Letter Vol. 45, No. 5 / 1 March 2020 / Optics Letters 1071

Hybrid femtosecond laser fabrication of asize-tunable microtrap chip with a high-trappingretention rateBing Xu,1 Shengyun Ji,1 Deng Pan,1 Wenjin Hu,1 Suwan Zhu,1 Yanlei Hu,1

Jiawen Li,1,3 Dong Wu,1,4 Jiaru Chu,1 AND Koji Sugioka2

1CASKey Laboratory ofMechanical Behavior andDesign ofMaterials, Department of PrecisionMachinery and Precision Instrumentation,University of Science and Technology of China, Hefei 230026, China2RIKENCenter for Advanced Photonics, 2-1 Hirosawa,Wako, Saitama 351-0198, Japan3e-mail: jwl@ustc.edu.cn4e-mail: dongwu@ustc.edu.cn

Received 16 December 2019; revised 13 January 2020; accepted 14 January 2020; posted 14 January 2020 (Doc. ID 386095);published 18 February 2020

In this Letter, we propose a new (to the best of our knowl-edge), promising concept of a hybrid femtosecond (fs)laser processing method composed of single-point scan-ning and holographic light modulation fabrication formanufacturing a tunable-size microtrap chip. The hybridmethod not only ensures key microfluidic device precisionbut also greatly improves the fabrication speed. By using anew asymmetry-bracket-shaped microtrap design with amechanical strain stretching method, real-time size-tunabletrapping is obtained, and a 100% particle trapping retentionis realized, ignoring the flow fluctuation. Finally, the micro-trap array is successfully applied to trap single yeast cellsand hold them for ∼ 10 h without escaping. © 2020 OpticalSociety of America

https://doi.org/10.1364/OL.386095

In recent years, a promising fabrication method, fem-tosecond (fs) laser two-photon polymerization (TPP), hasbeen developed to realize fantastic three-dimensional (3D)micro/nano-structures due to its distinct advantages, includingprogrammable designability, true 3D processing capability,and high spatial resolution [1–3]. TPP has been successfullyused to fabricate microbulls, micro-chains, and micro-needleson the surface [1]. For a lab-on-a-chip application, a variety ofmicro/nano-structures such as micromixers [4], microfilters[5], and microlens arrays [6] were also flexibly integrated insidea microfluidic chip for mixing of fluids, filtering of particles,and cell counting, respectively. However, due to its single-pointwriting scheme, the TPP method is limited by low processingefficiency. To improve the processing efficiency, several meth-ods have been developed such as multibeam interference [7],diffractive optical elements [8], and spatial light modulationtechnology [9]. In particular, the spatial light modulationtechnology has been widely used, as it can flexibly modulatethe laser beam into various light fields, including multifocusspots [10,11], flat-top beams [12], vortex beams [13], and

Bessel beams [14]. However, for complex microfluidic devicefabrication (e.g., microrotor, microvalve), the light modulationtechnology was not competent (e.g., failed to fabricate or thestructure was too rough).

In addition, the functions of microfluidic chips fabricatedby TPP are still limited to liquid mixing, cell filtering, and soon [4–6]. More high-functioning microchips are needed toachieve better applications, for example, single-cell analysis tostudy cellular heterogeneity at the single-cell level. Microfluidichydrodynamic trap arrays [15,16] have been widely utilized toseparate the cells of interest into individual ones for single-cellanalysis due to simple manipulation, high-density cell arrays,and high cell viability. The operation principle is that cells arecaptured randomly after infusing them into a microchip, whilecells (with polydisperse size distributions) with key informationmay be missed because of a lower cell-capture retention rateinduced by unmodified rigid feature sizes or shapes of the micro-trap. Therefore, a soft or size-tunable microtrap with bettertrapping performance is required.

To address the above issues, we demonstrate a hybrid fs-laserprocessing method, which is composed of single-point writ-ing and holographic light modulation scanning for efficientlyenhancing the fabrication speed. Single-point fs-laser writ-ing with high precision is utilized to fabricate the functionalmicrotrap structures and the liquid flow unit: a microchannelwith relatively low precision is fabricated by holographic multi-exposure technology. With the help of this hybrid method,the processing time can be reduced to ∼ 1 h, while the timeis ∼ 17 h by conventional single-point scanning. To achievetunable-size trapping, a new concept of stretchable asymmetry-bracket-shaped microtrap structure (SAMS) is proposed toachieve size-tunable trapping and a 100% particle-trappingretention rate. In the preliminary single-cell experiments, yeastcells are trapped and cultivated for ∼ 10 h without escaping.

