Laser Inscription of Microfluidic Devices for Biological Assays - ACS

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Biological and Medical Applications of Materials and Interfaces

Laser Inscription of Microfluidic Devices for Biological Assays Tawfiq Alqurashi, Muhammed Alnufaili, Muhammad Umair Hassan, Salman Aloufi, Ali K. Yetisen, and Haider Butt ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22400 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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ACS Applied Materials & Interfaces

Laser Inscription of Microfluidic Devices for Biological Assays Tawfiq Alqurashia,b *, Muhammed Alnufailic, Muhammad Umair Hassanb, Salman Aloufid, Ali K. Yetisene, and Haider Buttb,f* aDepartment

of Mechanical Engineering, School of Engineering, Shaqra University, Dawadmi,

P.O. Box 90 Zip Code 11921, Saudi Arabia bSchool

of Mechanical Engineering, University of Birmingham, Birmingham, B15 2TT, UK

cEngineering

dSchool

of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK

eDepartment

f

Services, Saudi Aramco, Dhahran, 31311, Saudi Arabia

of Chemical Engineering, Imperial College London, SW7 2AZ, London, UK

Department of Mechanical Engineering, Khalifa University, Abu Dhabi 127788, UAE

*Corresponding Author’s Email: [email protected]; [email protected]

Keywords: microfluidics; laser ablation; optics; biological assays; cell assays

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Abstract A rapid and direct CO2 laser ablation method was developed to create superhydrophilic surfaces and arrays of hydrophobic-superhydrophilic (HHA) patterns for application in bioassays. Here, a combination of superhydrophilic and hemiwicking wetting characteristics was exploited to create microfluidic slides that were used as biological assays that prevented cell aggregation. This feature allowed carrying out microscopic analyses at the individual cell level. This bioassay enabled controlling cell population in localized areas (15 cells cm-2). The device had 84% transparency, allowing direct fluorescence microscopy measurements in transmission mode. High adhesion of aqueous fluids on superhydrophilic areas surrounded by superhydrophobic boundaries provided selective retention and confinement. The adhered droplets maintained retention under 180° substrate tilt. These architectures provided a rapid self-partitioning of the liquid into an array of droplets. Hydrophobic-superhydrophilic patterned arrays may have application in microfluidic bioassays, high-throughput screening, and medical diagnostics.

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Introduction Surface engineering of glass and polymeric materials to control the wettability behavior has undergone a significant development 1-3. Precise control of interfacial water-repellent/attractive behavior via surface modification has opened up a myriad of applications in anti-icing and antifogging surfaces microarrays

9-10,

4-5,

self-cleaning windows

6-7,

enhanced heat transfer devices 8, droplet

microfluidic Lab-on-a-Chip (LOC) devices

11,

highly-hydrophilic contact

lenses 12, and cell-adhesive substrates 13. A wide range of fabrication techniques with varying level of complexity has been utilized to attain desired interfacial properties 4, 9, 14-15. To produce superhydrophilic soda-lime-silica (SLS) glass, glass surface was restructured by coating with a SiO2 sacrificial layer and subsequently etching the coated surface with CF4 plasma to form SiO2 nanopillars

16.

The surface possessed a roughness factor, r > 3, and a contact angle, θc < 5°.

Inductively-coupled plasma etching was employed to create randomly distributed nanoholes and nanopillars on glass substrates with r ≈ 7.3 that offered θc < 5.0° and 2.3°, respectively. Although these methods provided substantial hydrophilic properties, they were timeconsuming, high cost, complex to perform, and resulted in a simple and non-uniform surface topography, which deteriorated over time. Therefore, versatile and cost-effective fabrication techniques having a capability of permanent surface modification are needed in surface engineering

10, 17-19.

For example, direct femtosecond laser ablation has been used to create

microchannels on a substrate exhibiting r = 1.64 and θc = 0°

20.

