Microfabrication of Monolithic Microfluidic Platforms Using Low

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Microfabrication of monolithic microfluidic platforms using Low Temperature Co-Fired Ceramics suitable for fluorescence imaging Pedro Couceiro, and Julián Alonso-Chamarro Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01889 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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Analytical Chemistry

Microfabrication of monolithic microfluidic platforms using Low Temperature Co-Fired Ceramics suitable for fluorescence imaging Pedro Couceiro and Julián Alonso-Chamarro Sensors & Biosensors Group, Department of Chemistry, Autonomous University of Barcelona, Edifici Cn, 08193 Bellaterra, Catalonia, Spain; Tel: +34 935812149; E-mail: [email protected] In this work, the influence of laser ablation and lamination parameters in the fabrication of embedded microstructures using Low Temperature Co-Fired Ceramics, have been studied. First, the influence of laser ablation parameters in the dimensions of fabricated microchannels in Low Temperature Co-Fired Ceramics substrates was characterized and strategies for tailoring the microchannels aspect ratios are described. The influence of lamination conditions on the fabrication of monolithically embedded microstructures is presented. Thereafter, a ceramic microfluidic platform, was constructed using a multilayer approach. The ceramic microfluidic platforms incorporate three independent inlet channels and a microfluidic chamber with an monolithically integrated transparent optical windows. The construction procedure used ensures monolithic ceramic devices with homogeneous surface chemistry as well as homogeneous physical properties. Fluorescence dyes, where used in order to characterize the hydrodynamic focusing as a function of flow rate ratio of the microfluidic chamber inlets. The results obtained open the possibility of studying chemical process, in static or flow conditions using fluorescence imaging, within the traditional fields of LTCC technology, such as high-temperature or organic solvents applications, while using a simple fabrication procedure suitable for low cost mass production.

Introduction There are several advantages of scaling down benchtop systems in order to manipulate liquids in the microscale (10 to 100 µm)1. The high surface-to-volume ratio at the microscale allows increased heat transfer rates, allowing local heating and cooling of samples with low power consumption while the dramatic reduction of volume originate obvious advantages in the amount of reagents used and waste generated, but most importantly it constitutes the only tool which allows the efficient manipulation of extremely low sample volumes. This is of extreme importance for biological applications where the sample volumes can be extremely low. Nonetheless, miniaturization of benchtop systems poses challenges, such as the increased importance of microchannels surface chemistry control, leading to the necessity of fabrication devices with the same substrate material, or the need to use high sensitive detection systems, such has fluorescence, duo to optical path length reduction. Low Temperature Co-Fired Ceramics (LTCC) technology, emerged as an excellent substrate for the construction of such devices. Researches took advantages of the technology multilayer approach to develop three-dimensional (3D) sensors, actuators and microfluidic platforms, incorporating complex

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structures such as 3D microfluidic channels, for analytical applications2–18. The devices can be easily designed and fabricated without requiring special facilities (clean rooms), enabling rapid, low cost, prototyping. Furthermore, its compatibility with screen printing techniques has enabled the development of highly integrated devices which incorporate electrochemical detection (amperometric, potentiometric or conductiometric detection) as well as all the electronics for the signal acquisition and data processing8,9,11,12. In order to use optical detection, optical fibers19 and glass windows9,14,16,20 have been integrated in ceramic microfluidic devices. However, due to lack of transparency of the ceramic layers21, optical detection in monolithic microfluidic LTCC devices has been limited to absorbance measurements in optical microflow cells22 with optical windows constituted by a single ceramic layer. Since LTCC technology was originally developed for the fabrication of multilayer flat circuits, the fabrication methodology present in the data sheets, manly the structuring techniques and lamination conditions, are not suited for the construction of monolithic devices with embed microstructures. A number of different techniques have been used by researchers to fabricate microstructures in LTCC layers such as Milling, Hot Embossing Sacrificial Volume Materials and Laser Ablation. The use of Computer Numerical Control (CNC) milling3,23,24 presents same limitation when structuring flexible materials like green state LTCC, since the physical interaction between the cutting tool and the flexible LTCC sheets originate stretching which leads to structural deformation of the substrate and consequently alignment problems. Although it is possible to fabricate channels in the 10 to 100 µm range using Sacrificial Volume Materials25,26(SVM), the granularity of graphite powder is a critical parameter and the process is very design dependent. Hot embossing27,28 has also been used for LTCC microfabrication. However, due to the high cost of masters, this technique is not suited for prototyping but rather for high-volume lowcost production. Laser Ablation of LTCC substrates, was first reported by Susan D. Allen et al.29 using an Infrared laser. Since then a variety of Infrared26,30–33 (IR) and Ultraviolet33 (UV) lasers, have been reported. The technique is also suited for low cost rapid prototyping, enabling the fabrication of features in the 10 to 100 µm range. Studies of LTCC laser ablation have however been focused on the influence of laser parameters in the ablation of simple lines or the ability to laser cut electrical vias. The lamination process is crucial for the fabrication of monolithic microfluidic devices. Lamination parameters described in the data sheets are, however, optimized for the construction of flat, cavities free, devices for electronic purposes, and are not suited for designs that incorporate embedded cavities, originating wall sagging problems. Researchers have used different techniques in order to solve lamination sagging problems. Using the recommended lamination condition, sagging problems can be reduced by using SVM techniques. The results obtained using this approach are however very design dependent, as previously discussed. Pressure sensitive adhesives26 in LTCC layer lamination has been


