Ultrafast laser manufacturing of nanofluidic systems

1 Introduction – from micro- to nanofluidics

Microfluidics is the field that has been dedicated to the miniaturization and fluidic manipulation at microscale addressing integration of biological tools and functions in order to save systematic labor, large space, working times, and expensive costs [1, 2]. By developing complex microfluidic systems and control fluid dynamics by automatization, robotic workstations offer nowadays possibilities to solve essential issues for chemistry, biology and medicine. Lab-on-chip (LOC) devices and micro-total analysis systems (μTAS) have been then proposed to tackle specific applications, which typically involve microfluidics to be used for separation techniques [3], [4], [5], [6], [7], [8], micro-electro mechanical systems (MEMSs) [9], [10], [11], [12], clinical applications [13], [14], [15], forensic or molecular diagnostics [16], [17], [18], and proteomics [19], [20], [21] or for innovative tools enabling advanced research in the cancer field [22], [23], [24], [25], [26]. Fluid dynamics associated with microfluidics is dominated by inertial nonlinearity while the mass transport in such devices is controlled by viscous dissipation [27]. Specifically, viscous dissipation of mechanical energy induces fluidic resistance and transformation into heat due to internal friction [28]. Reynolds [29] and Peclet [30] are two dimensionless numbers often used in microfluidics to describe the physical phenomena within the microfluidic space. The Reynolds number, Re, measures the ratio between inertial and viscous force (Eq. 1), while Peclet number, Pe, defines the importance of convection in respect to diffusion (Eq. 2). Re could be related with Pe by Eq. (3).

where ρ is the fluid density, v is the average velocity, µ is the dynamic viscosity, l is the characteristic length, ʋ is vorticity difussivity and D is the diffusion coefficient. In microfluidic channels, since the flow velocity is low, the Re becomes small. This reflects the dominant role of the viscosity over inertial forces that implies the absence of eventual vortices or turbulences. Then, at such a low Re number, the Newton’s second law for fluid particles (known as Navier–Stokes flow state equation) which governs the fluid motion can be estimated as [31]:

where p is the fluid pressure. It evidences that the fluid velocity is given by pressure distribution only, the inertial force is negligible, the diffusion is dominant while the motion could be considered symmetric in time [32]. However, different derived flow states can be applied for topologically optimized configurations of microfluidic devices designed for specific applications [33].

Early works in fluidic microsystems used either silicon or glass as platform materials. In particular, microfabrication based on photolithographic techniques was applied to single crystalline silicon or glass for obtaining structures for flow injection analysis, electrophoresis and detection [4, 34]. Compared to glass, silicon is rather expensive and not suitable for the analysis by conventional optical methods due to opaque characteristics in a visible range. These materials have been recently replaced by polymers which contribute to rapid advancement of the microfluidics field. Indeed, the introduction of soft lithography for polymer microfabrication had a huge impact on microfluidics as it was a simple technique enabling fabrication of micro-systems without the need of using clean room equipment. Soft lithography employs the non-photolithographic strategy for micro- and nanofabrication. By using an elastomeric stamp with relief structures one may create patterns with micro or even nano scale feature sizes down to 30 nm [35]. These characteristics were then exploited to control patterning of complex molecules relevant to biology or to fabricate channel networks appropriate for use in microfluidics for cellular manipulation. The soft lithography is also the convenient, inexpensive, and rapid technology to produce configurations of large feature sizes used in biology (≥50 µm) without considerable effort [36]. In addition, as compared with rigid materials, the elastomers offer a simpler alternative to fabricate components required for microanalytical systems such as pumps and valves, and are also adequate to work with living mammalian cells as it offers good permeability to gases. Exploratory research in microfluidic systems has been carried out using poly(dimethylsiloxane), or PDMS, an optically transparent, soft elastomer.

As the microfluidics field advanced rapidly, it was further expected to downsize the scale to nanosizes for challenging the physics of fluid flow in ultrasmall channels and the possibility of single molecule analysis. Thus, a new field of nanofluidics has emerged and received considerable attention, especially oriented towards applications of small systems that include microarrays for DNA sequencing, microfluidic devices for polymerase chain reaction (PCR), LOC systems for synthesis and analysis of peptide and oligonucleotide libraries, microchips for drug screening or for single cell analysis. Nanofluidics can be also useful for sample manipulations in analytical chemistry, such as sample injections, separation, purifications for quantitative and qualitative analysis.

