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Dec 29, 2018 - The diffusive mass transfer between the two fluids was estimated and the optimal chip ... for low diffusion rate of the components was defined.
micromachines Article

Laser-Inscribed Glass Microfluidic Device for Non-Mixing Flow of Miscible Solvents Valeria Italia 1 , Argyro N. Giakoumaki 2,3 , Silvio Bonfadini 1,2 , Vibhav Bharadwaj 3 , Thien Le Phu 2,3 , Shane M. Eaton 3, *, Roberta Ramponi 2,3 , Giacomo Bergamini 4 , Guglielmo Lanzani 1 and Luigino Criante 1, * 1 2 3 4

*

Center for Nano Science and Technology, Istituto Italiano di Tecnologia, 20133 Milano, Italy; [email protected] (V.I.); [email protected] (S.B.); [email protected] (G.L.) Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy; [email protected] (A.N.G.); [email protected] (T.L.P.); [email protected] (R.R.) Istituto di Fotonica e Nanotecnologie-Consiglio Nazionale delle Ricerche (IFN-CNR), Piazza Leonardo da Vinci 32, 20133 Milano, Italy; [email protected] Department of Chemistry Giacomo Ciamician University of Bologna Via Selmi 2, I-40126 Bologna, Italy; [email protected] Correspondence: [email protected] (S.M.E.); [email protected] (L.C.); Tel.: +39-320-092-1952 (S.M.E.); +39-022-399-9812 (L.C.)

Received: 24 October 2018; Accepted: 24 December 2018; Published: 29 December 2018

 

Abstract: In recent years, there has been significant research on integrated microfluidic devices. Microfluidics offer an advantageous platform for the parallel laminar flow of adjacent solvents of potential use in modern chemistry and biology. To reach that aim, we worked towards the realization of a buried microfluidic Lab-on-a-Chip which enables the separation of the two components by exploiting the non-mixing properties of laminar flow. To fabricate the aforementioned chip, we employed a femtosecond laser irradiation technique followed by chemical etching. To optimize the configuration of the chip, several geometrical and structural parameters were taken into account. The diffusive mass transfer between the two fluids was estimated and the optimal chip configuration for low diffusion rate of the components was defined. Keywords: optofluidics; lab-on-a-chip; femtosecond laser; laser micromachining; diffusion

1. Introduction The recent introduction of microfluidics in chemistry and biology has led to a paradigm shift in both fields. Lab-on-a-Chip is now a commonly known concept and significant efforts have been made for the realization of multifunctional integrated systems for chemical analysis [1], cell culture, and biochemical systems investigation [2], but most importantly for multiphase chemical reactions even for miscible solutions. Microfluidic reactors have already been proven valuable due to their high surface-to-volume ratio, the scale-out capabilities for industrial applications, the higher yield over batch reactors, and the versatility of the microfluidic chip set-ups [3]. By manufacturing a microfluidic chip with a suitable geometry, it is possible to manage simultaneously two or more fluids and create dynamic interfaces between them while avoiding active mixing due to laminar flow [4,5]. The present approach exploits (mimics) liquid–liquid interfaces which could not be accessed in batch situations. Diffusive mixing between two interfacing laminar flows is a theoretically, and in some cases experimentally, well-defined process [6,7]. It depends mainly on the area of interaction, the laminarity of the flow, the time of interaction, and the concentration gradient between two or more streams. A wide variety of materials and techniques have been employed for the fabrication of microfluidic systems [8]. The most popular process is the soft lithographic fabrication of 2D chips on Micromachines 2019, 10, 23; doi:10.3390/mi10010023