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reported. Delamination of fired devices is however a recurrent problem using this procedure. Different research groups have modified the pressure, temperature and time conditions used in the fabrication of their LTCC devices17,26,34,35. The reported data is however very scattered and, at times, casuistic, not allowing to a compressive understand of the conditions best suited for fabricating embedded structures. In the present work, a systematic study on the influence of laser ablation and lamination parameters in the fabrication of embedded microstructures, using LTCC technology, was carried out and strategies for tailoring microchannel aspect ratio, where developed. Thereafter, a ceramic microfluidic platform, incorporating three independent inlet channels and a microfluidic chamber with a monolithically integrated transparent optical window, was fabricated and the hydrodynamic focusing was characterized, as a function of the inlets flow rate ratio, using fluorescence imaging of fluorescent dyes. The construction procedure used ensures monolithic ceramic devices with homogeneous surface chemistry as well as homogeneous physical properties. To our knowledge, this is the first time that a microfluidic platform with a monolithically integrated transparent optical window, suitable for fluorescence imaging, has been fabricated using the same ceramic substrate. Moreover, the simple fabrication procedure presented is suitable for low cost mass production.

2.Experimental 2.1 Materials and methods DuPont 951 Green Tapes with 115 µm unfired thickness (DuPont 951 PT) and 254 µm unfired thickness (DuPont 951 PX) were employed as ceramic substrates. The ablation of the ceramic substrates was performed using a IR Nd:YAG laser equipment (LPKF Protolaser 200, Garbsen, Germany), using constant laser power (1.3 kW) and pulse frequency (40 KHz). Ceramic substrates were aligned in aluminium plates and laminated in a uniaxial hydraulic press (Talleres Francisco Camp S.A., Granollers) at 70 ºC and 30 bars. The ceramic substrates were sintered in a programmable box furnace (Carbolite CBCWF11/23P16, Afora, Spain), applying the temperature profile consisting of 5 stages. In the first stage the temperature was increased from room temperature to 350 ºC, at 10º C min-1. A 30 min stabilization stage at 350 ºC followed by a second heating ramp to 850 ºC, at 10ºC min-1, was carried out. Finally, after 30 min of stabilization at 850ºC, cooling to room temperature was performed. Measurements of sintered ceramic substrates were performed using a profilometer (KLA Tencor P-15) and stereomicroscope (Leica S6D) equipped with a digital CCD camera (Leica DFC29). Channel dimension presented for unfired LTCC substrates was calculated taking into account DuPont 951 substrate shrinkage of 12.7% in the X and Y axis and 15% in the Z axis, after sinterization.



