Nanophotonics 2021; 10(9): 2389–2406

Review

Felix Sima* and Koji Sugioka* Ultrafast laser manufacturing of nanofluidic systems https://doi.org/10.1515/nanoph-2021-0159 Keywords: glass; lab-on-chip; nanofluidics; polymers; ul- Received April 12, 2021; accepted May 25, 2021; trafast lasers. published online June 11, 2021

Abstract: In the last decades, research and development 1 Introduction – from micro- to of microfluidics have made extraordinary progress, since they have revolutionized the biological and chemical fields nanofluidics as a backbone of lab-on-a-chip systems. Further advance- ment pushes to miniaturize the architectures to nanoscale Microfluidics is the field that has been dedicated to the in terms of both the sizes and the fluid dynamics for some miniaturization and fluidic manipulation at microscale specific applications including investigation of biological addressing integration of biological tools and functions in sub-cellular aspects and chemical analysis with much order to save systematic labor, large space, working times, improved detection limits. In particular, nano-scale chan- and expensive costs [1, 2]. By developing complex micro- nels offer new opportunities for tests at single cell or fluidic systems and control fluid dynamics by automati- even molecular levels. Thus, nanofluidics, which is a zation, robotic workstations offer nowadays possibilities to microfluidic system involving channels with nanometer solve essential issues for chemistry, biology and medicine. dimensions typically smaller than several hundred nm, has Lab-on-chip (LOC) devices and micro-total analysis been proposed as an ideal platform for investigating systems (μTAS) have been then proposed to tackle specific fundamental molecular events at the cell-extracellular applications, which typically involve microfluidics to be milieu interface, biological sensing, and more recently for used for separation techniques [3–8], micro-electro studying cancer cell migration in a space much narrower mechanical systems (MEMSs) [9–12], clinical applications than the cell size. In addition, nanofluidics can be used for [13–15], forensic or molecular diagnostics [16–18], and sample manipulation in analytical chemistry, such as proteomics [19–21] or for innovative tools enabling sample injections, separation, purifications or for quanti- advanced research in the cancer field [22–26]. Fluid tative and qualitative determinations. Among the nano- dynamics associated with microfluidics is dominated by fabrication technologies, ultrafast laser manufacturing is a inertial nonlinearity while the mass transport in such promising tool for fabrication of nanofluidics due to its devices is controlled by viscous dissipation [27]. Specif- flexibility, versatility, high fabrication resolution and three ically, viscous dissipation of mechanical energy induces dimensional (3D) fabrication capability. In this paper, we fluidic resistance and transformation into heat due to review the technological advancements of nanofluidic internal friction [28]. Reynolds [29] and Peclet [30] are two systems, with emphasis on fabrication methods, in dimensionless numbers often used in microfluidics to particular ultrafast laser manufacturing. We present the describe the physical phenomena within the microfluidic challenges for issues concerning channel sizes and fluid space. The Reynolds number, Re, measures the ratio dynamics, and introduce the applications in physics, between inertial and viscous force (Eq. 1), while Peclet biology, chemistry and engineering with future prospects. number, Pe, defines the importance of convection in respect to diffusion (Eq. 2). Re could be related with Pe by Eq. (3).

*Corresponding authors: Felix Sima, CETAL, National Institute for ρ v l R = Laser Plasma and Radiation Physics, Atomistilor 409, 077125, e μ (1) Magurele, Romania; and Koji Sugioka, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan, E-mail: felix.sima@inflpr.ro v l P = (F. Sima), [email protected] (K. Sugioka). https://orcid.org/0000- e D (2) 0001-5911-0288 (F. Sima)

Open Access. © 2021 Felix Sima and Koji Sugioka, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 2390 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

Pe D with living mammalian cells as it offers good permeability Re = (3) ʋ to gases. Exploratory research in microfluidic systems has been carried out using poly(dimethylsiloxane), or PDMS, where ρ is the fluid density, v is the average velocity, µ is the an optically transparent, soft elastomer. dynamic viscosity, l is the characteristic length, ʋ is As the microfluidics field advanced rapidly, it was vorticity difussivity and D is the diffusion coefficient. In further expected to downsize the scale to nanosizes for microfluidic channels, since the flow velocity is low, the Re challenging the physics of fluid flow in ultrasmall becomes small. This reflectsthedominantroleoftheviscosity channels and the possibility of single molecule analysis. over inertial forces that implies the absence of eventual Thus, a new field of nanofluidics has emerged and vortices or turbulences. Then, at such a low Re number, the received considerable attention, especially oriented Newton’ssecondlawforfluid particles (known as Navier– towardsapplicationsofsmallsystemsthatinclude Stokes flow state equation) which governs the fluid motion microarrays for DNA sequencing, microfluidic devices can be estimated as [31]: for polymerase chain reaction (PCR), LOC systems for μ∇2v = ∇p (4) synthesis and analysis of peptide and oligonucleotide libraries, microchips for drug screening or for single where p is the fluid pressure. It evidences that the fluid cell analysis. Nanofluidics can be also useful for sample velocity is given by pressure distribution only, the inertial manipulations in analytical chemistry, such as sample force is negligible, the diffusion is dominant while the injections, separation, purifications for quantitative and motion could be considered symmetric in time [32]. qualitative analysis. However, different derived flow states can be applied for The nanofluidics was defined as the study of fluid topologically optimized configurations of microfluidic transport in or around nanosized objects or, more devices designed for specific applications [33]. concretely, the microfluidic system with at least one Early works in fluidic microsystems used either silicon characteristic dimension below 100 nm [37, 38] while or glass as platform materials. In particular, micro- extended nanofluidics is considered to include the engi- fabrication based on photolithographic techniques was neering of fluids in channels of submicrometer dimensions applied to single crystalline silicon or glass for obtaining [39]. A channel surface usually gets electrostatic charges in structures for flow injection analysis, electrophoresis and contact with aqueous solutions. It then binds counter-ions detection [4, 34]. Compared to glass, silicon is rather from liquid, developing an electric double layer (EDL) close expensive and not suitable for the analysis by conventional to the surface. The excess counter-ions will travel, inducing optical methods due to opaque characteristics in a visible liquid motion by viscosity consequence. This motion is range. These materials have been recently replaced by known as electroosmotic flow [40]. The characteristic polymers which contribute to rapid advancement of the thickness of the EDL, known as Debye length, is given by microfluidics field. Indeed, the introduction of soft lithography for polymer microfabrication had a huge 1 λD = , (5) impact on microfluidics as it was a simple technique k enabling fabrication of micro-systems without the need of where k is a constant related to the ionic composition and using clean room equipment. Soft lithography employs defines the equilibrium distribution of ions diffused in the non-photolithographic strategy for micro- and nano- the EDL. In contrast to the microscale channels where the fabrication. By using an elastomeric stamp with relief thickness of the EDL can be completely negligible, this structures one may create patterns with micro or even nano becomes critical for the flow in nanochannels in which its scale feature sizes down to 30 nm [35]. These characteris- dimensions may be comparable or even smaller than the tics were then exploited to control patterning of complex Debye length. Thus, the mass transport in nanofluidic molecules relevant to biology or to fabricate channel net- systemsisdifferentascomparedwiththatinmicrofluidic works appropriate for use in microfluidics for cellular systems since the electric potential barrier given by a manipulation. The soft lithography is also the convenient, large EDL affects transport properties of ionic species. inexpensive, and rapid technology to produce configura- However,formostmicrofluidic applications, the EDL tions of large feature sizes used in biology (≥50 µm) without thickness may be negligible in the study of electroos- considerable effort [36]. In addition, as compared with rigid motic flow, if λD/h ≪ 1, where h is channel height. materials, the elastomers offer a simpler alternative to Nevertheless, the Navier–Stokes flow state equation fabricate components required for microanalytical systems (Eq. 4) was also accepted to the nanoscale while specific such as pumps and valves, and are also adequate to work transport phenomena appears due to electrostatics and F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2391