A hybrid fs-laser fabrication technology is presented inFigs. 1(a) and 1(b). Single-point scanning is applied to fabricate

0146-9592/20/051071-04 Journal © 2020 Optical Society of America

1072 Vol. 45, No. 5 / 1March 2020 /Optics Letters Letter

the more precise part of the microfluidic chip, here the micro-trap array structures [Fig. 1(a)]. First, commercially availablezirconium silicon hybrid sol-gel material SZ-2080 (providedby IESL-FORTH, Greece) was mixed in acetone with a volumeratio of 1:6. Then a 10 µL drop of the resist was dipped on acover glass surface resulting in the resist thickness of ∼ 10 µm.After 20 min heating, the resulting resist was moved to the laserprocess system (Chameleon Vision-S; Coherent, central wave-length of 800 nm, repetition rate of 80 MHz, and pulse durationof 75 fs) for inducing TPP. To reduce the processing time of themicrochannel, a holographic fs-laser modulation method [12]was applied [Fig. 1(b)]. First, the fs laser passed a beam colli-mation system and a 4f configuration; then a 60× microscopeobjective lens with numerical aperture (NA) of 1.35 was usedto focus the phase-modulated fs-laser beam into the sample.Multiple computer generated holograms (CGHs) [here, 20CGHs] were loaded onto a reflection type liquid crystal spatiallight modulator (SLM, Pluto NIR II, Holoeye, 1920 × 1080pixels, pixel pitch of 8 µm, and diagonal of 1.78 cm) to con-tinuously generate a large rectangle [10 µm length and 10 µmwidth, Fig. 1(b)] light field. The refresh rate of the CGHs was20 Hz in our experiments. The patterns were shifted from thecenter by loading blazed grating on the SLM, and a high-passfilter is placed at the focal plane to block the 0 order beam. Inthis hybrid process, the scanning pitch was 300 nm and 2.5µm,respectively, for the single-point process and holographicprocess [Fig. 1(f )]. The laser power is 15 mW for single-pointdirect-writing and 300 mW for the laser beam shaping method.The scanning speed is 0.50 and 0.01 mm/s, respectively, for theholographic process and for single-point scanning. The process-ing time for the whole microchannel mold was about 1 h bythe hybrid fs-laser fabrication method, while it was about 17 hwhen using single-point writing [Fig. 1(f )]. This proves that thehybrid fabrication method has great potential in high-efficiencymicrofluidic chip preparation. From the SEM and microscopeimages, the resulting mold [Figs. 1(c)–1(e)] has good precisionand a high surface quality. After the sample was developed in1-propanol for 30 min, the microfluidic chip was realized by softlithography using polydimethylsiloxane (PDMS) [Sylgard 184,Dow Corning]. Specifically, we spin-coated the PDMS mixtureat a speed of 200 r/min for 1 min, which formed the PDMS filmwith a thickness of 300µm. In order to easily peel off the PDMSmicrochannel, the mold was soaked in the 1 H,1 H,2 H,2 H-perfluorodecyltriethoxysilane (PFDTES) for 12 h. AnotherPDMS sheet (∼ 200 µm thickness) with one inlet and outletwas also prepared. The PDMS microchannel was then bondedto the PDMS sheet [Fig. 1(h)] after exposing both surfaces tooxygen plasma (Mingheng PDC-MG).

The rapid holographic light modulation shapes a singleGaussian distributed beam into a rectangle focus, but it resultsin speckle noise [12,17] which leads the fabricated rectangleto present several flawed and discontinued structures (Fig. 2).The simulated intensity distribution of one CGH at the focalplane of the objective lens also proves the existence of specklenoise [Fig. 2(a)]. CGHs calculated by different initial phaseslead to different distributions of speckle noise. To improvethe optical uniformity, we use multi-CGHs calculated by aweighted Gerchberg–Saxton algorithm [12] with differentinitial phases to average the speckle noise [Fig. 2(a)]. The opticalintensity distribution of these CGHs contains the same designpattern (10 µm × 10 µm). The optical uniformity increases

Fig. 1. Hybrid fs-laser fabrication. (a) Schematic diagram ofthe single-point direct-writing. (b) Holographic light modulationscanning; the inset images are the light fields by simulation andmeasurement. The scale bar is 5 µm in (a) and 20 µm in (b). (c) and(d) SEM images of the fabricated microtrap array and microchannel,respectively; (e) resulting microchip and (f ) contrast of processing timeby single-point writing and the hybrid process.

