thus enabling magnetic alignment and guidance by a remote magnet during the chemical

propulsion. The time-lapse images in Figure 4d–f, corresponding to Supplementary Movie

3, illustrate remote guidance of the microfish achieved by rotating a nearby magnet. The

swimming microfish exhibits rapid alignment to the external magnetic field. Combined, the

nanoparticle encapsulation methods can readily convert these polymeric microfish into

efficient and controllable microswimmers for diverse applications.

To explore a potential application of the 3D printed microfish, we further incorporated

functional toxin-neutralizing nanoparticles into the hydrogel matrix of the fish body as a

proof-of-concept demonstration of its use in a detoxification context. Specifically, we used

PDA nanoparticles, made from the self-assembly of

10,12

-pentacosadiynoic acid (PCDA),

for the detoxification of melittin.

[

]

The PDA nanoparticles have the structure of

nanovesicles, with the surface made of a

-conjugated polymer. This unique surface

structure can be used to mimic the biological membrane of a cell to effectively capture and

neutralize pore-forming toxins via binding interactions between the nanoparticles and the

toxins. The bound toxins will disrupt the ordered

-conjugated chain structure of the PDA

nanoparticle surface, leading to fluorescence emission, which enables us to use these

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particles as toxin sensors as well. Change of the fluorescence intensity can serve as a good

indicator of the detoxification efficiency during the experiment.

To evaluate the detoxification capability of the PDA nanoparticle enhanced microfish, we

designed three test groups (Figure 5). In the control group, we incubated these

functionalized microfish with 5% H

solution at 37 °C for 10 mins, and no visible

fluorescence was detected (Figure 5a). In the second group, we incubated the microfish in a

5% H

solution containing 2.5 mg/ml melittin at 37 °C for 10 mins. No Pt nanoparticles

were incorporated into this group, so the microfish were stationary during the incubation

period. As shown in Figure 5b, a low intensity of fluorescence was observed. Comparing

these two groups, we confirmed that the interaction between the melittin toxin and the PDA

nanoparticles led to the red fluorescence emission. In the third group, Pt nanoparticles were

incorporated within the tails of the functionalized microfish to make them mobile when

exposed to the peroxide solution. These microfish were also incubated in a 5% H

solution containing 2.5 mg ml

−1

melittin at 37 °C for 10 mins. Figure 5c indicates that the

motion of the microfish significantly enhanced the interaction between the melittin toxin and

the PDA nanoparticles encapsulated in the microfish. The fluorescence intensity was further

quantified to compare the detoxification efficacy (Figure 5d) using ImageJ, as previously

described.

[

]

Statistical analysis was performed to compare sample means by ANOVA

followed by Tukey’s post-hoc test, and

< 0.05 was considered statistically significant.

These data indicate that there is a significant difference between the mobile and stationary

microfish, highlighting the importance of the microswimmer movement for enhancing

detoxification process.

The natural locomotion of aquatic and airborne species within fluids has motivated scientists

and engineers who aim to develop devices and robotics with efficient locomotive properties

for use in transportation, delivery and other applications.

[

]

Engineering nature-inspired

structures and functionalities requires hierarchical design over several orders of magnitude,

both in spatial and temporal domains. Biomimetic microswimmers have emerged over the

past decade with the potential to be useful in biomedical applications or as rheological

probes

in vivo

. Manufacturing such microswimmers with optimal design and swimming

capacity is a critical step towards achieving these goals. We have demonstrated as proof of

concept that functional microfish can be created by patterning biocompatible hydrogels with

functionalized nanoparticles using our advanced 3D printing technology. With the

COP

system, diverse and complex 3D microswimmer designs can be rapidly fabricated in large,

uniform arrays, demonstrating the enhanced scalability and high-throughput capacity of our

approach when compared to conventional strategies. Biocompatible hydrogels are

specifically chosen as the matrix material to encapsulate functional nanoparticles due to

their wide use in medical implants, drug delivery devices, and tissue culture. PEG-based

hydrogels, in particular, have proven extremely versatile for tissue engineering applications

since they exhibit high biocompatibility and little or no immunogenicity. In addition, PEG-

based hydrogels display tunable mechanical properties over a wide range for various tissues/

organs. Importantly for patterning applications, PEG-based hydrogels are intrinsically

resistant to protein adsorption and cell adhesion, thus providing biological “blank slates”

upon which desired biofunctionality can be built.