The nanofluidics was defined as the study of fluid transport in or around nanosized objects or, more concretely, the microfluidic system with at least one characteristic dimension below 100 nm [37, 38] while extended nanofluidics is considered to include the engineering of fluids in channels of submicrometer dimensions [39]. A channel surface usually gets electrostatic charges in contact with aqueous solutions. It then binds counter-ions from liquid, developing an electric double layer (EDL) close to the surface. The excess counter-ions will travel, inducing liquid motion by viscosity consequence. This motion is known as electroosmotic flow [40]. The characteristic thickness of the EDL, known as Debye length, is given by

where k is a constant related to the ionic composition and defines the equilibrium distribution of ions diffused in the EDL. In contrast to the microscale channels where the thickness of the EDL can be completely negligible, this becomes critical for the flow in nanochannels in which its dimensions may be comparable or even smaller than the Debye length. Thus, the mass transport in nanofluidic systems is different as compared with that in microfluidic systems since the electric potential barrier given by a large EDL affects transport properties of ionic species. However, for most microfluidic applications, the EDL thickness may be negligible in the study of electroosmotic flow, if λD/h≪1, where h is channel height. Nevertheless, the Navier–Stokes flow state equation (Eq. 4) was also accepted to the nanoscale while specific transport phenomena appears due to electrostatics and interplay between flow and ionic transport in small spaces [41].

On the other hand, to study nanofluidic systems as well as to provide their functionality, it is required to connect them to the macroscale. The interface is given by microfluidics which further becomes necessary to assemble multifunctional miniaturized devices [42]. Mimicking nature functionality at nanoscale in which we can reproduce complexity of biology will challenge the artificial biotic systems [43]. We show in Figure 1 a schematic description of the length scales and lithographic techniques used to fabricate micro- and nanosystems.

Figure 1:

Description of the length scales and common processing methods used in micro and nanosystems.

In the following sections we focus on the fabrication techniques of nanofluidic systems by conventional methods and ultrafast laser processing. We then address the challenges for issues concerning sizes and fluid dynamics in nanofluidic systems and present some applications. At the end, we summarize our ideas and give some prospective.

2 Conventional fabrication technologies for nanofluidic systems

Nanochannels, nanopipettes and nanopores are the most known nanofluidic platforms. Most of the fabrication technologies are focusing to obtain such platforms with the aim of proposing functional devices with specific applications, in general for biological purposes. One of the targets is to improve a resolution with a reasonable cost so that research in laboratories can afford to advance science in nanofluidics field. However, each technique has limitation in materials that can be processed.

As alternatives to photolithographic methods [35, 44, 45], a lot of efforts for development of soft lithographic techniques have been made in micro- and nanofabrication for surface chemistry, materials science, optics, MEMS, microelectronics and, in particular, in biology and biochemistry [36, 46, 47]. The soft lithography also allowed improving the resolution down to tens of nanometers and developing at the same time microfluidics and nanofluidics domains [48]. Soft lithography techniques involve the fabrication or replication of various structures by using elastomeric stamps or molds. The most used material with these techniques is PDMS, which is cast on a master mold (various processed materials, e.g. SU-8) to build elastomeric stamps. For rendering 3D aspect to the assembling, these stamps can be further sealed to glass substrates by using oxygen plasma or alternative methods.

Photolithography, on the other hand, is well established and the most settled technology for device manufacturing. Originally, Mercury lamps are used as excitation radiation sources while conventional photoresists of alkali-soluble resin mixed with a photoinitiator or photosensitizer are sensitive to energy spectrum between 250 and 450 nm. A KrF* excimer laser was the first laser source used in photo lithography for mass production of 256-Mb DRAM with a feature size of 250 nm. The resolution is given by the resolving power of the photolithography (Rayleigh limit):

where k is the process factor (typically 0.61), λ is wavelength of the light source, NA is the numerical aperture of the projection lens. The equation is effective for monochromatic light sources while the resolution for larger spectral-width light sources is inferior. Thus, a laser source will have an advantage over lamp sources due to shorter wavelength as well as narrower linewidth. The much higher intensity supplied from laser can also increase throughput.

The optical resolution has been continuously improved by breaking the barrier of Rayleigh limit using different strategies. For example, using phase-shifting masks makes it possible to improve the resolution by 40% [49]. Another method for resolution improvement was the immersion lithography. It uses a high index filling medium in the space between the optics and substrate which allows the reduction of λ in Eq. (6) to an effective wavelength of λeff=λ0/n , where λ₀ is the actual employed laser wavelength and n the refractive index of filling medium [50]. In this scheme, ArF* excimer laser (193 nm wavelength) lithography in water achieved better resolution as compared with 70 nm resolution by F₂ laser (157 nm wavelength) [51, 52] due to the refractive index of 1.44 for water at 193 nm [53]. Scanning near-field optical microscope coupled to a UV laser has been developed to create molecular patterns with dimensions nearly 15 times smaller than the Rayleigh limit [54]. For example, molecular features with widths of only 20 nm have been fabricated in self-assembled monolayers of alkanethiols on gold using this method [55]. Interferometric lithography (IL) typically using two coherent light beams at +θ, −θ angles to expose photoresists offers a maskless, large-area, nanoscale processing with an interference pattern of period λ/2sinθ [56]. Development of special photoresists enables to get nanofeatures down to ∼λ/4 with the combination of ArF* excimer laser, which is as small as 48 nm as compared with the laser wavelength of 193 nm [57]. One may further decrease the resolution by using IL in immersion media down to 33 nm [58] or by using IL of X-rays down to 3 nm [59].