interplay between flow and ionic transport in small As alternatives to photolithographic methods [35, 44, spaces [41]. 45], a lot of efforts for development of soft lithographic On the other hand, to study nanofluidic systems as techniques have been made in micro- and nanofabrication well as to provide their functionality, it is required to for surface chemistry, materials science, optics, MEMS, mi- connect them to the macroscale. The interface is given croelectronics and, in particular, in biology and biochem- by microfluidics which further becomes necessary to istry [36, 46, 47]. The soft lithography also allowed assemble multifunctional miniaturized devices [42]. improving the resolution down to tens of nanometers and Mimicking nature functionality at nanoscale in which we developing at the same time microfluidics and nanofluidics can reproduce complexity of biology will challenge the domains [48]. Soft lithography techniques involve the artificial biotic systems [43]. We show in Figure 1 a sche- fabrication or replication of various structures by using matic description of the length scales and lithographic elastomeric stamps or molds. The most used material with techniques used to fabricate micro- and nanosystems. these techniques is PDMS, which is cast on a master mold In the following sections we focus on the fabrication (various processed materials, e.g. SU-8) to build elastomeric techniques of nanofluidic systems by conventional methods stamps. For rendering 3D aspect to the assembling, these and ultrafast laser processing. We then address the chal- stamps can be further sealed to glass substrates by using lenges for issues concerning sizes and fluid dynamics in oxygen plasma or alternative methods. nanofluidic systems and present some applications. At the Photolithography, on the other hand, is well estab- end, we summarize our ideas and give some prospective. lished and the most settled technology for device manufacturing. Originally, Mercury lamps are used as excitation radiation sources while conventional photore- sists of alkali-soluble resin mixed with a photoinitiator or 2 Conventional fabrication photosensitizer are sensitive to energy spectrum between technologies for nanofluidic 250 and 450 nm. A KrF* excimer laser was the first laser source used in photo lithography for mass production of systems 256-Mb DRAM with a feature size of 250 nm. The resolution is given by the resolving power of the photolithography Nanochannels, nanopipettes and nanopores are the most (Rayleigh limit): known nanofluidic platforms. Most of the fabrication kλ technologies are focusing to obtain such platforms with R = NA (6) the aim of proposing functional devices with specific applications, in general for biological purposes. One of where k is the process factor (typically 0.61), λ is wave- the targets is to improve a resolution with a reasonable length of the light source, NA is the numerical aperture cost so that research in laboratories can afford to advance of the projection lens. The equation is effective for mono- science in nanofluidics field. However, each technique chromatic light sources while the resolution for larger has limitation in materials that can be processed. spectral-width light sources is inferior. Thus, a laser source

Figure 1: Description of the length scales and common processing methods used in micro and nanosystems. 2392 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

will have an advantage over lamp sources due to shorter nanoscale feature sizes with high-accuracy and flexibility wavelength as well as narrower linewidth. The much in replication. It employs electron sources such as therm- higher intensity supplied from laser can also increase ionic or field electron emission sources, which are tightly throughput. focused by electrostatic and magnetic lenses to direct the The optical resolution has been continuously electron beam usually towards a resist material. The finely improved by breaking the barrier of Rayleigh limit using developed structures are then usually transferred to a different strategies. For example, using phase-shifting desired substrate for specific applications by etching, masks makes it possible to improve the resolution by 40% typically reactive ion etching. Most common electron- [49]. Another method for resolution improvement was the sensitive resist materials are based on poly(methyl meth- immersion lithography. It uses a high index filling medium acrylate) (PMMA) or so called chemically amplified resists in the space between the optics and substrate which al- (CARs). The most common EBL resists along with their lows the reduction of λ in Eq. (6) to an effective wavelength lithography properties are presented in detail in Ref. [64]. It of λeff = λ0/n ,whereλ0 is the actual employed laser wave- is possible to achieve sealed nanofluidic channels with length and n the refractive index of filling medium [50]. In cross-sections of 10 × 50 nm, which are essential for this scheme, ArF* excimer laser (193 nm wavelength) confining molecules of biological relevance [65]. By EBL it lithography in water achieved better resolution as compared was also feasible to create sub-10 nm PMMA tranches by with 70 nm resolution by F2 laser (157 nm wavelength) [51, resist development at low temperatures [66]. In addition, 52] due to the refractive index of 1.44 for water at 193 nm [53]. by applying EBL and subsequent etching, sub-10 nm