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polydimethylsiloxane (PDMS) [9] and other elastomers. The technique is easy, fast, and has a low cost, but although PDMS’ porosity is a virtue for long-term cell cultures, it becomes a drawback when it comes to chemical analysis or organic synthesis because it cannot be defined as chemically inert. PDMS can undergo swelling due to solvent adsorption in the pores, creating deformations on the microfluidic channels [10]. Extensive deformation can create leakages, thus compromising the chip. Combined with the incompatibility with many organic solvents, PDMS has a limited range of applications in synthetic chemistry. Even though a plethora of water-based biological applications have been demonstrated for PDMS microfluidic chips, the use of other solvents is prohibited due to the incompatibility of PDMS. It has been demonstrated that water-based chemical reactions [4] are able to be performed on the interface of two interacting laminar flows in a PDMS microfluidic chip. This high yield and recyclable approach could also be applied in organic chemistry, especially for reactions that involve toxic or expensive reagents. Lee et al. [10] performed an extensive study on the compatibility of PDMS with a variety of organic solvents, and it is evident that, for a broader application of microfluidics in organic chemistry, PDMS is not the optimal material. One of the most promising materials that can overcome the above mentioned challenges is fused silica. Fused silica microfluidic devices have been well established, but the exploitation of a liquid–liquid interface that is present in a Y-shaped fluidic system requires an extensive study on the behavior of pressure-driven flows. Glass chips differ from PDMS as they are rigid, and they do not exhibit deformation upon high-pressure-driven flow [11]. Fused silica is compatible with a wide variety of organic solvents as well as water, and since it is not gas-permeable, it can be used as a material for the fabrication of microreactors for a wide variety of reactions, including water splitting for hydrogen production. In this paper, we report the fabrication and characterization of a double Y-branch fused silica microfluidic device for the introduction, interaction, and separation of two miscible solutions characterized by laminar flow, taking advantage of the femtosecond laser irradiation followed by the chemical etching (FLICE) method [12–16]. There are many advantages attributed to this microfabrication technique in comparison with traditional photolithography, including the ability to quickly realize 3D monolithic structures completely buried in the substrate without the requirement of masks or a clean room. With FLICE, there is no need to create complex microfluidic chips in two halves to be subsequently welded as it is a process which often leads to sealing problems. As a preliminary investigation, the laser-fabricated, fused silica microfluidic device was used for the study of the diffusion of Rhodamine 6G (R6G) in the ethanol–ethanol interface. The angle between the two inlets and the height of the chamber were varied, and the diffusion was qualitatively determined for different flow rates. R6G was used as a colorant for one of the two streams due to its optical properties and its well-established diffusivity in ethanol. 2. Materials and Methods 2.1. Methods Fused silica glass is ideally suited for this application as it possesses several important characteristics: it is chemically inert to a variety of solvents, hydrophilic, thermally and mechanically stable, and optically transparent in a wide range of wavelengths [12,17]. To fabricate the optofluidic device in the bulk of glass, we exploit the FLICE method [12,13], which requires two steps: (1) tightly focused, femtosecond, laser pulses drive a permanent and localized periodic redistribution of material density, which defines the desired structure on the surface or in the bulk of fused silica [18]; (2) chemical etching of the laser-modified volume by a strong acid or a strong base (typically HF or KOH, respectively) to remove the irradiated zone, producing the hollowed-out, microfluidic device [15]. The femtosecond laser used for device fabrication in fused silica was a generatively amplified Yb:KGW system (Pharos, Light Conversion, Vilnius, Lithuania) with 230-fs pulse duration, 515-nm