Extreme ultraviolet (EUV) or X-ray lithography uses optical radiation of very short wavelengths of 10–14 nm emitted by either laser-produced plasma or synchrotrons sources. A typical setup also includes optics and a reflective mask or a proximity mask to direct the patterned radiation beam onto a special photoresist material. The resolution given by Eq. (6) can thus achieve sub-100 nm features [60]. EUV lithography is expected to improve the processing resolution down to 10 nm by the appearance of new stable sources with sub-10 nm wavelengths [61], [62], [63]. The drawback for EUV and X-ray lithography is that conventional refractive lenses cannot be used in typical setups and must be developed while a special attention should be paid due to the high radiation energy that can damage most of the masks or lenses.

Electron-beam lithography (EBL) is one of the most used technologies to create patterned structures of nanoscale feature sizes with high-accuracy and flexibility in replication. It employs electron sources such as thermionic or field electron emission sources, which are tightly focused by electrostatic and magnetic lenses to direct the electron beam usually towards a resist material. The finely developed structures are then usually transferred to a desired substrate for specific applications by etching, typically reactive ion etching. Most common electron-sensitive resist materials are based on poly(methyl methacrylate) (PMMA) or so called chemically amplified resists (CARs). The most common EBL resists along with their lithography properties are presented in detail in Ref. [64]. It is possible to achieve sealed nanofluidic channels with cross-sections of 10 × 50 nm, which are essential for confining molecules of biological relevance [65]. By EBL it was also feasible to create sub-10 nm PMMA tranches by resist development at low temperatures [66]. In addition, by applying EBL and subsequent etching, sub-10 nm nanoholes in Si, SiN or SiO₂ membranes can be fabricated with high precision [67].

Another high energy beam processing method at nanoscale is use of focused ion beam (FIB). A typical FIB technology uses a liquid metal ion source and electrostatic lenses to focus the ion beam onto the sample for lithography or direct substrate milling due to sputtering of atoms from the substrate. It is a very precise maskless technique and attains a processing resolution below 10 nm, which is the minimum of focused ion beam size [68]. FIB can be applied in various setups, from the site-specific preparation and even for composite samples, to sectioning at submicrometric level and nanostructuring or to ion-induced chemical vapor deposition (FIB-CVD) [69]. FIB can thus create 3D nanostructures made of metal or insulator materials starting from organic precursors [70]. FIB was also applied to create nanopores with nanometer control in SiN membranes [71].

Nanoimprint lithography has been developed as a need to produce large-area patterning with nanoscale feature sizes on casting materials [72, 73]. This technology was implemented to support conventional lithography for high-throughput, low-cost printing. Specifically, by producing compact stamps or molds with desired geometry and nanoscale features using optical lithography, EBL or FIB techniques one may further apply to stamp them on a printable polymer. In this process, critical aspects are related to the proper choice of polymers for nanoprinting [74]. Indeed, in order to obtain stamping resolutions of about 10 nm, requirements involve good mechanical behaviors and antisticking properties, and biocompatibility is further demanded, if they are used for biological purposes.

3 Ultrafast laser processing for nanofluidic systems

Ultrafast lasers are a relatively new category of pulsed lasers with ultrashort pulse duration from few picoseconds to several tens of femtoseconds [75]. Such lasers exhibit extremely high peak intensities (>10 TW/cm²) that allow modification of transparent materials by multiphoton absorption with resolutions beyond the diffraction limit as given in Eq. (6) [76] (Figure 2). Additionally, the multiphoton absorption provides 3D fabrication capability of the transparent materials. The transparency at the laser irradiation wavelength permits photons travelling through the material. However, an intense laser field given by the high peak intensity of ultrafast laser allows to spatiotemporally concentrate the photon energy in a material volume within a focal area of laser beam capable of exciting electrons over the forbidden energy gap due to simultaneous absorption of multiple photons (multiphoton absorption) (Figure 2a). The multiphoton absorption can improve the fabrication resolution beyond the diffraction limit because the probability of multiphoton absorption depends on the laser intensity and the ultrafast laser has typically a Gaussian beam profile (Figure 2a and b). The effective spot size w of m-photon absorption is given by

where w₀ is an actual spot size of focused ultrafast laser beam. Combination of the threshold effect for reaction with the multiphoton absorption achieves the fabrication resolution far beyond the diffraction limit [77].

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]).

The focused laser beam positioning in respect to the transparent sample with high precision 3D translational stages will then allow obtaining complex 3D configurations, which otherwise are impossible to be obtained by planar technologies.

In addition, the thermal diffusion to the outside of processing area is significantly eliminated since the process can be completed before thermal diffusion occurs due to the pulse duration shorter than electron–phonon coupling time from several ps to few tens of ps. This thermal diffusion suppression is essential for laser processing of materials with high accuracy and high resolution. Micro- to nanofeatures can be thus assembled into novel architectures and arrangements with features down to sub-100 nm [78]. In the following we show the potential of ultrafast laser processing for nanofluidics fabrication by two different schemes: additive and subtractive processing.