Scanning near-field optical microscope coupled to a UV nanoholes in Si, SiN or SiO2 membranes can be fabricated laser has been developed to create molecular patterns with with high precision [67]. dimensions nearly 15 times smaller than the Rayleigh limit Another high energy beam processing method at [54]. For example, molecular features with widths of only nanoscale is use of focused ion beam (FIB). A typical FIB 20 nm have been fabricated in self-assembled monolayers of technology uses a liquid metal ion source and electrostatic alkanethiols on gold using this method [55]. Interferometric lenses to focus the ion beam onto the sample for lithog- lithography (IL) typically using two coherent light beams at raphy or direct substrate milling due to sputtering of atoms +θ, −θ angles to expose photoresists offers a maskless, large- from the substrate. It is a very precise maskless technique area, nanoscale processing with an interference pattern of and attains a processing resolution below 10 nm, which is period λ/2sinθ [56]. Development of special photoresists the minimum of focused ion beam size [68]. FIB can enables to get nanofeatures down to ∼λ/4 with the combi- be applied in various setups, from the site-specific prepa- nation of ArF* excimer laser, which is as small as 48 nm as ration and even for composite samples, to sectioning at compared with the laser wavelength of 193 nm [57]. One may submicrometric level and nanostructuring or to ion- further decrease the resolution by using IL in immersion induced chemical vapor deposition (FIB-CVD) [69]. FIB media down to 33 nm [58] or by using IL of X-rays down to can thus create 3D nanostructures made of metal or insu- 3 nm [59]. lator materials starting from organic precursors [70]. FIB Extreme ultraviolet (EUV) or X-ray lithography uses was also applied to create nanopores with nanometer optical radiation of very short wavelengths of 10–14 nm control in SiN membranes [71]. emitted by either laser-produced plasma or synchrotrons Nanoimprint lithography has been developed as a sources. A typical setup also includes optics and a reflec- need to produce large-area patterning with nanoscale tive mask or a proximity mask to direct the patterned feature sizes on casting materials [72, 73]. This technology radiation beam onto a special photoresist material. The was implemented to support conventional lithography for resolution given by Eq. (6) can thus achieve sub-100 nm high-throughput, low-cost printing. Specifically, by pro- features [60]. EUV lithography is expected to improve the ducing compact stamps or molds with desired geometry processing resolution down to 10 nm by the appearance of and nanoscale features using optical lithography, EBL or new stable sources with sub-10 nm wavelengths [61–63]. FIB techniques one may further apply to stamp them on a The drawback for EUV and X-ray lithography is that printable polymer. In this process, critical aspects are conventional refractive lenses cannot be used in typical related to the proper choice of polymers for nanoprinting setups and must be developed while a special attention [74]. Indeed, in order to obtain stamping resolutions of should be paid due to the high radiation energy that can about 10 nm, requirements involve good mechanical be- damage most of the masks or lenses. haviors and antisticking properties, and biocompatibility Electron-beam lithography (EBL) is one of the most is further demanded, if they are used for biological used technologies to create patterned structures of purposes. F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2393

3 Ultrafast laser processing for The focused laser beam positioning in respect to the transparent sample with high precision 3D translational nanofluidic systems stages will then allow obtaining complex 3D configura- tions, which otherwise are impossible to be obtained by Ultrafast lasers are a relatively new category of pulsed planar technologies. lasers with ultrashort pulse duration from few picoseconds In addition, the thermal diffusion to the outside of to several tens of femtoseconds [75]. Such lasers exhibit processing area is significantly eliminated since the 2 extremely high peak intensities (>10 TW/cm ) that allow process can be completed before thermal diffusion occurs modification of transparent materials by multiphoton due to the pulse duration shorter than electron–phonon absorption with resolutions beyond the diffraction limit coupling time from several ps to few tens of ps. This as given in Eq. (6) [76] (Figure 2). Additionally, the multi- thermal diffusion suppression is essential for laser photon absorption provides 3D fabrication capability of processing of materials with high accuracy and high the transparent materials. The transparency at the laser resolution. Micro- to nanofeatures can be thus assembled irradiation wavelength permits photons travelling through into novel architectures and arrangements with features the material. However, an intense laser field given by the down to sub-100 nm [78]. In the following we show the high peak intensity of ultrafast laser allows to spatiotem- potential of ultrafast laser processing for nanofluidics porally concentrate the photon energy in a material volume fabrication by two different schemes: additive and sub- within a focal area of laser beam capable of exciting elec- tractive processing. trons over the forbidden energy gap due to simultaneous absorption of multiple photons (multiphoton absorption) (Figure 2a). The multiphoton absorption can improve the 3.1 Additive laser processing fabrication resolution beyond the diffraction limit because the probability of multiphoton absorption depends on the Two photon polymerization (TPP) is the most known op- laser intensity and the ultrafast laser has typically a tical lithographic technique using ultrafast lasers that can Gaussian beam profile (Figure 2a and b). The effective spot fabricate 3D structures with nanoscale features. The poly- size w of m-photon absorption is given by merization is typically induced by a femtosecond laser beam which is focused through a high NA objective in a w = w /m1/2 , (7) 0 small volume of photocurable resin or negative-tone where w0 is an actual spot size of focused ultrafast laser photoresist both containing photoinitiator. TPP is based beam. Combination of the threshold effect for reaction on two-photon absorption (TPA) phenomena, in which two − with the multiphoton absorption achieves the fabrication photons are absorbed practically in the same time (∼10 5 s) resolution far beyond the diffraction limit [77]. by a photoiniator molecule to excite it to a higher energy