Yb:KGW system (Pharos, Light Conversion, Vilnius, Lithuania) with 230-fs pulse duration, 515-nm wavelength (frequency doubled), and 500-kHz repetition rate focused with a 0.42-NA microscope objective (M Plan Apo SL50X Ultra-Long Working Distance Plan-Apochromat, Mitutoyo, Kawasaki, Japan). Computer-controlled, 3-axis motion stages (ABL-1000, Aerotech, Pittsburgh, PA, USA) Micromachines 2019, 10, 23 3 of 10 interfaced by CAD-based software (ScaBase, Altechna, Vilnius, Lithuania) with an integrated acousto-optic modulator were used to translate the sample relative to the laser irradiation desiderate patch. An average power (pulse energy) of 200 mW (400 nJ) and a scan with speeda of 5 mm/smicroscope were used wavelength (frequency doubled), and 500-kHz repetition rate focused 0.42-NA to laser-pattern the microfluidic device shown in Figure 1a. A multiscan writing procedure with 7 objective (M Plan Apo SL50X Ultra-Long Working Distance Plan-Apochromat, Mitutoyo, Kawasaki, μm spacing between transverse scans adopted to form the microfluidic The thickness of Japan). Computer-controlled, 3-axiswas motion stages (ABL-1000, Aerotech,device. Pittsburgh, PA, USA) the fused silica windows was 1 mm, and the buried microfluidic chips were laser-inscribed at a depth interfaced by CAD-based software (ScaBase, Altechna, Vilnius, Lithuania) with an integrated of 0.5 mm. Themodulator overall fabrication time of a singlethe chip varied between and 69 min for chamber acousto-optic were used to translate sample relative to 57 themin laser irradiation desiderate heights between 100 μm and 500 μm. The sample was etched in a sonication bath of HF (20% vol in patch. An average power (pulse energy) of 200 mW (400 nJ) and a scan speed of 5 mm/s were used to water), with a feedback-controlled temperature of 37 °C. The etching rate of the laser-exposed laser-pattern the microfluidic device shown in Figure 1a. A multiscan writing procedure with 7area µm of the fused silicatransverse was 500 μm/h, for the non-exposed area, thedevice. etchingThe ratethickness was 20 μm/h. spacing between scans whereas was adopted to form the microfluidic of the The resulting rectangular chamber’s internal dimensions were 2 mm × 200 μm (length width) with fused silica windows was 1 mm, and the buried microfluidic chips were laser-inscribed×at a depth of a0.5 height h that varied from 100 μm to 500 μm (Figure 1c). mm. The overall fabrication time of a single chip varied between 57 min and 69 min for chamber Thebetween suitable 100 structure for500 theµm. diffusion study in a microfluidic is the double configuration heights µm and The sample was etched in a chip sonication bath ofYHF (20% vol in as shown in Figure 1, showing optical microscopic images of the device after femtosecond laser ◦ water), with a feedback-controlled temperature of 37 C. The etching rate of the laser-exposed area of irradiation (Figure then after chemical etching (Figure The final device the fused silica was 1a) 500 and µm/h, whereas for the non-exposed area, 1b). the etching ratemicrofluidic was 20 µm/h. The consists two inlets and two outlets at both ends of a long which the interface interaction resultingofrectangular chamber’s internal dimensions werechamber 2 mm ×in 200 µm (length × width) with a occurs. height h that varied from 100 µm to 500 µm (Figure 1c).

Figure of the Figure 1. 1. Microfluidic Microfluidic chip chip geometry; geometry; image image from from optical optical microscope microscope of the chip chip after after (a) (a) femtosecond femtosecond (fs) laser irradiation and subsequent; (b) chemical etching; (c) schematic of chip design, h is (fs) laser irradiation and subsequent; (b) chemical etching; (c) schematic of chip design, where where h is the the theseparation separationangle. angle. chamber height and θθ isisthe

Thecomplete suitable structure for the diffusion a microfluidic chip is themass double Y configuration as To the characterization of thestudy chip,inthe behavior of diffusive transfer was studied shown in Figure 1, showingangle opticalbetween microscopic images of the device after femtosecond laserpressure irradiation by varying the separation the inlet/outlet channels, θ, and the pumping of (Figure 1a) and then afterturbulence, chemical etching (Figure 1b). Thethe final microfluidic device were consists of two the fluids, p. To avoid the inlet tubes and interaction chamber designed inlets and two at both ends of a long chamber in which the interface interaction occurs. considering theoutlets continuity of the fabrication process and the equality in resistance for both the inlets To complete characterization of the chip, the behavior diffusive masswas transfer was studied and outlets of thethe chip. A microfluidic pump (OB1, Elveflow,ofParis, France) connected to the by varying of thethe separation angle between the inlet/outlet channels, and the pumping of the reservoirs solutions. Polytetrafluoroethylene (PTFE) tubingθ, was inserted into pressure the reservoirs fluids, p. Tothe avoid turbulence, the inlet and the interaction chamber were with designed considering and drove fluids into the chips by tubes Polyether ether ketone (PEEK) tubing an outer (inner) the continuity the(150 fabrication process the equality in resistance for both the and outlets diameter of 360ofμm μm) by using anand appropriate adapter. The latter tubing wasinlets connected to the of thechip chip. A microfluidic pump (OB1, France) wasasconnected the reservoirs of glass using ultraviolet (UV) glue. TheElveflow, materialsParis, for both tubings well as thetoreservoirs and the the solutions. Polytetrafluoroethylene (PTFE)chemical tubing was inserted the solvents. reservoirs and drove the glue were selected due to their extraordinary inertness to into organic fluids into the chips by Polyether ether ketone (PEEK) tubing with an outer (inner) diameter of 360 µm 2.2. Materials (150 µm) by using an appropriate adapter. The latter tubing was connected to the glass chip using ultraviolet (UV) glue. The materials for both the tubings as wellflows, as thean reservoirs and the glue were To visualize the interaction zone between two parallel optical technique was used. selected due to their extraordinary chemical inertness to organic solvents. Using a coloured (1 mM Rhodamine 6G in ethanol) and a transparent solution (pure ethanol) it was 2.2. Materials To visualize the interaction zone between the two parallel flows, an optical technique was used. Using a coloured (1 mM Rhodamine 6G in ethanol) and a transparent solution (pure ethanol) it was possible to understand how the geometric and microfluidic parameters influence the diffusive mass transfer between two interfacing laminar flows.