3.2 Subtractive laser processing

There are two main approaches for fabrication of nanofluidic systems by subtractive laser processing: laser ablation and laser assisted etching. The former one can be applied to various materials while the latter one is limited to few materials only. Femtosecond laser nanoablation of GaN was demonstrated with surface modification at a sub-diffraction resolution [82]. In this study, a laser wavelength of 387 nm was employed to achieve nanohole arrays with sub-200 nm diameters due to collaboration of multiphoton absorption and the threshold effect. By femtosecond laser direct write ablation in water, nanochannels with diameters of about 700 nm with a complex 3D geometry have been fabricated in fused silica [83]. Further employing periodic nanograting formation in the water-assisted femtosecond laser ablation, which was a specific phenomenon for femtosecond laser processing, enabled fabrication of nanofluidic channels with transverse widths narrower than 50 nm [84]. More details of this technique are given in Section 6. In the water-assisted ablation, the water medium has an important role to remove the ablated residues from the nanochannels and eventually to create long channels with complex 3D geometries.

The use of nondiffractive beams such as Bessel beams became motivating for femtosecond laser processing, in particular for high aspect ratio (>10:1) nanochannels, with widths of 200 nm and lengths of 20 µm [85]. Such beams have a specific characteristic so as to make the intensity profile constant along the propagation [86]. They became thus challenging alternatives to other nanoprocessing technologies for high efficiency nanochannel fabrication due to highly localized and controlled energy deposition in transparent materials, although they can create only straight channels [87].

The other well-known subtractive ultrafast laser based technology for the fabrication of typical microfluidic channels is femtosecond laser assisted etching (FLAE) [88]. This technique is usually applied to photosensitive glass or fused silica, which consists of femtosecond laser direct writing followed by chemical etching [89]. For the photosensitive glass, thermal treatment is necessary before the etching. The channel fabrication resolution is determined by the etching selectivity (typically ∼50) between the laser exposed and unexposed regions in the subsequent etching so that embedded microchannels are formed with resolutions, of few micrometers, inferior to the water-assisted ablation. However, a new hybrid technique that combines FLAE and TPP should allow compensation of a drawback of FLAE in terms of fabrication resolution, simultaneously offering enhanced functionality and robustness to the fabricated device [90]. In this hybrid process, FLAE is first carried out to create microfluidic channels inside glass volume which are then filled with a negative-tone photoresist. A subsequent TPP in the glass channels enables to integrate the polymeric nanochannels with a sub-200 nm resolution in the glass microchannels.

Recently, it was demonstrated that a glass microchannel size obtained by FLAE was able to be reduced via a post-thermal treatment [91]. This treatment at the temperature slightly below the glass melting point can induce glass deformation and create architectures down to nanoscale inside channels due to melting of the shallow glass surface layers that exhibit lower surface energy than bulk. Then, the surface is reformed while channel width dimensions can be controllably reduced down to several hundreds of nanometers (Figure 3).

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].

It is worth to mention that downsizing dimensions to obtain nanofluidics in microfluidic systems is an advantage to link the macro-world to nanoscale. This allows fluid communication as well as a facile analysis of the entire system. Some issues that will be mentioned in the next section can be solved by a soft transition from macro- to nanoscale.

4 Challenges for issues concerning sizes and fluid dynamics in nanofluidic systems

It has been suggested that water hydrodynamics can be considered valid for nanofluidic applications so that Navier–Stokes equations should be considered for the fluid transport down to a continuum limit of 1 nm [41, 92]. However, with the confinement increase, the surface to volume ratio increases so that the surface characteristics play critical roles. The Debye length, λ_D, explained in Section 1, Eq. (5), which characterizes the EDL, ranges from 0.3 to 30 nm, and is dependent on the ion concentration in the fluid. It is thus important to carefully evaluate fluid dynamics in confinements down to 30 nm. In consequence, it becomes critical for applications in which nanopores exhibit dimensions that equals twice the Debye length due to Debye layer overlapping [93]. This issue affects interfacial transport phenomena such as electroosmosis or electrophoresis due to ion dynamics within EDL.

Another issue is revealed by the so called Navier boundary condition that complements Navier–Stokes equation, which reveals that the friction coefficient and slip length strongly depend on the strength of interaction at fluid-solid interfaces [94]. It has been shown that large slip lengths of 10–50 nm are dominant when the contact angle of the liquid increases (weak interaction) while “no slip” boundary condition is possible at very low contact angles. In this context, it has been also evidenced that the roughness decreases the slippage while it can amplify the non-wetting surface characteristics. It is thus necessary to cautiously consider these aspects at nanoscale in order to propose engineered interfaces that reduce flow friction.