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Microfluidic platforms where mounted in an in-house build multichannel connector and connected to 1mL syringes (Hamilton series GASTIGHT 1000 TLL) using Teflon tubes (0.8 mm i.d.) fitted with 1/4"28 fluidic connectors (Fisher Scientific) and Luer-Lock-to-1/4-28 fittings (Fisher Scientific). The syringes were mounted on 3 syringe pumps (TSE systems 540060). Fluorescence imaging of microfluidic platforms was performed using a UV-A lamp (Philips PL-S 9W UV-A/2P 1CT) as a light source, and a CCD Camera (Nikon D90) as an optical detector. The CCD camera and the syringe pumps where controlled using a PC. Fluorescein

sodium

(Fl),

4-methylumbelliferone

(4MU)

and

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) were purchased from Sigma-Aldrich (Madrid, Spain). Fluorescent dye solutions of 0.5 mM 4MU in 10 mM HEPES and 0.5 mM of Fl in 10 mM HEPES were prepared in double distilled water.

3.Results and discussion 3.1 Laser Ablation of LTCC substrates. For the the laser ablation studies of LTCC substrates, presented in Figure 1, LTCC blocks, consisting on two DuPont 951 PX layers (254 µm unfired thickness) laminated at 70 ºC and 30 bars for 5 min, were laser ablated according with a pre-defined design.

3.1.1 Laser mark speed influence on open channel dimensions. In order to characterized the influence of the laser mark speed on the dimensions of ablated open channels, a design consisting of 22 parallel line was created using CAD software. The line-to-line separation used was 200 µm in order to ensure independent line ablations. Using constant laser parameter of laser power (1.3 kW) and pulse frequency (40 KHz), each line was laser ablated according with a pre-defined mark speed into the LTCC blocks. After sinterization, the substrate surface was characterized by profilometry. The profilometry data, summarized in Figure 1b and 1c, denotes a decrease in channel depth as mark speed increases. For a constant laser power and pulse frequency, each laser pulse has a constant energy. The laser pulse number can therefore define the total energy used for ablation. The number of laser pulses used for each ablation can be calculated by dividing the ablation time, obtained by dividing the constant line length (10 mm) by the mark speed used, by the constant pulse frequency (40 KHz), considering however the number of laser pulses has to be a natural number. The equation used can be written has:


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=

ℎ . =

Where A is a constant. As seen in Figure 1b, the channel depth and the laser pulse number used for ablation, follow the same inversely proportional relation to laser mark speed. Small inconsistencies in channel depth decrease with laser mark speed are probably a consequence of the non-planarity of unfired LTCC substrate.



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Figure 1. a) Image of the Gaussian shaped microchannels fabricated in LTCC using a single line ablation strategy with different laser mark speeds. b) Effect of laser mark speed on fabricated microchannel depth. c) Effect of laser mark speed on fabricated microchannel width. d) Image of microchannels fabricated in LTCC using superposed lines ablation strategy. e) Influence of superposed lines ablations in channel width and depth f) Influence of superposed lines ablation in the channels aspect ratio. f) Image of microchannels fabricated in LTCC using parallel lines ablation strategy. g) Influence of the number of parallel lines in channel width and depth. i) Influence of the number of parallel lines ablations in the channels aspect ratio.


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since unfired LTCC substrates are flexible materials. For the desired application however, such variations are negligible. The same inversely proportional tendency is found between channel width and laser mark speed. There seems to be, however, a channel's width threshold of 45 µm for unfired LTCC substrates (40 µm in the fired state). The 25 µm diameter laser spot, operating in Gaussian mode, concentrates the maximum energy which spread in a Gaussian-like shape over space originating gauss shaped channels wider than the laser spot. A direct consequence of channel's width threshold is the channel’s aspect ratio, increase with laser mark speed.

3.1.2 Decreasing open channel aspect ratio Since laser ablation is a direct write technique, ablated channel dimensions and consequently open channel aspect ratio, are determined by the laser parameters. Therefore, in order to vary the aspect ratio of the fabricated channels, design strategies must be used. A straightforward strategy to decrease the channels aspect ratio is to design structure with superposed lines. In order to evaluate the effect of having increasing superposed lines in the fabricated channels dimensions, a design consisting of 22 parallel line was created using CAD software. The line to line space was maintained at 200 µm, having each line one additional superpose line than the previous one. As seen in Figure 1d, data obtained from imaging analysis show a linear increased of channels depth with the increment of superposed line. Channel width also increases with the increment of superposed line, reaching however a maximum threshold after 3 ablated lines. As a rule of thumb, the width threshold corresponds to a 25% increase on the width of 1 ablated line. Consequently, the increment of superposed line originates a decrease in channel aspect ratio. Channels with aspect ratio as low as 1:10 can be fabricated in LTCC substrates using this technique.