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possible to understand how the geometric and microfluidic parameters influence the diffusive mass Micromachines 2019, 10, 23 4 of 10 transfer between two interfacing laminar flows. The dye solution was prepared starting from R6G powder (Sigma-Aldrich) dissolved in filtered ethanol. was prepared filtered once more to prevent undissolved dye particles and impurities The The dye solution solution was starting from R6G powder (Sigma-Aldrich) dissolved in filtered from entering the chipwas andfiltered creating turbulence. The Rhodamine solution was stored glass vials in ethanol. The solution once more to prevent undissolved dye particles and in impurities from aentering dry, cool, and dark environment to prevent degradation. the chip and creating turbulence. The Rhodamine solution was stored in glass vials in a dry, cool, and dark environment to prevent degradation. 3. Results 3. Results 3.1. Data Analysis 3.1. Data Analysis Starting from the work of Werts et al. [7], we developed a simple and useful method to extract Starting data fromusing the work of Werts et al. [7], we developed aWe simple and useful method to extract and analyze a conventional optical microscope. obtained an empty channel imageand to analyze data using a conventional optical microscope. We obtained an empty channel image to use use as a reference (Figure 2a), and subsequently we imaged the flowing colorants, varying the as a reference (Figure(Figure 2a), and2b). subsequently we imaged the management flowing colorants, varying(developed the pumping pumping pressure Using a custom image algorithm in pressure (Figure 2b).aforementioned Using a custom data image management algorithm (developed in MATLAB), MATLAB), the two images can be subtracted to indicate the difference the i.e.,two the aforementioned data images can be subtracted indicate theby difference i.e., the dye solution flow, and dye solution flow, and to eliminate electronic to noise caused the intrinsic roughness of the device. to eliminate electronic noise caused by the intrinsic roughness of the device. The visible roughness of The visible roughness of the chip, as well as the discontinuation between the chamber and the outlets, theFigures chip, as 2a,b well is as caused the discontinuation chamber anddevice, the outlets, Figure 2a,b it is caused in by the laserbetween writing the pattern of the and in even though can be by the laser writing pattern of the device, and even though it can be improved by post-fabrication improved by post-fabrication annealing, there was no visible turbulence in the chip due to this effect. annealing, therereported was no visible turbulence in the chip to this effect. As it has reported in the As it has been in the past, the roughness of due microchannels formed by been this technique have past, the roughness of microchannels formed by this technique have sub-micrometer roughness [19]. sub-micrometer roughness [19].

Figure 2. Visual Visualanalysis analysis technique of laser-fabricated microfluidic (a) reference (b) Figure 2. technique of laser-fabricated microfluidic chip: chip: (a) reference image, image, (b) colored colored image with the flowing dye solution; (c) negative image obtained by subtracting (a) from (b). image with the flowing dye solution; (c) negative image obtained by subtracting (a) from (b). The red The red line dashed line indicates theofoutput of position the chip chosen positiontochosen extract the profile. intensityScale profile. dashed indicates the output the chip extractto the intensity bar Scale bar corresponds corresponds to 200 µm.to 200 μm.

way, we obtained a clear negative negative image image (Figure (Figure 2c) 2c) from from which which the the cross-section cross-section intensity intensity In this way, outlet of of the the channel channel (red (red dashed dashed line line in in Figure Figure 2c). 2c). profile is extracted at the fixed position near the outlet image (Figure 2c)2c) is shown in Figure 3a. The is then The typical typical intensity intensityprofile profileofofthe thebi-colour bi-colour image (Figure is shown in Figure 3a. signal The signal is processed with with a low-pass filter filter (Figure 3b) and (Figure 3c) to 3c) be comparable with other then processed a low-pass (Figure 3b)normalized and normalized (Figure to be comparable with intensity profiles. other intensity profiles. At this point, in order to qualitatively define the conditions for the lowest diffusion of R6G inside the channel, we calculated the slope of the cross-section intensity profile with linear interpolation. The left region of high intensity in the normalized profile in Figure 3c indicates the presence of the dye, while the region of low intensity on the right indicates the absence of it. The transitional region highlighted in red between the high and low intensity regions represents the diffusion zone. It is important to note that the value of the slope in the diffusion region is inversely proportional to the amount of diffused dye between the liquids (ideally infinite slope indicates zero diffusion).