Nanobubbles have spherical or irregular shapes with diameters up to 100 nm, which are rather developed on surfaces with increased hydrophobic characteristic due to the long-range strong attractive force in aqueous media [95], [96], [97]. They may also coalesce by a gaseous bridge capillary force [98]. By molecular dynamics simulations it has been indicated that the bubble nucleation in confined nanochannels is homogenous on rather hydrophilic surfaces, and becomes heterogeneous with surface hydrophobicity while no bubble nucleation may be possible on the non-wetting surface [99]. A critical aspect of the nanobubbles is their life-time which can last up to days in certain conditions [100]. It has been also suggested that nanobubbles may be stable in water for long periods of time, due to both concentration gradient and repulsion forces at liquid-gas interface that prevent coalescence [101]. As consequence, due to attractive forces between nanobubbles, they may drastically influence velocity slip and disturb the flow in nanochannels [102].

The above-mentioned issues are main factors for appearance of more fluctuations when decreasing the sizes in nanochannels or for the non-linear transport in nanopores or nanoslits [92]. In addition, problems with molecule adsorption on the walls should be considered as the space becomes narrower. On the other hand, a positive aspect may be the precise correlation between ion transport and electrostatics in nanochannels, which can take full advantage when the height is small enough to overlap Debye layer.

As many applications of nanofluidics concern chemical or physical analysis, another important aspect is the detection and observation inside the nanochannels. For chemical analysis, detection of low concentration analytes is difficult in small volumes so that expensive single molecule detection methods become necessary. On the other hand, for the observation one may use conventional two-photon or other sub-diffraction limited microscopy, in which the image is then captured by point scanning of a tightly focused pulsed laser beam. Alternatively, temporal focusing with widefield illumination could represent a benefit for a scanless scheme, already applied to super-resolution imaging or even photolithography [103].

5 Applications of nanofluidics

In this Section, we refer to some concrete applications of nanofluidics including nanopores, nanopipettes and nanochannels. We, however, put more emphasize on the use of nanochannels for the biological field, because the ultrafast lasers have been limited to be proposed for this application so far. The nanochannels can be categorized in three geometric configurations: square, planar and high-aspect ratio nanochannels [104]. In all applications, nanofluidics is rather an engineering tool to produce molecules confined in ultra-small spaces and then expose them to controlled forces for exploring fundamental knowledge, especially in biological systems, at the molecular level.

5.1 Nanofluidics fabricated by conventional technologies

Nanopores correspond to sub-100 nm or smaller diameter structures with lengths below 10 µm. Solid-state synthetic nanopores became alternatives to the biological counterparts as they are more stable, controllable in dimensions with tailored properties specific to the applications. They are necessary tools for single molecule analysis as the passage of individual DNA, RNA or protein molecules through such a nanoscale space is essential in many biological processes [105]. In particular, processes such as DNA translocation through the nanopore may help in unfolding and linearization of the molecule with great use in its sequence reading for genomics applications [106]. Pores fabricated by FIB and EBL techniques were proposed with the initial studies for DNA translocation [71, 107]. It was then found out that the singles-strand DNA (ssDNA) could pass through sub-2 nm pores while double-strand DNA (dsDNA) was blocked but could pass through the larger pores [108]. Other proteins can be as well translocated through pores of various dimensions so that controllable unfolding may be possible. Ultrasmall and ultrathin nanopores with pore diameters and thickness below 2 nm were fabricated by laser-assisted SiNx etching coupled with dielectric breakdown [109]. Such pores were used to discriminate DNA length as well as sense DNA homopolymer sequence identification with very high sensitivity. A laser-etching technique recently developed was employed for in situ nanopore formation inside a sealed microfluidic system [110]. In this study a near-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 channel (∼2.5 μm wide and 200 nm deep) were first prepared for funneling linearized molecules to the nanopore. By pressure-induced flow, DNA concentration as low as 50 fM was delivered to the nanopore. Ultralong DNA molecule can be then translocated in a graded micro/nanofluidic platform for sensing of biomolecules.

The main potential of nanopores is expected by single-molecule DNA sequencing and could represent a key technology for reading genome at single cell level [111]. Some other applications have been proposed for electrochemistry [112, 113] and single molecule conformation analysis [114], bioseparations in electroanalytical chemistry and biosensing [115], [116], [117], biomimetic stimuli-responsive membranes, and energy generation from salinity gradient and light [118].

Nanopipettes are defined as structures with needle-like geometry of sub-200 nm diameters with various ratios between outer and inner diameters, dependent on applications [119]. Different from the nanopores, due to relatively larger dimensions, they are more robust, exhibit good wettability and can be easily integrated in microfluidic devices. Besides the common applications with the nanopores, they may be further used for nanoinjections (dispensers or aspirators of ultrasmall volumes), nanobiopsies with subcellular precision, or probes for microscopy. They are usually made of glass or quartz, commonly by a laser pipette puller instrument capable to downsize diameters to nanometer scale, depending on glass thickness, temperature, or pulling force. It achieved fabrication of nanopipettes by sequential heating and pulling with diameters below 80 nm [120] in borosilicate glass or even sub-10 nm in quartz [121]. It was thus possible by electrophoretic and dielectrophoretic forces to pull and trap proteins and DNA with a higher concentration closer to physiological conditions [120]. This approach could be very useful for ultrasensitive detection in miniaturized bioassays.