Figure 2: Scheme of laser-material interaction along the beam axis (a) and at the focal spot in a plane perpendicular to the beam axis in a transparent material (b) to achieve subdiffraction-limited fabrication resolution by multiphoton absorption. In (b), thick dashed line, solid line, and thin dashed line correspond to spatial distributions of laser energy with a Gaussian beam profile absorbed by transparent materials by single-, two- and three-photon absorption, respectively. The solid horizontal line indicates the reaction threshold (adapted from [76]). 2394 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

level [79]. The excited photoinitiator molecules interact particular for high aspect ratio (>10:1) nanochannels, with with monomers during propagation to generate monomer widths of 200 nm and lengths of 20 µm [85]. Such beams radicals, and eventually photo-polymerization takes place have a specific characteristic so as to make the intensity when monomer radicals are chained in the termination profile constant along the propagation [86]. They became processes. This reaction is known as radicalic polymeri- thus challenging alternatives to other nanoprocessing zation [80]. There is also photo-polymerization based on technologies for high efficiency nanochannel fabrication cation generation instead of radicals. In this case, a cata- due to highly localized and controlled energy deposition lytic photoacids are formed that initiate polymerization in transparent materials, although they can create only [81]. TPP is categorized into additive processing because straight channels [87]. the polymerization occurs along the scanning trajectory of The other well-known subtractive ultrafast laser focused laser beam and non-irradiated areas are washed based technology for the fabrication of typical micro- away by specific solvents to construct 3D structures. For fluidic channels is femtosecond laser assisted etching TPP there is theoretically no limitation of resolution due to (FLAE) [88]. This technique is usually applied to photo- material threshold effect when laser intensity is precisely sensitive glass or fused silica, which consists of femto- controlled, so that sub-100 nm fluidic structures can be second laser direct writing followed by chemical etching achieved [76]. Nevertheless, in order to create nano- [89]. For the photosensitive glass, thermal treatment is channels by TPP technique one may mention the difficulty necessary before the etching. The channel fabrication of removing non-polymerized residues in the channel as resolution is determined by the etching selectivity (typi- the channels become narrower. cally ∼50) between the laser exposed and unexposed re- gions in the subsequent etching so that embedded microchannels are formed with resolutions, of few mi- 3.2 Subtractive laser processing crometers, inferior to the water-assisted ablation. How- ever, a new hybrid technique that combines FLAE and TPP There are two main approaches for fabrication of should allow compensation of a drawback of FLAE in nanofluidic systems by subtractive laser processing: terms of fabrication resolution, simultaneously offering laser ablation and laser assisted etching. The former enhanced functionality and robustness to the fabricated one can be applied to various materials while the latter device [90]. In this hybrid process, FLAE is first carried out one is limited to few materials only. Femtosecond laser to create microfluidic channels inside glass volume which nanoablation of GaN was demonstrated with surface are then filled with a negative-tone photoresist. A subse- modification at a sub-diffraction resolution [82]. In this quent TPP in the glass channels enables to integrate the study, a laser wavelength of 387 nm was employed to polymeric nanochannels with a sub-200 nm resolution in achieve nanohole arrays with sub-200 nm diameters the glass microchannels. due to collaboration of multiphoton absorption and the Recently, it was demonstrated that a glass micro- threshold effect. By femtosecond laser direct write channel size obtained by FLAE was able to be reduced via a ablation in water, nanochannels with diameters of post-thermal treatment [91]. This treatment at the temper- about 700 nm with a complex 3D geometry have been ature slightly below the glass melting point can induce fabricated in fused silica [83]. Further employing peri- glass deformation and create architectures down to odic nanograting formation in the water-assisted nanoscale inside channels due to melting of the shallow femtosecond laser ablation, which was a specificphe- glass surface layers that exhibit lower surface energy than nomenon for femtosecond laser processing, enabled bulk. Then, the surface is reformed while channel width fabrication of nanofluidic channels with transverse dimensions can be controllably reduced down to several widths narrower than 50 nm [84]. More details of this hundreds of nanometers (Figure 3). technique are given in Section 6. In the water-assisted It is worth to mention that downsizing dimensions ablation, the water medium has an important role to to obtain nanofluidics in microfluidic systems is an remove the ablated residues from the nanochannels advantage to link the macro-world to nanoscale. This and eventually to create long channels with complex 3D allows fluid communication as well as a facile analysis geometries. of the entire system. Some issues that will be mentioned The use of nondiffractive beams such as Bessel beams in the next section can be solved by a soft transition became motivating for femtosecond laser processing, in from macro- to nanoscale. F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2395

Figure 3: Optical microscope images of the sample after the thermal treatment at 645 °C (a, b). Imaging of submicrometer channels inside the microfluidic biochip using Rhodamine Red: (c) Magnified image in the xy plane. (d) Side view of (c). Reproduced from [91].