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Figure 3. Intensity profile of the flow behavior processed with MATLAB algorithm. The x-axis Figure 3. Intensity profile of the flow behavior processed with MATLAB algorithm. The x-axis represents the position in the image [pixels], while the y-axis is the fluorescence intensity in arbitrary represents the position in the image [pixels], while the y-axis is the fluorescence intensity in arbitrary units (a.u.). The red region indicates the diffusion zone between the two liquids. (a) original, (b) units (a.u.). The red region indicates the diffusion zone between the two liquids. (a) original, processed by a low-pass filter, and (c) normalized intensity profile. The slope of the red line represents (b) processed by a low-pass filter, and (c) normalized intensity profile. The slope of the red line the diffusion behavior. represents the diffusion behavior.

3.2. Preliminary AtConsiderations this point, in order to qualitatively define the conditions for the lowest diffusion of R6G inside the channel, we calculated thenumber slope of(Pe) the are cross-section intensityvalues profilethat withdefine linearfluidic interpolation. Reynolds number (Re) and Peclet two dimensionless The left region of high intensity in the normalized profile in Figure 3c indicates the presence and diffusive mixing characteristics of a microfluidic system, respectively. They are described as of the [20,21] dye, while the region of low intensity on the right indicates the absence of it. The transitional region highlighted in red between the high regions represents the diffusion zone. It is 𝜌𝜐̅ ℓ and low intensity 𝜐̅ ℓ (1) Re of the , slope Pe in the diffusion region is inversely proportional important to note that the value to the 𝜇 𝐷 amount of diffused dye between the liquids (ideally infinite slope indicates zero diffusion). where, 𝜌 is the density, 𝜐̅ is the mean flow velocity, μ is the dynamic viscosity of the fluid, ℓ is the characteristic length of the microfluidic channel, and D is the diffusivity. A flow with Re lower than 3.2. Preliminary Considerations 2300 is considered laminar, while a high Pe number defines the number of channel widths required Reynolds number (Re) and Peclet number (Pe) are two dimensionless values that define fluidic to completely mix two fluids by diffusion. and diffusive mixing characteristics of a microfluidic system, respectively. They are described as [20,21] For ethanol, 𝜌 = 789 kg/m3 and μ = 1.2 mPa·s. For Rhodamine 6G, D = 3 × 10−10 m2/s [22]. The −4 characteristic length of the rectangular channel was calculated to be ρυ` υ` 1.33 × 10 m for h = 100 μm and Re = , Pe = (1) −4 2.85 × 10 m for h = 500 μm. µ D In a microfluidic device, we can assume the Hagen–Poiseuille equation to describe the where, ρ is the υ is the(ΔP mean velocity, µ is therate dynamic of the fluid, relationship between thedensity, pressure drop = Pflow in – Pout ) and the flow (Q) of viscosity pressure-driven flow ` is the characteristic length of the microfluidic channel, and D is the diffusivity. A flow with Re lower than [23]: 2300 is considered laminar, while a high Pe number defines the number of channel widths required to (2) 𝛥𝑃 𝑅 𝑄 𝑅 𝜐̅ 𝑆 completely mix two fluids by diffusion. 2 /s [22]. The where R is the hydrodynamic resistance of the system and S is6G, theDcross-sectional For ethanol, ρ = 789 kg/m3 and µ =microfluidic 1.2 mPa·s. For Rhodamine = 3 × 10− 10 marea − 4 of the microfluidic chamber. single microfluidic chip,calculated we can consider constants, characteristic length ofFor thearectangular channel was to be 1.33R×and 10 S as m for h = 100 µm and − 4 meaning2.85 that×the increases 10fluid m velocity for h = 500 µm. with the pumping pressure. Starting In from Stokes equations, it iscan possible calculate the fluidicequation resistance a fluid a microfluidic device, we assumetothe Hagen–Poiseuille to for describe thewith relationship viscositybetween μ flowing a [23]: theinside pressure drop (∆P = Pin – Pout ) and the flow rate (Q) of pressure-driven flow [23]:

•

cylindrical channel (tubings) with length L and internal radius r, ∆Ptotal = Rtotal Q = Rtotal υS (2) 8𝜇𝐿 (3) 𝑅 where R is the hydrodynamic resistance of the𝜋𝑟 microfluidic system and S is the cross-sectional area

•

of the microfluidic For a single we can rectangular channelchamber. (glass chip) with lengthmicrofluidic L, height h, chip, and width w, consider R and S as constants, meaning that the fluid velocity increases with the pumping pressure. 12𝜇𝐿 1 Starting from Stokes equations, it is possible to fluid with 𝑅 ∙ calculate the fluidic resistance for a(4) ℎ ℎ 𝑤 viscosity µ flowing inside a [23]: 1 0.63 𝑤

• present cylindrical channel with length L and internal radiuswe r, can consider 𝑅 In the work, since (tubings) all the elements are connected in series, 𝑅 𝑅 . Considering that 𝑅 ≫𝑅 due to the tubing’s comparable radius to the chip 8µL but a comparatively much greater L, we can neglect theRresistance tubing = of4 the glass chip and calculate from πr Equation (3) that 𝑅 = 1.11 × 1011 mbar∙s/m3 for r = 75 μm and L = 11.5 cm. Assuming that the pressure given by the pump is equal to 𝛥𝑃 , we can calculate the flow rate and subsequently, the flow velocity of the chips with different h and varying pumping pressures, as reported in Table 1.

(3)

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rectangular channel (glass chip) with length L, height h, and width w, Rchip =

1 12µL  · 3 h h w 1 − 0.63 w

(4)

In the present work, since all the elements are connected in series, we can consider Rtotal = Rtubing + Rchip . Considering that Rtubing  Rchip due to the tubing’s comparable radius to the chip but a comparatively much greater L, we can neglect the resistance of the glass chip and calculate from Equation (3) that Rtotal = 1.11 × 1011 mbar·s/m3 for r = 75 µm and L = 11.5 cm. Assuming that the pressure given by the pump is equal to ∆Ptotal , we can calculate the flow rate and subsequently, the flow velocity of the chips with different h and varying pumping pressures, as reported in Table 1. Micromachines 2018, 9, x FOR PEER REVIEW

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Table 1. Calculated flow rates, velocities, Reynolds number (Re), and Peclet number (Pe) for different Table 1. Calculated rates, Reynolds number (Re),ofand Peclet (Pe) forofdifferent pumping pressures.flow υ1 and υ2 velocities, correspond to the flow velocity glass chipsnumber with heights 100 µm pumping pressures. 𝜐 and 𝜐̅ correspond to the flow velocity of glass chips with heights of 100 μm and 500 µm, respectively. and 500 μm, respectively. ∆P (mbar) 25 50 100 200

¯ Q (µL/min) ΔP (mbar) Q (μL/min)υ1 (mm/s) 𝝊𝟏 (mm/s) 13.5 22.5 25 13.5 22.5 45.045.0 50 27.0 27.0 90.090.0 100 54.1 54.1 108.1 180.0 200 108.1 180.0

¯

2 (mm/s) Re 𝝊𝟐υ(mm/s) 4.54.5 2 9.09.0 3 18.0 18.0 6 36.0 36.0 12

Re Pe 2 7507.5 3 15015.0 6 30030.0 12 60060.0

Pe 7507.5 15,015.0 30,030.0 60,060.0

3.3. The Effect Effect of 3.3. The of Pumping Pumping Pressure Pressure The flow velocity velocityis isdirectly directly dependent on pumping the pumping pressure according to the The flow dependent on the pressure according to the Hagen– Hagen–Poiseuille equation in Equation (2), and is the only parameterother otherthan than the the geometrical Poiseuille equation in Equation (2), and it is itthe only parameter geometrical characteristics of the chip that can affect the diffusion of R6G in ethanol. Diffusion is a time-dependent characteristics of the chip that can affect the diffusion of R6G in ethanol. Diffusion is a time-dependent process, process, and and it it is is obvious obvious that that aa slowly slowly flowing flowing solution solution (i.e., (i.e., in in the the case case of of ∆P ΔP == 25 25 mbar) mbar) exhibits exhibits greater diffusion, as seen in Figure 4. greater diffusion, as seen in Figure 4.