Nanochannel-based nanofluidics integrated in functional devices are essential for applications that involve biomolecule transport, bioseparation, and biodetection [122]. Due to their dimensions with the order of biomolecules, they initially offer potential in DNA and other molecule separation, detection, and sensing [123]. Conventional DNA detection based on gel uses porous media with limited pore sizes and poor mechanical properties. In contrast, electrical field applied in nanochannels can provide precise control on size dependent separation of DNA and exhibit capacity to probe the conformational properties of DNA, sort the molecules within confining environment and define the spatial location of genetic information along the molecule [124, 125]. An important biosensing application of dedicated devices is the mapping and observation of genomic and epigenomic DNA information during stretching of DNA in nanoscale channels [126], [127], [128].

Photolithography is the most used conventional method for fabricating planar nanochannels as the depth of channel can be precisely controlled down to few tens of nm by the subsequent etching process. The channels with narrow constrictions and wider regions were proposed in pioneering work for the separation of long DNA molecules. Size-dependent trapping of DNA and electrophoretic mobility differences allowed separating long DNA molecules in 15-mm-long channels without the use of a gel matrix or pulsed electric fields [129]. Interference lithography is complementary to the standard lithography technique as it can be used for fabricating deep nanochannels [57]. One may easily generate periodical narrow nanochannels on large areas that can be applied to DNA transport and stretching. EBL was also applied for fabricating nanochannel-based nanofluidic systems for DNA analysis. EBL followed by reactive ion etching fabricated arrays of 100 nm wide nanochannels in fused silica [130]. Specific DNA orientation was found and transport direction of DNA was controlled under DC electrophoresis. Nanochannels of sub-5 nm lateral dimensions with smooth surfaces were fabricated by FIB milling and then sealed with a cover plate, which were also found suited for single-molecule DNA transport studies [131, 132]. Integrated multi-level lithographic processes consisting of EBL, UV stepper and printer were combined with dielectric deposition by plasma-enhanced chemical vapor deposition, plasma etch and chemical mechanical polishing for the fabrication of a functional nanofluidic device [133]. This consisted of stacked multi-layers on a silicon single chip with channels of lateral dimensions from <20 nm to 1 mm and vertical from 40 nm to >2 μm. This scale up-scale down architecture allowed fast fluidic transport and controlled biomolecular manipulation demonstrated by regulated λ-DNA straddling and stretching in an array of nanochannels and nanopillars. In addition, damage assays of single-molecule DNA are possible in a nanofluidic chip capable to stretch the molecule in constrictive channels of ∼50 nm in width [134]. In this case, the nanofluidic biochip was fabricated on thermoplastics by nanoimprint lithography.

Most of developed nanochannels for DNA analysis suffer, however, from complex fabrication procedures together with issues of DNA overloading at the nanochannel entrance which obstruct solution exchange. New strategies are under development targeting simplicity and relevant hierarchical or graded architectures that can controllably downsize dimensions and create a functional interface of micro and nanofluidic systems. FIB-milling was found useful for 3D nanofunnels connected with microscale reservoirs that define dynamics of DNA molecules with increased confinement. Then, electrokinetically driven introduction of the DNA molecules into a nanochannel was facilitated by incorporating the 3D nanofunnels at the nanochannel entrance [135].

Another study proposed light-induced local heating of liquids inside micro- and nanofluidic channels fabricated by thermal imprint in low molecular weight PMMA [136]. It was demonstrated thermophoretic manipulation of genomic-length DNA in micro- and nanochannels as well as compression or stretching of DNA in nanochannels by steep temperature gradients created by the light-induced local heating.

The main challenge with fabrication technologies for such applications is the construction of a reliable device that can target single-cell trapping and then allow subsequent single-genome analysis. The bottleneck resides in interfacing microscale confinement for single-cell manipulation with nanoscale confinement for single-molecule DNA linearization.

Recently, a rather simple multilayer soft lithography corroborated with controllable elastomeric collapsing allowed the fabrication of a uniform nanochannel array with confining spaces down to 20 nm and lengths up to sub-millimeters. In this study, the authors obtained in a single micro/nanofluidic biochip with complex scale up–scale down architectures that were used to either cell isolation, lysis, DNA extraction, purification, labeling, or linearization for single-cell genomic analysis (Figure 4) [137].

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].

Another integrated micro- and nanofluidic device was fabricated by FIB milling and UV nanoimprint lithography [138]. Microchannels and nanochannels of different depths and layouts were combined to facilitate and smoothen the flow (Figure 5). It then showed an improved stretching of DNA molecules in long nanochannels whose widths and depths were gradually decreased. It was further evidenced that the inlets captured more molecules as they exhibited smooth graded transitions due to low entropic barrier. Eventually, a nanochannel with squared transient configuration collected twice as many molecules as that with the abrupt transition.