as the number of CGHs increases [Fig. 2(a)]. For example,the optical uniformity is only 0.55 for 1 CGHs while, for 20CGHs, it is 0.87 [Fig. 2(a)]. The higher optical uniformitycan produce fewer flawed microstructures. Thus, more CGHsresult in better optical uniformity and produce more precisemicrostructures. In addition, with the help of scanning overlap,the resulting microstructures are smoother by eliminating theflawed microstructures [Fig. 2(b)]. Meanwhile, the middle partof the microstructures is more precise than the edge part due tothe former scanning with more overlaps [Fig. 2(c)]. To betterunderstand the holographic scanning, we have investigatedthe scanning parameters, including the laser power, scanningspeed, scanning pitch (overlap level), and refresh rate of theSLM. In our experiments, a 40 µm × 40 µm rectangle is shownas an example. Lower laser power and faster speed result in alower surface quality. The suitable scanning parameters (powerand speed) with a good surface quality are labeled [Fig. 2(c)].The scanning pitch is another key parameter which affectsthe surface precision greatly. 2.5 (overlap of 75%) and 5.0 µm(overlap of 50%) pitches lead to a smooth surface while 7.5pitch (overlap of 25%) and a larger one (e.g., 10.0 µm with 0%overlap) produce a rough microstructure. Here a 2.5 µm pitchis chosen due to better precision and an acceptable process time(∼ 2.7 min). The refresh rate of the SLM is the loading time ofone CGH. For example, a refresh rate of 20 Hz indicates thateach CGH remains on the SLM for 50 ms. In our experiments,all the refresh rates from 5 to 30 Hz lead to a good surface quality[Fig. 2(d)]. Combining the holographic light modulation tech-nique with high overlap scanning, the flawed and discontinuedmicrostructures are reduced largely.

To improve the trapping retention rate of the target cellsand achieve size-tunable trap, here we propose a new conceptdesign of a stretchable SAMS. In a conventional microtrap lab-on-a-chip (LoC) system [15], a half-closed trap structure [e.g.,“U”-shaped trap structure; see Fig. 3(a)] with a similar size to thecells was designed and fabricated. It suffered from cell loss [18]because of the flow perturbations (flow can act as a virtual closedgate) induced by the liquid injection system [Fig. 3(a)]. Ourdesign presented a “quasi-full-closed” trap structure [Fig. 3(b)]without cell loss in the whole flow process. The differencebetween our design and the conventional one is that the latter

Letter Vol. 45, No. 5 / 1March 2020 /Optics Letters 1073

Fig. 2. Parameter investigation for better microstructure pre-cision. (a) Simulated optical field for different numbers of CGHs.(b) Schematic diagram of the holographic scanning with differentscanning overlaps. Using a smaller scanning overlap, the flawed anddiscontinuously microstructures can be eliminated effectively, whichproduces a smoother rectangle. (c) and (d) Investigation of the rela-tionship between the surface quality and experimental parameters[(c) laser power and scanning speed; (d) scanning pitch, number ofCGHs, and refresh rate of the SLM]. The checkmarks represent thesuitable fabrication parameters.

is like a bandpass filter with a front gap that is larger than theparticles and a back gap that is smaller, while our design hastwo gap sizes which are both smaller than the target particles[Figs. 3(a) and 3(b)]. To characterize the tunable performanceof the SAMS, 5.0 and 2.5 µm microbeads with a mixing rate of1:1 were chosen as the test particles. 2.5 µm microbeads werefirst trapped inside the SAMS, and 5.0µm microbeads flowed tothe outlet [Figs. 3(c) and 3(d)]. After stretching the microchip,the size of both the front and back gaps gradually increased untilthe back gap increased to 3.0 µm which is larger than the sizeof the smaller particle; the 2.5 µm beads were released from thetraps, inducing the microtrap resetting [Fig. 3(d)]. By furtherincreasing the stretching rate, the front gap size enlarged to 6µm(>5 µm); meanwhile, the back one was still smaller than 5.0µm(e.g., 4 µm). Then 5.0 µm microbeads entered the traps andwere confined [Fig. 3(d)]. Figure 3(d) shows the entire flowingprocess. From the optical microscopic images, it was clear thatall small particles flowed away, meaning that the trap array had agood capability of resetting (the resetting efficiency is ∼100%).

Meanwhile, the SAMS can be flexibly stretched to differentsizes by different mechanical forces. Figure 4(a) showed thelinear dependence between the microchip elongation and theresulting gap size. The stretching repeatability of the microchipwas also tested, as shown in Fig. 4(b). Five repeated cycles weresuccessively applied to stretch and release the microchip. Thisshowed the robustness of the trapping-size tunability.

The front gap size is enlarged from 4.4 to 6.2 µm in thestretching process, and the back gap is enlarged from 2.2 to4.0 µm. Additionally, the front gap size is reduced from 6.2 to4.4 µm in the releasing process, and the back gap is reducedfrom 4.0 to 2.2 µm. Usually, the stretch rate in a PDMS sheet isthe same while, in this experiment, the front gap (stretch rate,1.4) and the back gap (stretch rate, 1.8) have different stretch

Fig. 3. (a) and (b) show the trap performance contrast; the newquasi-full-closed trap can completely capture the target cells withoutmissing; (c) and (d) demonstrate the tunable-size trap. The scale bar in(d) is 20µm.