High wettability was

emphasized by the extensive water spread across the substrate, even flowing uphill on a vertically aligned substrate defying gravity. Creation of superhydrophilic spot-array pattern on glass substrates through direct CO2 laser ablation has been shown to exhibit a reduced contact angle, θc= 10° as compared to its pristine value of 13° 21. Alternating arrays of hydrophobic and hydrophilic surfaces have been created to restrain fluid at predefined locations and geometries without the need for physical walls around the restrained liquid drops 9, 22-23. The water repellency of the hydrophobic coating promoted self3 ACS Paragon Plus Environment


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partitioning of microdroplets into the superhydrophilic micro-spots/channels. The liquid patterns were used as microchannels to refill micro fuel cells

24

and microfluidic devices

25.

Such fabrication has also a significant impact on the amount of data acquired in a broad spectrum of applications including high-throughput bioassays of single cells

21, 26-27,

drug

screening 23, 28, genome libraries 29, and cell culture microenvironments 30-31. Here, we demonstrate the HHAs as a biological assay by spreading cells on biomicrofluidic substrates. The HHAs were patterned on glass slides by direct CO2 laser texturing for the first time. These surface-engineered slides provided localized cell separation by preventing their overlap and facilitated a high cell distribution. A dispersion of 15 cells cm-2 was measured as probed by the fluorescence microscopy. A systematic analysis of the relationship between laser parameters and laser-induced wettability of the irradiated glass was conducted to determine the optimum conditions that yield high surface hydrophilicity. To evaluate the wettability of glass, θc was measured and its behavior was correlated with the topographic properties of the textured glass. Light transmission through ablated glass slides was examined to assess the laser-induced deterioration of their transparency. A range of HHAs was also fabricated by coating the SLS surface with hydrophobic films and subsequently ablating them from selective areas using the CO2 laser. To establish the practicality of these microarrays, the behavior of dispersed dyed droplets was studied on HHA geometries. Results and Discussion Characterization of biomicrofluidic assays The surface profile of the laser modified SLS glass slides was studied for different laser parameter ranges: 9  P  12 W, and 200  S  800 mm.s-1, (Figure 1, Supporting Information Table S1, S2 and Figure S1). The microscopic studies revealed that deeper grooves were formed with lower laser speeds and higher power values due to increased imparted energy. The groove depth, h ~ 80 μm, was the deepest at P = 12 W and S = 200 mm.s-1, and the lowest value of h ~ 25 μm was achieved at P = 9 W and S ~ 800 mm.s-1. The groove profile at HD = 400, P = 4 ACS Paragon Plus Environment

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9.6 W and S~ 500 mm.s-1 is shown in Supporting Information Figure S2. Surface cracking and redeposition of the glass at ablated edges and groove sides were also detected especially for higher powers. However, the overall integrity of the desired geometry did not suffer significant damage in most of the cases as topographic patterns were accurate and microstructures remained intact.

Figure 1. CO2 laser modification of soda-lime glass substrates. (a) Schematic of CO2 laser setup utilized to fabricate the groove arrays. (b,c) Illustration of a plain substrate modification via laser ablation having a 2D pattern and related geometric parameters: pillar height (h), width (w), and spacing (b) and hatch distance (HD = w+b). (d) Optical microscopic images of patterned glass substrates at a constant hatch distance of 400 μm at different laser powers and scanning speeds (black) with their 3D simulated images (blue). Scale bar = 200 μm. (e,f) The effects of changing laser power and scanning speed on the groove depth.



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Untreated SLS glass slides were hydrophilic and showed an average of contact angle α of 20±2° based on measurements, consistent with the literature values

32.