4 Challenges for issues concerning surfaces with increased hydrophobic characteristic due to the long-range strong attractive force in aqueous media sizes and fluid dynamics in [95–97]. They may also coalesce by a gaseous bridge nanofluidic systems capillary force [98]. By molecular dynamics simulations it has been indicated that the bubble nucleation in confined It has been suggested that water hydrodynamics can be nanochannels is homogenous on rather hydrophilic sur- considered valid for nanofluidic applications so that faces, and becomes heterogeneous with surface hydro- Navier–Stokes equations should be considered for the fluid phobicity while no bubble nucleation may be possible on transport down to a continuum limit of 1 nm [41, 92]. the non-wetting surface [99]. A critical aspect of the However, with the confinement increase, the surface to nanobubbles is their life-time which can last up to days in volume ratio increases so that the surface characteristics certain conditions [100]. It has been also suggested that nanobubbles may be stable in water for long periods of play critical roles. The Debye length, λD, explained in Section 1, Eq. (5), which characterizes the EDL, ranges from time, due to both concentration gradient and repulsion 0.3 to 30 nm, and is dependent on the ion concentration in forces at liquid-gas interface that prevent coalescence the fluid. It is thus important to carefully evaluate fluid [101]. As consequence, due to attractive forces between fl dynamics in confinements down to 30 nm. In consequence, nanobubbles, they may drastically in uence velocity slip fl it becomes critical for applications in which nanopores and disturb the ow in nanochannels [102]. exhibit dimensions that equals twice the Debye length The above-mentioned issues are main factors for due to Debye layer overlapping [93]. This issue affects appearance of more fluctuations when decreasing the interfacial transport phenomena such as electroosmosis or sizes in nanochannels or for the non-linear transport in electrophoresis due to ion dynamics within EDL. nanopores or nanoslits [92]. In addition, problems with Another issue is revealed by the so called Navier molecule adsorption on the walls should be considered as boundary condition that complements Navier–Stokes the space becomes narrower. On the other hand, a positive equation, which reveals that the friction coefficient and aspect may be the precise correlation between ion trans- slip length strongly depend on the strength of interaction at port and electrostatics in nanochannels, which can take fluid-solid interfaces [94]. It has been shown that large slip full advantage when the height is small enough to overlap lengths of 10–50 nm are dominant when the contact angle Debye layer. of the liquid increases (weak interaction) while “no slip” As many applications of nanofluidics concern chemi- boundary condition is possible at very low contact an- cal or physical analysis, another important aspect is the gles. In this context, it has been also evidenced that the detection and observation inside the nanochannels. For roughness decreases the slippage while it can amplify the chemical analysis, detection of low concentration analytes non-wetting surface characteristics. It is thus necessary to is difficult in small volumes so that expensive single cautiously consider these aspects at nanoscale in order to molecule detection methods become necessary. On the propose engineered interfaces that reduce flow friction. other hand, for the observation one may use conventional Nanobubbles have spherical or irregular shapes with two-photon or other sub-diffraction limited microscopy, in diameters up to 100 nm, which are rather developed on which the image is then captured by point scanning of a 2396 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

tightly focused pulsed laser beam. Alternatively, temporal sensitivity. A laser-etching technique recently developed focusing with widefield illumination could represent a was employed for in situ nanopore formation inside a benefit for a scanless scheme, already applied to super- sealed microfluidic system [110]. In this study a near- resolution imaging or even photolithography [103]. ultraviolet laser (375 nm, 10–15 mW) was used to form nanopores precisely located at the center of the narrow channel. Central silicon nitride pillar array and a narrow 5 Applications of nanofluidics channel (∼2.5 μm wide and 200 nm deep) were first pre- pared for funneling linearized molecules to the nanopore. In this Section, we refer to some concrete applications By pressure-induced flow, DNA concentration as low as of nanofluidics including nanopores, nanopipettes and 50 fM was delivered to the nanopore. Ultralong DNA nanochannels. We, however, put more emphasize on the molecule can be then translocated in a graded micro/ use of nanochannels for the biological field, because the nanofluidic platform for sensing of biomolecules. ultrafast lasers have been limited to be proposed for this The main potential of nanopores is expected by application so far. The nanochannels can be categorized single-molecule DNA sequencing and could represent a in three geometric configurations: square, planar and high- key technology for reading genome at single cell level aspect ratio nanochannels [104]. In all applications, [111]. Some other applications have been proposed for nanofluidics is rather an engineering tool to produce electrochemistry [112, 113] and single molecule conforma- molecules confined in ultra-small spaces and then expose tion analysis [114], bioseparations in electroanalytical them to controlled forces for exploring fundamental chemistry and biosensing [115–117], biomimetic stimuli- knowledge, especially in biological systems, at the responsive membranes, and energy generation from molecular level. salinity gradient and light [118]. Nanopipettes are defined as structures with needle- like geometry of sub-200 nm diameters with various ra- 5.1 Nanofluidics fabricated by conventional tios between outer and inner diameters, dependent on technologies applications [119]. Different from the nanopores, due to relatively larger dimensions, they are more robust, exhibit Nanopores correspond to sub-100 nm or smaller diameter good wettability and can be easily integrated in micro- structures with lengths below 10 µm. Solid-state synthetic fluidic devices. Besides the common applications with the nanopores became alternatives to the biological counter- nanopores, they may be further used for nanoinjections parts as they are more stable, controllable in dimensions (dispensers or aspirators of ultrasmall volumes), nano- with tailored properties specific to the applications. They biopsies with subcellular precision, or probes for micro- are necessary tools for single molecule analysis as the scopy. They are usually made of glass or quartz, commonly passage of individual DNA, RNA or protein molecules by a laser pipette puller instrument capable to downsize through such a nanoscale space is essential in many diameters to nanometer scale, depending on glass thick- biological processes [105]. In particular, processes such as ness, temperature, or pulling force. It achieved fabrication DNA translocation through the nanopore may help in of nanopipettes by sequential heating and pulling with unfolding and linearization of the molecule with great use diameters below 80 nm [120] in borosilicate glass or even in its sequence reading for genomics applications [106]. sub-10 nm in quartz [121]. It was thus possible by electro- Pores fabricated by FIB and EBL techniques were proposed phoretic and dielectrophoretic forces to pull and trap with the initial studies for DNA translocation [71, 107]. It proteins and DNA with a higher concentration closer to was then found out that the singles-strand DNA (ssDNA) physiological conditions [120]. This approach could be very could pass through sub-2 nm pores while double-strand useful for ultrasensitive detection in miniaturized DNA (dsDNA) was blocked but could pass through the bioassays. larger pores [108]. Other proteins can be as well trans- Nanochannel-based nanofluidics integrated in func- located through pores of various dimensions so that tional devices are essential for applications that involve controllable unfolding may be possible. Ultrasmall and biomolecule transport, bioseparation, and biodetection ultrathin nanopores with pore diameters and thickness [122]. Due to their dimensions with the order of bio- below 2 nm were fabricated by laser-assisted SiNx etching molecules, they initially offer potential in DNA and other coupled with dielectric breakdown [109]. Such pores were molecule separation, detection, and sensing [123]. Con- used to discriminate DNA length as well as sense DNA ventional DNA detection based on gel uses porous media homopolymer sequence identification with very high with limited pore sizes and poor mechanical properties. In F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2397