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 sensing, other applications using the nanochannels include nucleic acid biopsy in precision medicine [139], nanochannel chromatography for single-cell analyses [140], bio-inspired nanochannels for molecular filters, biosensors, energy conversions [141], platform for detection and characterization of individual sub-100 nm particles with applications in semiconductor manufacturing, environmental monitoring, biomedical diagnostics and drug delivery [142].

5.2 Micro- to nanofluidics fabricated by ultrafast laser processing

The development of ultrafast lasers with high-repetition rates (GHz to MHz) has now made this process to be used for many applications, including in industry and medical clinics. Ultrafast laser processing with capabilities of 3D and nano fabrication demonstrated to be a very useful tool to shape the material with topography, morphology, and structural arrangement. This technology alone or in combination with other conventional processing was applied to develop functional biochips containing 3D nano-components for integrated optics and lab-on-a-chip applications. Graded or hierarchical configurations with dimensions from hundreds of micrometers to tens of nanometers were then developed for various materials with specific surface characteristics that can be proposed for a wide range of micro- and nanofuidic applications.

UV lithography was combined with TPP to integrate nanochannels with microfluidic channels [143]. Specifically, TPP was applied to fabricate nanofluidic channels of various heights down to sub-100 nm using conventional photoresists, which were connected to two microchannels fabricated by the UV lithography. For TPP, a Menlo System C-Fiber 780 HP Er:doped fiber oscillator (120 fs, 100 MHz) integrated with amplifier was used to generate a second harmonic. SU-8 photoresist was used for both the UV lithography and TPP. A microfluidic pattern of UV light was projected using a mask, followed by TPP for creation of the nanochannel. The post baking at 95 °C for development followed by washing left the mold of nanofluidics, which was used for replication by soft lithography with PDMS. The test of nanofluidic device was carried out by aqueous dye solution. In this case, a total internal reflection illumination was used to observe single fluorescent molecules diffusing into the nanochannels from the reservoirs. TPP allows integration of nanopatterns with complex shapes and various sizes into microfluidic devices. The combination of EBL and UV lithography is also conceivable; however, TPP exhibits flexibility for the potential integration of nanofluidic functionalities into volumes of transparent microfluidic devices.

As already introduced in Section 3, sub-50 nm wide nanofluidic channels were successfully obtained in silicate glass volume by ultrafast laser direct write ablation in water [84]. In this study, a focused femtosecond laser beam (Coherent, Inc., 800 nm, 100 fs, 250 kHz) was employed to fabricate hollow 3D micro- and nano-channels inside a glass substrate (Figure 6a–c). To achieve sub-50 nm nanochannels, a porous silicate glass was used as a substrate. The pores of 10 nm were uniformly distributed in glass volume to create a 3D connective network, which had an important role to efficiently supply water to the ablation site inside glass. After the ablation, thermal treatment was carried out to consolidate the porous glass, which transformed it to compact silicate glass while the micro and nanofluidic structures inside the glass remained formed. A computer-controlled XYZ translation stage was used to allow the fabrication of complex desired 3D geometries in the glass substrate. The fabrication resolution far beyond the diffraction limit achieved relied on a periodic nanograting formation, which is specific for femtosecond laser processing. A periodic nanograting can be formed inside the glass with irradiation of a linearly polarized beam [144], which is ascribed to spatially modulated energy deposition with nanoscale periodicity inside the glass. By intentionally decreasing the intensity of femtosecond laser beam with a Gaussian profile, only a single cycle of the modulated energy distribution at the center of beam can exceed the threshold intensity for ablation to create a single nanochannel (Figure 6d–f). Using this strategy, an integrated micro-nanofluidic system in which two microfluidic channels were connected with an array of nanochannels could be obtained. Stretching of DNA molecules was observed in these nanochannels to evidence their potential for investigation of single molecular behaviors (Figure 6g).

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].

A new concept of hybrid technique referred to as “ship-in-a-bottle” laser fabrication was proposed [90, 145], in which subtractive FLAE of glass and additive TPP were sequentially performed for integration of polymeric nanostructures in glass microfluidic devices. This hybrid technique exploits the advantages of both processes while compensates each other’s disadvantages. The fabrication resolution of TPP inside the glass microchannel was improved by using a spatial light modulator for wave front correction [146]. This hybrid approach was thus proposed to create 3D environments at the submicrometric scale inside a closed glass microfluidic chip (Figure 7a and b). The second harmonic of a Yb-fiber laser beam (532 nm; 360 fs, 200 kHz) was used for 3D direct writing of glass, followed by thermal treatment and etching to develop microfluidic channels. TPP of SU-8 photoresist filled in the glass microfluidic channels was carried out to develop polymer nanochannels inside the fabricated glass microchannels (Figure 7c–e).

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].