4. Diffusion behavior inside a microfluidic chip with 30 30°◦ incident angle and h = 500 µm, μm, at Figure 4. ΔP == 25 25 mbar; mbar; (b) (b) ∆P ΔP == 75 75 mbar; mbar; (c) (c) ∆P ΔP == 200 200 mbar. mbar. Scale bars increasing pumping pressure (a) ∆P μm. Arrow Arrow indicates indicates the the flow flow direction. direction. correspond to 500 µm.

3.4. The Effect of Angle Between Inlets We studied the effect of the angle between the two inlets. All the microfluidic chips were fabricated with a constant chamber height (h = 500 μm). Three different angles θ = 30°, 60°, and 80°

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3.4. The Effect of Angle Between Inlets Micromachines 2018, 9, the x FOR PEERofREVIEW We studied effect the angle between the two inlets.

7 of 9 All the microfluidic chips were fabricated with a constant chamber height (h = 500 µm). Three different angles θ = 30◦ , 60◦ , and 80◦ were chosen flows enter into are thereported chamberinwith minimum reason, we determined and the results Figure 5. Anglesturbulence. greater thanFor 80◦this exhibited significant diffusionthat dueato separation angle of θ = 30° is the most suitable for minimum diffusion flow. the trajectory of the fluids, so we excluded them for the purposes of this work.

Figure 5. Slope of the linear interpolation of the intensity profile of the visual diffusion analysis Figure 5. Slope3c) of in thethe linear of the visual diffusion analysisangles (see (see Figure chipinterpolation as a functionofofthe theintensity pair inletprofile pressure at the different separation ◦ , 60 Figure in◦the chip θ = 303c) , and 80◦as . a function of the pair inlet pressure at the different separation angles θ = 30°, 60°, and 80°.

For each angle value, the slope of the intensity profile in the diffusion region increases 3.5. The Effect of Chamber Height monotonously with the (identical) pumping pressure at the two inlets: the greater the pressure, theIn greater the velocity of theslope fluids, to an increased value ofheights the Pe of coefficient andand reduced Figure 6, we report the ofleading the diffusion zone for channel h = 100 μm 500 diffusive mixing. It is clear that for a given pumping pressure, a smaller separation angle results μm for chips with a length of 2 mm and an angle of 30° between inlets. It is evident that the channelin reduced between the two parallel flowing fluids. height hasmixing a significant impact on the diffusion process in this microfluidic platform, with the slope Increasing the separation angle of the inlets, the fluids active mixing in the first part in the diffusion region for the 100 μm tall chamber being twice undergo that of the 500 μm tall channel in any of the chamber due In to aother greater change trajectory is upon the main chamber. the case of pressure measured. words, thein diffusion lessentering pronounced in the shorterIn chamber, ◦ and 80◦ , a saturation of the slope value is observed. However, in the case of smaller angles, the 60 irrespective of the pumping pressure. A reasonable explanation for this observation is that by parallel flows into with turbulence. determined that a decreasing the enter height ofthe thechamber chamber, theminimum interaction area of For the this tworeason, fluids we decreases as well. ◦ separation angle of θ =of30theischamber the most is suitable diffusion flow.fluids is 0.2 mm2 for the Considering the length 2 mm,for theminimum interaction area of the

100 μm high chamber and 1 mm2 for the 500 μm. 3.5. The Effect of Chamber Height In Figure 6, we report the slope of the diffusion zone for channel heights of h = 100 µm and 500 µm for chips with a length of 2 mm and an angle of 30◦ between inlets. It is evident that the channel height has a significant impact on the diffusion process in this microfluidic platform, with the slope in the diffusion region for the 100 µm tall chamber being twice that of the 500 µm tall channel in any pressure measured. In other words, the diffusion is less pronounced in the shorter chamber, irrespective of the pumping pressure. A reasonable explanation for this observation is that by decreasing the height of the chamber, the interaction area of the two fluids decreases as well. Considering the length of the chamber is 2 mm, the interaction area of the fluids is 0.2 mm2 for the 100 µm high chamber and 1 mm2 for the 500 µm.