contrast, electrical field applied in nanochannels can Most of developed nanochannels for DNA analysis provide precise control on size dependent separation of suffer, however, from complex fabrication procedures DNA and exhibit capacity to probe the conformational together with issues of DNA overloading at the nanochannel properties of DNA, sort the molecules within confining entrance which obstruct solution exchange. New strategies environment and define the spatial location of genetic in- are under development targeting simplicity and relevant formation along the molecule [124, 125]. An important hierarchical or graded architectures that can controllably biosensing application of dedicated devices is the mapping downsize dimensions and create a functional interface of and observation of genomic and epigenomic DNA infor- micro and nanofluidic systems. FIB-milling was found mation during stretching of DNA in nanoscale channels useful for 3D nanofunnels connected with microscale [126–128]. reservoirs that define dynamics of DNA molecules with Photolithography is the most used conventional increased confinement. Then, electrokinetically driven method for fabricating planar nanochannels as the depth introduction of the DNA molecules into a nanochannel was of channel can be precisely controlled down to few tens of facilitated by incorporating the 3D nanofunnels at the nm by the subsequent etching process. The channels with nanochannel entrance [135]. narrow constrictions and wider regions were proposed Another study proposed light-induced local heating of in pioneering work for the separation of long DNA liquids inside micro- and nanofluidic channels fabricated molecules. Size-dependent trapping of DNA and electro- by thermal imprint in low molecular weight PMMA [136]. It phoretic mobility differences allowed separating long was demonstrated thermophoretic manipulation of DNA molecules in 15-mm-long channels without the use genomic-length DNA in micro- and nanochannels as well of a gel matrix or pulsed electric fields [129]. Interference as compression or stretching of DNA in nanochannels by lithography is complementary to the standard lithog- steep temperature gradients created by the light-induced raphy technique as it can be used for fabricating deep local heating. nanochannels [57]. One may easily generate periodical The main challenge with fabrication technologies narrow nanochannels on large areas that can be applied for such applications is the construction of a reliable de- to DNA transport and stretching. EBL was also applied for vice that can target single-cell trapping and then allow fabricating nanochannel-based nanofluidic systems for subsequent single-genome analysis. The bottleneck DNA analysis. EBL followed by reactive ion etching resides in interfacing microscale confinement for single- fabricated arrays of 100 nm wide nanochannels in fused cell manipulation with nanoscale confinement for single- silica [130]. Specific DNA orientation was found and molecule DNA linearization. transport direction of DNA was controlled under DC Recently, a rather simple multilayer soft lithography electrophoresis. Nanochannels of sub-5 nm lateral corroborated with controllable elastomeric collapsing dimensions with smooth surfaces were fabricated by FIB allowed the fabrication of a uniform nanochannel array milling and then sealed with a cover plate, which were with confining spaces down to 20 nm and lengths up to also found suited for single-molecule DNA transport sub-millimeters. In this study, the authors obtained in a studies [131, 132]. Integrated multi-level lithographic single micro/nanofluidic biochip with complex scale processes consisting of EBL, UV stepper and printer were up–scale down architectures that were used to either cell combined with dielectric deposition by plasma-enhanced isolation, lysis, DNA extraction, purification, labeling, or chemical vapor deposition, plasma etch and chemical linearization for single-cell genomic analysis (Figure 4) mechanical polishing for the fabrication of a functional [137]. nanofluidic device [133]. This consisted of stacked multi- Another integrated micro- and nanofluidic device was layers on a silicon single chip with channels of lateral fabricated by FIB milling and UV nanoimprint lithography dimensions from <20 nm to 1 mm and vertical from 40 nm [138]. Microchannels and nanochannels of different depths to >2 μm. This scale up-scale down architecture and layouts were combined to facilitate and smoothen the allowed fast fluidic transport and controlled biomolecular flow (Figure 5). It then showed an improved stretching of manipulation demonstrated by regulated λ-DNA strad- DNA molecules in long nanochannels whose widths and dling and stretching in an array of nanochannels and depths were gradually decreased. It was further evidenced nanopillars. In addition, damage assays of single- that the inlets captured more molecules as they exhibited molecule DNA are possible in a nanofluidic chip capable smooth graded transitions due to low entropic barrier. to stretch the molecule in constrictive channels of ∼50 nm Eventually, a nanochannel with squared transient config- inwidth[134].Inthiscase,thenanofluidic biochip was uration collected twice as many molecules as that with the fabricated on thermoplastics by nanoimprint lithography. abrupt transition. 2398 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

Figure 4: An integrated micro/nanofluidic device fabricated by soft lithography collaborated with controllable elastomeric collapsing for single-cell genomic analysis: (a) Schematic of the device; optical images of (b) single-cell trapping and (c) cell lysis; (d) fluorescent image of the DNA around the micropillars. (e) The DNA strands extend more than 1 mm around the micropillars in the microchannel. (f) Long genomic DNA molecule extension in a 60 nm deep nanochannel under a control pressure. Reproduced from [137].