[

]

Last but not least, in the present

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work of microswimmers, PEGDA has demonstrated favorable stability of maintaining the

fabricated shapes of the microfish as well as the encapsulation of nanoparticles over an

extensive time period. The microfish can maintain the propulsion capability after more than

two hours (longest swimming time tested) of swimming in the peroxide solution, and after

the fabrication, they can be stored at room temperature for over one week before use.

The systematic engineering method reported here provides a general strategy for designing

nature-inspired swimmers in fluidic environments. Beyond the example of microfish shown

here, the digitized nature of the 3D

COP system can be applied to fabricate virtually any

other designs to optimize the locomotive performance of the microswimmers. With the

COP system, we have demonstrated in the previous work that we can readily alternate the

designs and fabricate a variety of structures with different materials encapsulating

nanoparticles, biochemical molecules and even living cells.

[

]

Applying this

approach to the micromotor field, we have the capability to achieve diverse 3D

microswimmer designs, ranging from simple cylindrical or tubular geometries to complex

biomimetic architectures. We integrated platinum and iron oxide nanoparticles for guided

chemical propulsion in hydrogen peroxide fuel, but other catalytic components, such as Ag,

Ir, MnO

or catalase, can also be integrated using the same approach for use with different

chemical fuels.

[

–

]

Alternatively, new 3D printed micromachines that can harvest energy

from their own surrounding environment, i.e., use a biological fluid or water as their fuel

source, are also expected to be developed by our current manufacturing platform for diverse

functionalities.

[

]

Furthermore, as a proof-of-concept, we integrated PDA nanoparticles

into the microfish and realized an improved detoxification platform as well as a real-time

sensing tool. This strategy can be readily extended to incorporate multiple other functional

nanoparticles into a fully-integrated microswimmer system as a powerful platform for

applications including but not limited to drug delivery, personal therapeutics, and

environmental conservation.

Experimental Section

3D printing of microfish

PEGDA (MW = 700 Da) was purchased from Sigma (USA). Photoinitiator lithium

phenyl-

2,4,6

-trimethylbenzoylphosphinate was synthesized according to the previous

work.

[

]

PEGDA (40 wt%) in H

O with lithium phenyl-

2,4,6

-trimethylbenzoylphosphinate

(1 wt%) was used as the matrix material. For the head of the microfish, magnetic Fe

nanoparticles were diluted in the same matrix material with a concentration of 5 mg/ml; for

the tail, catalytic Pt nanoparticles were diluted in the same matrix material with two different

concentrations of 8.0×10

Pt particles per ml and 4.0×10

Pt particles per ml to evaluate the

effect of Pt concentration on the swimming speed. For detoxification, PDA nanoparticles (3

mg ml

−1

) were loaded to the matrix material for the body of the microfish. The DMD chip

used in the set-up is DLP-07 XGA from Texas Instruments.

Preparation of PDA nanoparticles

Polydiacetylene (PDA) nanoparticles were prepared according to previous work.

[

]

10,12

pentacosadiynoic acid (PCDA) was purchased from Sigma (USA). PCDA was dissolved in

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dichloromethane and then filtered for purifying purpose. Dichloromethane was evaporated

by heating after the filtration. Distilled water (6 ml) was added to purified PCDA (36mg),

followed by probe sonication for 5 mins at ~80 °C. Then the self-assembled PDA

nanoparticle solution was refrigerated at 4 °C overnight for stabilization. Finally, before 3D

printing, the PDA nanoparticle solution was irradiated with ultraviolet light (285 nm) for 10

mins.

Characterization and propulsion experiments

The 3D microscopy images with height profile were obtained on microscope Keyence

VHX1000. High-resolution electroscope microscopy images and EDX spectroscopy images

were obtained on Phillips XL30 ESEM. In order to get self-propelled microfish, aqueous

solutions with concentrations ranging from 5% to 15% were used as chemical fuels.