Fig. 4. Tunable trapping performance. (a) Linear dependencebetween the microchip elongation and the resulting gap size, (b) thegood stretching repeatability of the trap, (c) percentages of the trappedparticles, and (d) trap occupancy of the two particles.

rates. This is because the microtraps are relief microstructuresand can be treated as rigid structures. It is believed that the rigidmicrotraps [Fig. 4(d)] induce different stretching rates. Wealso characterized the capability of trapping single particles, asshown in Fig. 4(c). The performance of trapping single particlesis similar to the traditional “U” shaped traps [15] in which thenumber of particles captured varied from none to one, to multi-ple. Single-trap (only one particle captured) efficiency is ∼ 50%for 2.5 µm particles and ∼ 40% for 5.0 µm particle. The trapoccupancy, which was defined as the ratio of the number of trapscontaining particles to the total number, was characterized. Itwas about 78% for the 2.5µm particles and 73% for the 5.0µmparticles. In fact, the trap occupancy could be enhanced if weinjected the samples for a longer time.

To study the trapping retention performance of this SAMS,5.0 µm beads were chosen to be the target. At first, the SAMS(front gap, 4.4 µm; back gap, 2.2 µm) was stretched to capture5 µm particles. Then it was recovered to its original size byreleasing the mechanical strain. A positive flow was applied totest the escaping possibility of the trapped particles [Fig. 5(a)].The result showed that all particles were kept. Even when a

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Fig. 5. Trap retention of the new trap design. (a)–(c) Particlesremaining inside the trap in a large flow fluctuation. (d)–(h)Performance of yeast cell trap retention: (d) yeast cell trap and caughtcells producing, (e) trap process, (f ) and (g) two bud positions, (h) yeastcells cultured and held inside for ∼ 10 h. The scale bar is 10 µm in allimages.

reverse flow [Fig. 5(c)] was applied, the particles still held sta-bly inside the SAMS. In fact, a reverse flow was always usedfor particle collection in the conventional microtraps. In ourexperiments, the particles can be restricted in the trap for a longtime, which shows that the SAMS offered an effective way toachieve a 100% particle retention ratio. Furthermore, we usedthe SAMS to achieve single-cell analysis with a high retentionrate which ignored the pressure fluctuation and cell division. Asshown in Fig. 5(d), the SAMS was first stretched until the yeastcells (∼ 5.0 µm) were trapped [Fig. 5(e)]; then the strain wasreleased to keep them inside [Fig. 5(e)], so the trapped cells wereable to be in-situ monitored by a charge-coupled device. Thetrapped yeast cell was then cultured in the Sabouraud mediumfor ∼ 10 h, and bright-field images captured every 5 min wereused to quantify the number of budding events and the growthof the daughter cell. Figures 5(f ) and 5(g) demonstrated thatthe mother cell budded from two positions: one was in the sideof large gap, and the other was close to the small gap. Once thebud was produced, the constant flow washed away the growingdaughter cells which could help observation and analysis. Asshown in Fig. 5(h), a mother cell produced four buds duringthe entire monitor time. From the observation, even though thebuds were produced from the same mother cell, the daughtercells showed different characteristics in terms of size, buddingtime, and growing speed. Note that during the whole monitortime, the yeast cells were always kept inside the SAMS, regardlessof the flow fluctuations caused by the cell division. this provesthat a SAMS can offer high single-cell retention performance insingle-cell analysis studies.

In this Letter, we have presented a high-efficiency hybridfs-laser process method toward a lab-on-a-chip research area.The processing time of a microtrap chip can be reduced to a fac-tor of 1/16 than that by the conventional single-point writing.In addition, a new concept of stretchable asymmetry-bracket-shaped microtrap endured 100% particles or cell–trappingretention rate. The significant advancement of the new trap

over a conventional one is that it can completely trap particles orcells, regardless of the flow fluctuations. Meanwhile, the SAMScan be reset and reused by a simple strain stretching method. Webelieve that the method present here is quite useful in the area ofsingle-cell/particle analysis, especially in the case that requireshigh cell-trapping retention rate.

Funding. Key Project of Equipment Pre-Research FieldFund of China (61409230310); National Natural ScienceFoundation of China (51675503, 51875544, 61475149,61805230); National Basic Research Program of China (973Program) (2018YFB1105400); China Postdoctoral ScienceFoundation (2019M662190); Fundamental Research Fundsfor the Central Universities (WK6030000103).

Acknowledgment. The authors acknowledge theExperimental Center of Engineering and Material Sciencesfrom USTC for the use of their equipment.

Disclosures. The authors declare no conflicts of interest.

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