Figure 5: DNA flow in nanochannels with different configurations of inlets in the same device fabricated by FIB milling and UV nanoimprint lithography. Sketch of the geometries, scanning electron microscopy images of the channels, and consecutive fluorescence images that show the translocation of a DNA molecule. Flow of a DNA molecule in a long nanochannel without inlets (a), connected to smooth, 3D tapered inlets (b), accessed with a 1 μm wide and 1 μm deep channel (abrupt micro-to-nano transition); (c), and with gradually decreased depths and widths (d). (e) Representation of the position of the DNA molecules along the nanochannel and inlets versus time for the different configurations shown in (a)–(d). Reproduced from [138].

Besides potential of DNA separation, detection and biosensors, energy conversions [141], platform for detec- sensing, other applications using the nanochannels tion and characterization of individual sub-100 nm par- include nucleic acid biopsy in precision medicine [139], ticles with applications in semiconductor manufacturing, nanochannel chromatography for single-cell analyses environmental monitoring, biomedical diagnostics and [140], bio-inspired nanochannels for molecular filters, drug delivery [142]. F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2399

5.2 Micro- to nanofluidics fabricated by substrate. The pores of 10 nm were uniformly distributed in ultrafast laser processing glass volume to create a 3D connective network, which had an important role to efficiently supply water to the ablation The development of ultrafast lasers with high-repetition site inside glass. After the ablation, thermal treatment rates (GHz to MHz) has now made this process to be used was carried out to consolidate the porous glass, which for many applications, including in industry and medical transformed it to compact silicate glass while the micro and fl clinics. Ultrafast laser processing with capabilities of 3D nano uidic structures inside the glass remained formed. A XYZ and nano fabrication demonstrated to be a very useful computer-controlled translation stage was used to tool to shape the material with topography, morphology, allow the fabrication of complex desired 3D geometries in and structural arrangement. This technology alone or in the glass substrate. The fabrication resolution far beyond combination with other conventional processing was the diffraction limit achieved relied on a periodic nano- fi applied to develop functional biochips containing 3D grating formation, which is speci c for femtosecond laser nano-components for integrated optics and lab-on-a-chip processing. A periodic nanograting can be formed inside applications. Graded or hierarchical configurations with the glass with irradiation of a linearly polarized beam [144], dimensions from hundreds of micrometers to tens of which is ascribed to spatially modulated energy deposition nanometers were then developed for various materials with nanoscale periodicity inside the glass. By intention- with specific surface characteristics that can be proposed ally decreasing the intensity of femtosecond laser beam fi for a wide range of micro- and nanofuidic applications. with a Gaussian pro le, only a single cycle of the modu- UV lithography was combined with TPP to integrate lated energy distribution at the center of beam can exceed nanochannels with microfluidic channels [143]. Specif- the threshold intensity for ablation to create a single – ically, TPP was applied to fabricate nanofluidic channels of nanochannel (Figure 6d f). Using this strategy, an inte- fl fl various heights down to sub-100 nm using conventional grated micro-nano uidic system in which two micro uidic photoresists, which were connected to two microchannels channels were connected with an array of nanochannels fabricated by the UV lithography. For TPP, a Menlo System could be obtained. Stretching of DNA molecules was C-Fiber 780 HP Er:doped fiber oscillator (120 fs, 100 MHz) observed in these nanochannels to evidence their potential integrated with amplifier was used to generate a second for investigation of single molecular behaviors (Figure 6g). harmonic. SU-8 photoresist was used for both the UV A new concept of hybrid technique referred to as “ ” lithography and TPP. A microfluidic pattern of UV light was ship-in-a-bottle laser fabrication was proposed [90, 145], projected using a mask, followed by TPP for creation of the in which subtractive FLAE of glass and additive TPP nanochannel. The post baking at 95 °C for development were sequentially performed for integration of polymeric followed by washing left the mold of nanofluidics, which nanostructures in glass microfluidic devices. This hybrid was used for replication by soft lithography with PDMS. technique exploits the advantages of both processes while The test of nanofluidic device was carried out by aqueous compensates each other’s disadvantages. The fabrication dye solution. In this case, a total internal reflection illu- resolution of TPP inside the glass microchannel was mination was used to observe single fluorescent molecules improved by using a spatial light modulator for wave front diffusing into the nanochannels from the reservoirs. TPP correction [146]. This hybrid approach was thus proposed allows integration of nanopatterns with complex shapes to create 3D environments at the submicrometric scale in- and various sizes into microfluidic devices. The combina- side a closed glass microfluidic chip (Figure 7a and b). The tion of EBL and UV lithography is also conceivable; how- second harmonic of a Yb-fiber laser beam (532 nm; 360 fs, ever, TPP exhibits flexibility for the potential integration of 200 kHz) was used for 3D direct writing of glass, followed nanofluidic functionalities into volumes of transparent by thermal treatment and etching to develop microfluidic microfluidic devices. channels. TPP of SU-8 photoresist filled in the glass As already introduced in Section 3, sub-50 nm wide microfluidic channels was carried out to develop polymer nanofluidic channels were successfully obtained in silicate nanochannels inside the fabricated glass microchannels glass volume by ultrafast laser direct write ablation in (Figure 7c–e). water [84]. In this study, a focused femtosecond laser beam The array of nanochannels acted as a nanoscale (Coherent, Inc., 800 nm, 100 fs, 250 kHz) was employed to diffusion-based gradient generator that allowed forming a fabricate hollow 3D micro- and nano-channels inside a stable gradient of biochemical epidermal growth factor glass substrate (Figure 6a–c). To achieve sub-50 nm (EGF) attractant. Prostate cancer (PC3) cells were culti- nanochannels, a porous silicate glass was used as a vated inside the hybrid 3D biochips and exposed to the 2400 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

Figure 6: Schematics of (a–c) procedure for fabrication of micro-nanofluidic system by ultrafast (femtosecond) laser direct write ablation of porous glass in water; (d) scheme and (e) mechanism of nanochannel fabrication; (f) scanning electron micrograph of nanochannel; (g) fluorescence image of DNA in an array of nanochannels with a width of 50 nm. Reproduced from [84].