3.1.3 Increasing open channel aspect ratio. In order to increase the aspect ratio of fabricated open channels a strategy of using multiple parallel lines can be used. In order to study the influence of increasing parallel lines in the fabricated channels dimensions, a CAD design consisting on parallel lines sets was ablated into LTCC substrate. Each set had an increment of 1 parallel line when compared with the previous, and the line-to-line distance was defined as half the width of the channel fabricated using the determine laser mark speed. As seen in Figure 1g, the channel depth increases with the number of parallel lines until a maximum depth threshold is obtained for channels fabricated using 3 or more parallel lines. A channels depth increase of 50% is obtained, in this condition, relatively to single line ablated channel. From this point on the channels depth is constant,



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Figure 2. a) Image of embedded channels fabricated in LTCC. b) Image of square micropillars fabricated in LTCC using parallel line strategy. c) Image of embedded channel fabricated in LTCC with 1:10 aspect ratio d) Graphical representation of the influence of lamination time in embedded channel depth e) Image of multidepth circular micropillar matrix fabricated, combining parallel and superposed line strategies, in LTCC. f) Sagging problem in embedded channel fabricated in LTCC with aspect ration higher then 1:10.

while channels width increases linearly width with parallel line increments, originating channels with increasing aspect ratio.

3.2 Lamination of LTCC substrates For the lamination studies of LTCC substrates, presented in Figure 2, DuPont 951 PT (114 µm unfired thickness) where laser ablated according with a pre-defined design and laminated with LTCC blocks, consisting of two DuPont 951 PX (254 µm unfired thickness sheets), originating embedded channels. The laminated LTCC substrates with embedded channels were laser cut through, perpendicularly to the channel direction, before being sintered. The LTCC embedded channels were investigated based on crosssectional observations.

3.2.1 Influence of lamination time in embedded channel dimensions. In order to characterize the influence of lamination time in the dimensions of fabricated embedded channels, channels design, composed of 1 to 10 parallel lines, with line-to-line separation of 50 µm, were ablated using 100 mm/s mark speed into DuPont 951 PT layers. The ablated DuPont 951 PT layers were then laminated with pre-laminated LTCC block for 30s, 4 min, 16 min and 32 min at 70 ºC and 30 bars. As seen in Figure 2d, the data obtained for the variation of the depth of ablated channels with lamination time show that channel depth decreases with time, reaching a minimum depth after 4 minutes of lamination. The same behaviour is observed in the variation of channel width over lamination time. From this point on, no channel deformation was observed. The results suggest that there is a layer-to-layer


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interpenetration threshold, probably dependent on temperature and laminating pressure. The layer-to-layer threshold, for the presented methodology, is 20 µm (fired state). The results imply that embedded channel dimensions can be tailored using the laser ablation parameter, since the lamination of LTCC substrates originate a constant decrease in channels depth. Also, since from 4 minutes to 32 minutes no channel deformation is observed, the presented lamination methodology can be used in the construction of complex multilayer structured LTCC devices. Device design can be break down into multiple LTCC blocks, fabricated using 4 minutes as standard lamination time. Finally, LTCC blocks assembled and laminated into a final device, since consecutives lamination will not originate any embedded channels deformation.

3.3.3 Aspect ratio of embedded channels. The presented design strategies allow easy fabrication of channels in ceramic substrates. As seen in Figure 2b, complex fluidic networks can be designed, where fabricated channels are laser ablated into the ceramic substrates, while pillars are generated in the non-ablated areas. As seen in Figure 2e, the shape and size of micropillars, as well as channels depth and width, can be easily tailored. In order to accurately design monolith ceramic microfluidic networks, the maximum aspect ratio of embedded channels, fabricated using LTCC, that must be determined. As seen in Fig 2c embedded channel with aspect ratio as high as 10:1 can be fabricated using LTCC. Channels with higher aspect ratios present sagging of the top cover, as seen in Figure 2f.