Figure 6. Theoretical and experimental slope of the intensity profile in the diffusion region versus the pumping pressure for two different chamber heights: 100 μm and 500 μm.

Another parameter that justifies this drastic change in the slope is the fluid velocity, which in the case of h = 500 μm is much lower than that of h = 100 μm (Table 1) at constant pressure. As

in the diffusion region for the 100 μm tall chamber being twice that of the 500 μm tall channel in any pressure measured. In other words, the diffusion is less pronounced in the shorter chamber, irrespective of the pumping pressure. A reasonable explanation for this observation is that by decreasing the height of the chamber, the interaction area of the two fluids decreases as well. Considering Micromachinesthe 2019,length 10, 23 of the chamber is 2 mm, the interaction area of the fluids is 0.2 mm2 for 8the of 10 2 100 μm high chamber and 1 mm for the 500 μm.

Figure 6. Theoretical and experimental slope of the intensity profile in the diffusion region versus the Figure 6. Theoretical andtwo experimental slope of heights: the intensity profile the diffusion region versus the pumping pressure for different chamber 100 µm and in 500 µm. pumping pressure for two different chamber heights: 100 μm and 500 μm.

Another parameter that justifies this drastic change in the slope is the fluid velocity, which in the Another parameter thatlower justifies this drastic the slope is the fluid velocity, in case of h = 500 µm is much than that of h =change 100 µmin (Table 1) at constant pressure. As which mentioned the case of h = 500 μm is much lower than that of h = 100 μm (Table 1) at constant pressure. As before, the diffusion is proportional to the residence time of the interacting fluids in the chamber and consequently inversely proportional to the flow velocity. The parametric study of the effect of the height the diffusion was simulated by COMSOL, as well as the theoretical data, are also presented in Figure 6. For the purposes of this study, we assumed the same mean velocities as in Table 1. Considering the diffusion coefficient of R6G in ethanol, we were able to extract the slope of the concentration gradient, following the same procedure as for the experimental data. Although the same general trends are observed in the theoretical simulations, there is a discrepancy in the absolute values of slopes. The resistance of the chip and the roughness of the walls were neglected for the purposes of the theoretical study, which can explain the lower diffusive mixing that is predicted by the simulations compared to the experimental data in Figure 6. 4. Conclusions In this work, we performed a parametric study of the geometry of a double Y-shaped microfluidic chip in order to minimize the diffusive mass transfer between two laminar flows. This first approach on the characterization of such chips examines the behavior of two ethanolic solutions, but the results can be translated in any kind of application of multifunctional complex microfluidic systems with a similar configuration. The optical technique that was developed for the purposes of this study is simple, straightforward, and can be replicated using widely used and easily accessible equipment such as an optical microscope with a CCD camera and image processing software. The FLICE manufacturing technique has enabled the fabrication of complex 3D geometries of buried microfluidic chips in glass while avoiding the problematic process of sealing two substrates together by welding. Being able to use glass as a substrate for microfluidics removes the limitations that are created by the incompatibility of the majority of elastomers, such as PDMS, and allows for a wider variety of applications in flow chemistry. For the visualization of the diffusion, a R6G solution and a transparent solution were used, enabling the detection of mixing by a simple optical microscopy image analysis. As a result, we determined that an angle of 30◦ or lower between the two inlet streams is optimal for non-mixing flow. Also, we found that the height of the interaction chamber has a major impact on the diffusion, with the smaller height of 100 µm being preferable. In future work, we will perform chemical reactions at the interface of the parallel laminar flows in the laser-inscribed buried branching network.

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Author Contributions: G.B., S.M.E., L.C., G.L., and R.R. conceived the idea of laser forming the non-mixing microfluidic chip in glass. V.I., S.B., L.C., V.B., and S.M.E. designed the geometry of the microfluidic chip. V.I., S.B., L.C., A.N.G., and T.L.P. aided in the characterization and analysis of the microfluidic chip. All authors discussed the experimental implementation and results and contributed to writing the paper. Funding: This work was funded by the H2020 Marie Skłodowska-Curie ITN PHOTOTRAIN project, FP7 DiamondFab CONCERT Japan project and DIAMANTE MIUR-SIR grant. Acknowledgments: The authors thank Sara Lo Turco and Simone Varo for enlightening scientific discussions. Conflicts of Interest: The authors declare no conflict of interest.

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