For laser-treated

surfaces, wetted substrates indicated larger coverage area by water droplets, and a transition in the wetting behavior from Wenzel state to hemi-wicking state, which was an indication of high hydrophilicity 33. In the case of hemi-wicking spread, a thin film of water propagated from the deposited drop and filled the microstructure, occasionally leaving the top surface of the square posts as dry islands (Figure 2a). Subsequently, the remaining of the droplet was on a composite surface of deionized water and glass 34 and was pinned by the pillars’ top edges. Similar wetting behavior on the roughened glass slides was also reported 35. The wetting effect was visualized for laser-treated surface under an optical microscope (Figure 2b,2c). The wet region indicated with dashes consisted of an inner spot surrounded by an outer ring of finite extension, the spot being the droplet lying on the composite interface, whereas the ring represented the progressing water film. The advance of the water film through the micropillar array was monitored through the wicking distance measured from the droplet contact line to the wicking front. The wicking distance was measured from the border of the inner spot to the maximum distance covered by the water film. Generally, the wicking front did not uniformly progress in all directions as the film proceeded in finite portions where resistance and pinning of the film by surface features existed 36. Hence, the evaluation of total spreading area was carried out to evaluate the wicking performance. The spreading of a droplet (2 μL) on pristine SLS glass was 8.6±0.9 mm2. Hemi-wicking requires α < θc, such that cosθc = (1-∅)/(r-∅) and cosα > (1-∅)/(r-∅), where θc is the critical angle, and ∅ and r are the two geometrical variables, solid fraction ∅, and surface roughness factor r, respectively r = 1+(4ah)/(a+b)2

36, 39.

37-38.

For 2D square architectures, ∅ = a2/HD2,

The solid fraction Ø expresses the emerging islands: fraction of the

dry surface area above the film level (Figure 2a). To fulfill the hemiwicking criteria, the surface profile density and roughness must be controlled to a certain degree, which was achieved by


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laser texturing that satisfied the geometrical requirements; the minimum value of θc was calculated to be 30°.

Figure 2. Wettability analysis of the CO2 laser-textured glass substrates. (a) Schematic describing the dynamics of the hemi-wicking wetting state. (b) Images of wet glass slides arranged according to the laser scanning speed and laser power for a constant hatch distance of 200 μm. The spreading area covered by the water droplet in each ablated region is encompassed with dashes. (c) Effect of variation in hatch distance on wicking performance. (d-g) Graphical representations of the roughness factor, the cosine of the critical angle, spreading area, and wicking distance corresponding to different laser scanning speed and power in (b). (h-k) 7 ACS Paragon Plus Environment


Correlation between the pattern hatch distance and the measured values of the roughness factor, cosine of critical angle, spreading area, and the wicking distance. Scale bar = 5 mm. For a constant HD, and varying P and S, wicking distance and spreading area depended on roughness factor r (Figure 2). The spreading area, increases in wicking distance and cosine of θc are proportional to laser power and inversely proportional to scanning speed. The highest measured wetting area was 127.2±0.9 mm2 with a maximum wicking distance of 6.2 ±0.3 mm at S = 200 mm.s-1 and P = 10.2 W. The highest spreading area and wicking distance corresponding to an intermediate power level of 10.2 W because severe cracking occurred at higher power levels, specifically at P = 10.8 W (Figure 1d). Thus, these cracks may form crevices or delaminate layers into which water could seep. Consequently, the spreading area and wicking distance at S = 200 mm.s-1 and laser power of 10.8 W had a sudden decrease in value followed by a recovery. The increase in hatch distance from 200 μm to 400 μm resulted in a rapid decline in a solid fraction from 56% to 12%, at which point the textured surface was mostly submerged under the water film. This correlated to a considerable enhancement in wicking performance, where the spread area increased by a factor of 4.5 from 43.7 to 197.3 mm2 at HD of 400 μm and 200 μm, respectively (Figure 2j). The water film extended by a maximum value of 5.6±0.3 mm at HD of 200 μm as compared to 2.5±0.3 mm obtained at 400 μm. The laser parameters, HD of 200 μm, S of 400 mm.s-1 and P of 7.8 W yielded the highest wetting level in terms of spreading area. The increase in the cosine of θc and spread signifies the more favored wicking phenomenon (Figure 2g, j). Although r (1.22 μm) at HD of 200 was less than those of calculated for HD of 300 μm (1.28 μm) and 400 μm (1.23 μm), the increase in solid fraction had counterbalanced the difference in the overall wicking force and, subsequently, wicking performance. The solid surface energy at the solid-liquid-air interface transfers to the liquid and consumes in the permeation of the wicking front through the topological variations of the structure 34, 37. 8 ACS Paragon Plus Environment