The array of nanochannels acted as a nanoscale diffusion-based gradient generator that allowed forming a stable gradient of biochemical epidermal growth factor (EGF) attractant. Prostate cancer (PC3) cells were cultivated inside the hybrid 3D biochips and exposed to the chemo-attractant gradient to evaluate specific behaviors, such as migration and invasiveness. It was evidenced that the cancer cells could penetrate the submicrometric channels, migrate very fast and then split into vesicular fragments that eventually fused back into single bodies.

The fabrication resolution of FLAE was also improved by inducing glass deformation via post-thermal treatment (nanoscale glass deformation-NGD) to create nanochannels in an ultrathin glass substrate. The Yb-fiber laser beam (532 nm; 360 fs, 200 kHz) was used for glass irradiation. This process provides much more simple schemes in terms of the fabrication procedure and the material due to elimination of TPP process and the biochip consisting only of glass. More importantly, it can create an in vivo-like environment with architectures down to the nanoscale, providing narrow constrictive spaces for cancer cell migration. Using such biochips enabled to observe the capability of PC3 cells to penetrate consecutive narrow constrictions that mimic intravasation–extravasation events in the in vivo environment. Very long (∼50 µm) nucleus stretch was then confirmed during migration in the nanochannels without altering their viability (Figure 8). In addition, the ultrathin chip allowed fluorescence microscopy analysis with high resolution. It could be then observed that the cell body was compressed to a thin sheet-like shape to occupy the entire narrow constricted region in the nanochannel from the entrance to the exit, whereas the cell nucleus was deformed to a disk-like shape that was pulled into the constricted region of the channel later during the migration process.

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].

Some future applications of nanofluidic devices may include single-cell genomic analysis for personalized cancer therapies. Biopsies from cancer tumors harvested from patients could be analyzed in micro/nanofluidic biochips. The evaluation of cell population behavior could be correlated with the degree of migration and invasiveness and genomic analysis at single-cell level. The objective could be to test specific doses of radiation therapy, specific chemotherapy, immunotherapy, or a combinations.

6 Summary and future prospects

Microfluidic systems helped developing single-cell analysis methods in microspaces. Extension to nanospaces is increasingly necessary to provide a new generation of analytical tools for biological applications. Due to accurate control of liquid flow and of molecular behavior at the nanoscale, nanofluidic systems found a prominent role either in the analysis of individual cells or biomolecules. Analytical nanosystems in such small spaces could provide ultra-sensitive analyses at a single-cell and single-molecule level. It can be supposed that nanopores and nanochannels could offer the essential support of label free identification and characterization of single-stranded genomic DNA or RNA without conventional amplification while DNA sequencing becomes possible. Mass transport at this scale could reveal new phenomena as e.g. enhanced mass flow rate and highly selective molecular transport, similar to those in natural transmembrane proteins such as ion channels and aquaporin.

Since critical issues of nanofluidic systems consist of fouling and clogging, future studies may concentrate on design of smart geometries and development of fabrication methods used for efficient cell/molecule separations and DNA sequencing. Integration of nanofluidic systems with microchannels becomes compulsory to provide nanochannel connectivity. Indeed, it is necessary to connect laboratory tools with nanoscale spaces so that graded structures and tapered dimensions could provide adequate fluidic control. Large area hierarchical fluidic structures should be then reliable and robust to provide the necessary solution to nanofluidic application.

Nanofluidics will continue to trigger more interest when improved fabrication techniques generating even smaller critical dimensions with higher precision and repeatability are developed. Indeed, there is still a deficiency of cost-effective nanofabrication techniques that can offer device-to-device reproducibility. The laboratory equipment is rather expensive, which makes mass production of nanofluidic systems hard. The fabrication of nanochannels with controlled sizes and well-controlled surface properties responsive to fluidic transport is still a challenge.

There are few techniques capable to develop nanofluidic systems such as FIB, EBL, UV and deep UV lithography rendering structures with dimensions from hundreds of to tens of nanometers and well-controlled surface characteristics are still expensive, so that further development is awaited. They are also difficult to be employed for rendering functionality by creating graded or hierarchal configurations for specific applications, for which repeated use of multiple techniques is necessary.

Besides the nano-scale surface processing of a wide range of materials such as metals, semiconductors, ceramics, polymers and even soft materials by nanomachining, nanostructuring and nanoablation, ultrafast laser technology can be a viable alternative to fabricate highly functional biochips integrated with almost arbitrary shapes of 3D nano-components. Specifically, the multiphoton absorption of ultrafast lasers can induce nanoscale modifications and fabrication in volume of transparent materials with desired geometries and configurations by TPP, water-assisted ablation and FLAE. Additionally, the processed regions can be controllably functionalized in a space selective manner. It is then possible to integrate functional components and devices for integrated optics and lab-on-a-chip applications. New devices with graded or hierarchical configurations consisting of different dimensions from hundreds of micrometers down to few nanometers and with various surface properties can be developed in a wide range of materials. Such biochips may open new avenues for research on single-cell, single molecule analysis, drug screening, and the discovery or testing of personalized therapies using patient-derived cells.