Videos were captured by an inverted optical microscope (Nikon Instrument Inc. Ti-S/L100),

coupled with 10× and 4× objectives and a Hamamatsu digital camera C11440, using the NIS

Elements AR 3.2 software. The speed of the microfish was obtained by tracking and

analyzing their motion with the NIS Elements AR 3.2 software.

Detoxification experiments

Detoxification of melittin by the microfish was performed by loading the microfish to 100 μl

of different solutions at 37 °C for 10 mins. For Figure 5a, the solution was 5% H

without

melittin toxin; for Figure 5b and 5c, the solution was the same with melittin (2.5 mg ml

−1

) in

5% H

. The microfish in Figure 5b were not encapsulated with Pt nanoparticles to keep

them stationary (i.e., no movement). The detoxification efficiency was evaluated by

fluorescence microscopy imaging of the microfish after the neutralization process. ImageJ

(developed by National Institute of Health) was used to measure the relative fluorescence

intensity according to the previous work.

[

]

The relative fluorescence intensity is calculated

according to equation (1). Both the integrated density and area can be measured directly

with ImageJ from the fluorescence microscopy images. Data are reported as mean ±

standard deviation. Comparison of sample means was performed by ANOVA followed by

Tukey’s post-hoc test (Origin software),

< 0.05 was considered statistically significant.

(1)

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This project received support from the Defense Threat Reduction Agency-Joint Science and Technology Office for

Chemical and Biological Defense (Grants no. HDTRA1-14-1-0064 and HDTRA1-13-1-0002), the National Science

Foundation (Grants no. CMMI-1120795, CMMI-1332681) and National Institutes of Health (Grant no. EB012597,

EB017876). R.D. and Z.W. acknowledge the China Scholarship Council (CSC) for the financial support. We thank

Michael Galarnyk and John Warner for their assistance.

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Figure 1.

(a) Schematic illustration of the

COP method to fabricate microfish. UV light illuminates

the DMD mirrors, generating an optical pattern specified by the control computer. The

pattern is projected through optics onto the photosensitive monomer solution to fabricate the

fish layer-by-layer. (b) 3D microscopy image of an array of printed microfish. Scale bar, 100

μm.

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Figure 2.

Assorted SEM images of the microfish. (a) A uniform array of microfish. (b) Microfish of

various sizes and (c–h) fabricated fish of different designs: fish, shark, and manta ray,

respectively. Scale bar, 50 μm.

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Figure 3.

(a) Schematic illustration of the process of functionalizing a microfish for guided catalytic

propulsion. Pt nanoparticles are first loaded into the tail of the fish for propulsion via

catalytic decomposition of H

. Secondly, Fe

nanoparticles are loaded into the head of

the fish for magnetic control. (b) EDX spectroscopy images illustrating the iron-oxide head

and platinum tail with respect to the PEGDA microfish body. Scale bar, 50 μm.

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Figure 4.

Track lines of motion of (a) a microfish and (b) a micro manta ray, over 3 seconds in the

presence of 10 % hydrogen peroxide (highlighted from Supplementary Movie 1 and 2). (c)

Speed profiles of microfish with different shape designs and different Pt nanoparticle

concentrations at 5, 10, and 15% H

. Fish 1: common fish with 8.0×10

Pt particles per

ml; Fish 2: manta ray with 8.0×10

Pt particles per ml; Fish 3: common fish with 4.0×10

particles per ml. (d–f) Time-lapse images of the microfish movement in 15% H

performing sharp turns with magnetic guidance (Supplementary Movie 3). Scale bar, 100

μm.

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Figure 5.

Fluorescent images demonstrating the detoxification capability of the microfish containing

encapsulated PDA nanoparticles. (a) Control group of microfish incubated in 5% H

without melittin toxin. (b) Stationary microfish incubated in 2.5 mg ml

−1

melittin solution

(5% H

). (c) Mobile microfish incubated in 2.5 mg/ml melittin solution (5% H

). (d)

Relative fluorescence intensity measurements indicating the amount of melittin absorbed by

the microfish. Statistical analysis indicates that there is significant difference between any

two conditions among control, stationary or mobile,

<0.05.

Zhu et al.

Page 14

Adv Mater

. Author manuscript; available in PMC 2016 December 29.

Author Manuscript