3.4. Monolithic LTCC microfluidic platforms suitable for fluorescence imaging. Fluorescence imaging is a powerful tool in microfluidics since it allows spatio-temporal characterisation of chemical processes in static or flow conditions. Using the presented microfabrication techniques, a methodology for the fabrication of ceramic microfluidic platforms with integrated transparent optical windows, suitable for fluorescence imaging, was developed. As seen in Fig 3c, the methodology is based on the fabricating microfluidic network on DuPont 951 PT (114 mm unfired thickness) by laser ablation, while using the non-ablated substrate as transparent optical window. In order to exemplify the presented fabrication methodologies, a monolithic ceramic microfluidic platform, integrating a transparent optical window and AgPd electrodes, was constructed using LTCC technology, and a hydrodynamic focusing process was characterized using fluorescence imaging. The AgPd electrodes was not used in any of the experiments.



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Figure 3. a) Schematic representation of the layers CAD design arranged in lamination Blocks. b) Illustration of the three dimensional structure of the fabricated monolithic microfluidic platform. c) Schematic representation of the cross-section view of the laser ablation of Block A and the lamination process of Block A to Block B (Not to scale). d) Image of the unfired open microfluidic chamber showing, 500 µm and 250 µm diameter, pillar matrix e) Image of the final monolithic microfluidic platform fabricated with integrated AgPd electrodes.

3.4.1 Design and Fabrication. The ceramic microfluidic platform was designed layer by layer, as depicted in Figure 3a, using CAD software. DuPont 951 PX (254 µm unfired thickness) layers where used as ceramics substrate in the fabrication of the microfluidic platform except for Block A where DuPont 951 PT (114 µm unfired thickness) layer was employed. The DuPont 951 PX (254 µm unfired thickness) layers laser cut through using 2 superposed designs and 25 mm/s mark speed. Block A presented two designs that where ablated into the ceramic substrate using different laser parameters, in a single step. The ceramic substrate was laser cut through using 2 superposed designs at 25 mm/s mark speed while the microfluidic chamber was laser ablated using paralleled lines separated by 50 µm and 100 mm/s mark speed, generating a relief microstructure. The ceramic layer where aligned in aluminium plates and laminated originating blocks A


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Figure 4. Fluorescence imaging setup composed of UV lamp, CCD camera, microfluidic platform and in-house build microfluidic multi channels connector.

to E, as seen in Fig 3a. Each block was laminated for 5 min. at 70º C and 30 bar. All the ceramic blocks where aligned in aluminium plates and laminated for 5 min. at 70º C and 30 bar. After lamination, AgPd paste (DuPont 961) and glass After lamination, AgPd paste (DuPont 961) and glass encapsulant paste (DuPont 9615) were screen printed into the microfluidic platform using Block A Mylar as a mask. Finally, before sinterization, the Block A Mylar was peeled off. The final microfluidic platform, depicted in Fig 3e, consists of a 7.5 mm wide, 36.5 mm long and 60 µm deep microfluidic chamber, connected to inlet and outlet O-ring ports by three independent, 500 µm wide and 200 µm deep, microfluidic inlet channels and one microfluidic outlet channel, disposed three dimensionally. The microfluidic chamber, generated as a bas-relief of the DuPont 951 PT layer, presents 250 µm wide walls, two pillar matrixes, of 500 µm and 250 µm diameter spaced 1 mm centre to centre, as seen in Figure 3 d. The pillar matrix supports a 20 µm thick, integrated optical window, generated by the non-ablated DuPont 951 PT layer area. Since LTCC layer transmittance is thickness dependent22, in order to maximize transmittance, ceramic optical windows integrated in microfluidic platforms, should be as thin as possible.



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The fabrication of 20 µm thick ceramic layer represent a significant increase in optical window transmittance when compared to the thinnest commercial, DuPont 951 (50 µm unfired thickness), LTCC layer available. The transmittance of laser ablated ceramic layer should be further investigated. Using the construction procedure different scale structures can be fabricated, allowing to address different problems such as word-to-chip connectivity, three dimensional fluidic distributions of meso scale (1mm to 100 µm) fluidic channels or microfluidic networks (100 µm to 10 µm) fabrication with integrated transparent optical windows suitable for fluorescence imaging, into the monolithic ceramic microfluidic platform with homogeneous surface chemistry as well as homogeneous physical properties.