Figure 7: Biochip fabrication. Schematics of (a) FLAE of Foturan glass followed by (b) TPP of SU-8 inside glass microchannel, (c) optical microscopy image of the glass microfluidic platform with (d) details of the observation area, and (e) panpipe-shaped scaffold consisting of six nanochannels with lengths of 6, 8, 11, 14, 18, and 21 μm integrated by TPP at the bottom of glass microchannel. Reproduced from [146]. F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems 2401

chemo-attractant gradient to evaluate specific behaviors, nucleus stretch was then confirmed during migration in such as migration and invasiveness. It was evidenced the nanochannels without altering their viability that the cancer cells could penetrate the submicrometric (Figure 8). In addition, the ultrathin chip allowed fluores- channels, migrate very fast and then split into vesicular cence microscopy analysis with high resolution. It could be fragments that eventually fused back into single bodies. then observed that the cell body was compressed to a thin The fabrication resolution of FLAE was also improved sheet-like shape to occupy the entire narrow constricted by inducing glass deformation via post-thermal treatment region in the nanochannel from the entrance to the exit, (nanoscale glass deformation-NGD) to create nano- whereas the cell nucleus was deformed to a disk-like shape channels in an ultrathin glass substrate. The Yb-fiber laser that was pulled into the constricted region of the channel beam (532 nm; 360 fs, 200 kHz) was used for glass irradi- later during the migration process. ation. This process provides much more simple schemes in Some future applications of nanofluidic devices may terms of the fabrication procedure and the material due to include single-cell genomic analysis for personalized elimination of TPP process and the biochip consisting only cancer therapies. Biopsies from cancer tumors harvested of glass. More importantly, it can create an in vivo-like from patients could be analyzed in micro/nanofluidic environment with architectures down to the nanoscale, biochips. The evaluation of cell population behavior providing narrow constrictive spaces for cancer cell couldbecorrelatedwiththedegreeofmigrationand migration. Using such biochips enabled to observe the invasiveness and genomic analysis at single-cell level. capability of PC3 cells to penetrate consecutive narrow The objective could be to test specific doses of radiation constrictions that mimic intravasation–extravasation therapy, specific chemotherapy, immunotherapy, or a events in the in vivo environment. Very long (∼50 µm) combinations.

Figure 8: Cell stretching inside the nanochannels created by FLAE-NGD: (a, b) Grayscale fluorescence microscopy images of cell nuclei invading the narrow regions of the nanochannels. (a) Obtained by merging fluorescence and optical microscope images of cells. The white arrow in (b) shows an elongated cell inside the nanochannel. (c) 2D and (d) 3D confocal fluorescence microscopy images of cells additionally stained for a membrane marker (green). Reproduced from [91]. 2402 F. Sima and K. Sugioka: Ultrafast laser manufacturing of nanofluidic systems

6 Summary and future prospects hierarchal configurations for specific applications, for which repeated use of multiple techniques is necessary. Microfluidic systems helped developing single-cell anal- Besides the nano-scale surface processing of a wide ysis methods in microspaces. Extension to nanospaces is range of materials such as metals, semiconductors, ceramics, increasingly necessary to provide a new generation of polymers and even soft materials by nanomachining, nano- analytical tools for biological applications. Due to accu- structuring and nanoablation, ultrafast laser technology can rate control of liquid flow and of molecular behavior at be a viable alternative to fabricate highly functional biochips the nanoscale, nanofluidic systems found a prominent integrated with almost arbitrary shapes of 3D nano- role either in the analysis of individual cells or bio- components. Specifically, the multiphoton absorption of ul- molecules. Analytical nanosystems in such small spaces trafast lasers can induce nanoscale modifications and fabri- could provide ultra-sensitive analyses at a single-cell and cation in volume of transparent materials with desired single-molecule level. It can be supposed that nanopores geometries and configurations by TPP, water-assisted abla- and nanochannels could offer the essential support of tion and FLAE. Additionally, the processed regions can be label free identification and characterization of single- controllably functionalized in a space selective manner. It is stranded genomic DNA or RNA without conventional then possible to integrate functional components and devices amplification while DNA sequencing becomes possible. for integrated optics and lab-on-a-chip applications. New Mass transport at this scale could reveal new phenomena devices with graded or hierarchical configurations consisting as e.g. enhanced mass flow rate and highly selective of different dimensions from hundreds of micrometers down molecular transport, similar to those in natural trans- to few nanometers and with various surface properties can be membrane proteins such as ion channels and aquaporin. developed in a wide range of materials. Such biochips may Since critical issues of nanofluidic systems consist of open new avenues for research on single-cell, single molecule fouling and clogging, future studies may concentrate on analysis, drug screening, and the discovery or testing of design of smart geometries and development of fabrication personalized therapies using patient-derived cells. methods used for efficient cell/molecule separations and DNA sequencing. Integration of nanofluidic systems Acknowledgements: F.S. is grateful for the support of with microchannels becomes compulsory to provide the projects PCE8/2021 by UEFISCDI and LAPLAS VI nanochannel connectivity. Indeed, it is necessary to con- (16 N/2019). nect laboratory tools with nanoscale spaces so that graded Author contributions: All the authors have accepted structures and tapered dimensions could provide adequate responsibility for the entire content of this submitted fluidic control. Large area hierarchical fluidic structures manuscript and approved submission. should be then reliable and robust to provide the necessary Research funding: This work has received funding from solution to nanofluidic application. the European Union’s Horizon 2020 research and innovation Nanofluidics will continue to trigger more interest program under grant agreement no. 871124 Laserlab-Europe. when improved fabrication techniques generating even Conflict of interest statement: The authors declare no smaller critical dimensions with higher precision and conflicts of interest regarding this article. repeatability are developed. Indeed, there is still a defi- ciency of cost-effective nanofabrication techniques that can offer device-to-device reproducibility. The laboratory References equipment is rather expensive, which makes mass production of nanofluidic systems hard. The fabrication of [1] G. M. 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