3.4.2 Hydrodynamic focusing In order to characterized the hydrodynamic focusing as a function of flow rate ratio (α)of the inlets of the fabricated microfluidic chamber, a fluorescence experimental setup that would maintain the relative position of the ceramic microfluidic platform, the light source and the optical detector was assembled. As seen in Figure 4 an in-house build microfluidic multichannel connector, that connected the syringes, mounted in syringe pumps, to ceramic microfluidic platform, was mounted to a breadboard, where a commercial UV-A lamp (365 nm peak), used as a light source, had been screwed using PMMA adapters. In the same breadboard a commercial CCD camera, used as an optical detector, was mounted using an adapter. Hydrodynamic focusing is a technique in which an injection stream is squeezed between two side stream, allowing the generation of injection width streams down to the nanometrical scale while using microchannels36. For a fix geometry, hydrodynamic focusing width of the injection stream is dependent only on the ratio between the pressure of the side streams (Ps) and the injection stream (Pi): α=Ps/Pi. Since hydrodynamic focusing is observed only within a range of α, Fl solution was injected into the separation chamber, through inlet channel 1 (IC1) at 10 µL/min, and through inlet channel 3 (IC3) at 20 µL/min, while the flow rate of the 4MU solution, injected through inlet channel 2 (IC2) was varied, as seen in Figure 5. Since no filters were used in the optical detection setup, the contrast of the fluorescent images is far from optimum. This simple imaging setup allowed, however, the continuous monitoring of the focusing of the injection stream between 1< α 10 the side stream periodically entered the inlet channel 2 (IC2), interrupting the injection flow stream. Although the injection stream was hydrodynamic focused, the stream presented a threshold width defined by the pillars of the microfluidic chamber. Pillars originate broadening of injection stream and, depending on the application, the pillars design should be taken into account.


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Figure 5. Fluorecence imaging, using blue (4MU) and green (Fl) fluorescent dyes, of the microfludic chamber hydrodynamic focusing for inlet flow rate ratio (α), a) α=1 and b) α=10.

This is, as far as we know, the first characterization of flow processes using fluorescence imaging in a monolithic ceramic microfluidic device opening the possibility of studying chemical processes in flow conditions in traditional application fields of LTCC technology, such as high-temperature or organic solvents applications, while using a simple fabrication procedure suitable for low-cost mass production.

Conclusions All-ceramic microfluidic platforms with monolithically integrated optical window, suitable for fluorescence imaging, has been fabricated for the first time, to our knowledge. The described procedure allows the fabrication of hermetically sealed microfluidic platforms with homogeneous surface chemistry and physical properties. A systematic study on the influence of laser ablation parameters in the dimensions of microchannels, fabricated using LTCC technology, has been carried out. Design strategies that allow tailoring microchannel aspect ratio have been presented. Open microchannels with depth in the 40 to 96 µm and width in the 10 to 160 µm range (fired dimensions) were fabricated varying the laser mark speed parameter and microchannels with aspect ratios as low as 1:10 have fabricated using superposed lines design.



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The influence of lamination time in fabricated embedded microchannel dimensions has been studied. The results show a channel depth loss threshold of 20 µm (fired dimensions), for lamination times higher than 4 minutes. The results suggest that embedded channel dimensions can be tailored using the laser ablation parameter, since the lamination of LTCC substrates originate a constant decrease in channels depth. Embedded microchannels with aspect ratios as high as 10:1 have been constructed using LTCC technology. Fluorescence dyes where used to characterize the hydrodynamic focusing, as a function of the inlets flow rate ratio, of a microfluidic chamber with a monolithically integrated, 20 µm thick, transparent optical window. This is, as far as we know, the thinnest ceramic layer fabricated using LTCC. The results demonstrate the possibility of obtaining robust microfluidic platforms with monolithically integrated transparent optical windows suitable for fluorescence imaging, opening the possibility of studying chemical processes in static or flow conditions within the traditional fields of LTCC technology, such as high-temperature or organic solvents applications. Furthermore, the fabrication procedure described is suitable for low-cost mass production.

Acknowledgements This work was funded by Spain's Ministry of Economy and Competitivity (Project CTQ2012-36165), FEDER and the Catalonian Government (Project 2014SGR837).

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82x44mm (300 x 300 DPI)


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Microfabrication of Monolithic Microfluidic Platforms Using Low

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