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The wicking driving force improves as the rate of energy transfer increases with higher roughness. The advancing of the water film is limited by the equilibrium of surface tensions 𝛾𝑆𝐿, 𝛾𝐿𝐴 and 𝛾𝑆𝐴, as the wicking driving force vanishes at a certain distance. Viscosity driven hydraulic resistance dissipates the energy stored in the liquid and reduces film progress velocity due to the acting shear force

40.

Subsequently, the momentum of the fluid and its ability to

advance to further distances are minimized. In contrast, the liquid impregnation efficiency is improved with lower solid fraction, as it enhances the fluid diffusivity through the microstructure and reduces the pinning of the fluid meniscus to the pillar sides

41.

Hence,

maximum spreading area in the fabricated device was achieved with Ø of 12.30% at HD of 200 μm. The θc was not governed by Wenzel’s model since the droplet laid on a combination of flat solid and liquid interfaces which, in turn, reduced the influence of surface roughness. The current r values were well above the threshold value of 1.07 required to reach θc = 0° in accordance with cosθ = r cosα. The measured apparent α for all the samples ranged between 8.87° to 9.51° – superhydrophilicity was achieved. The droplet contact line conformed mainly to the solid fraction Ø. For a fixed density of microstructures, the droplet spreading area (inner circle) did not change. The increase in density led to the increased expansion of the droplet (Figure 2h). The maximum percentage increase in spreading area due to change in laser beam speed, power and hatch distance was 39%, 45%, and 350%, respectively. The maximum percentage difference in wicking distance was ~ 100% for all parameters. Thus, modifying the hatch distance yielded the best wicking performance. An important challenge related to the instability of the droplets in the HHA was highlighted while slight movements caused the droplets to cross the hydrophobic borders and coalesce with adjacent droplets.23 In the present work, this challenge was overcome at a tilt angle of 90° and 180°, the droplets were retained in their confined regions and did not intermingle (Figure 3). Thus, the array arrangement showed a high slide resistance and exhibited a selective confinement that kept the droplets intact regardless of the orientation of the channel. In 9 ACS Paragon Plus Environment


Figure 3d, different HHA geometries have been shown to exhibit the droplet retention feature of such patterned glass slides. Hence, individual droplets can be utilized as microenvironments, for example, in cell culture, in which multiple cell types can be cultured in adjacent but isolated microreservoirs to separately monitor their growth without cross-contamination. In applications where intercellular communication is of an interest of study, either the distance between the microenvironments can be shortened or linking channels can be fabricated to facilitate the interaction among neighboring cells. The cell migration may occur due to cell overpopulation of the superhydrophilic regions 42 (Supporting Information Table S4).


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Figure 3. Fabrication of hydrophobic-superhydrophilic arrays (HHA) on a pretreated soda-lime glass slide using CO2 laser ablation. (a) Illustration of an aqueous droplet dropped on the HHA with adjacent plain and laser-patterned surfaces: The wettability contrast leads to selfpartitioning of liquid from hydrophobic surface into the channel. (b) Contact angle on the processed glass substrate coated with a hydrophobic emulsion. (c) Droplet partitioning into the hydrophilic area at horizontal, normal and 180° flip orientations. (d-g) Different geometries of HHA filled with aqueous solutions containing food coloring. The flipping at 180 did not cause any slippage or cross-contamination as droplets were held within the predefined areas.

HHA arrays exhibited selective wettability contrast between the two differently processed sections, that is, the laser ablated hydrophilic (fluidic channel) and thin-film coated hydrophobic (confinement geometry) parts on glass substrates. The sharp contrast facilitated an efficient self-partitioning of the aqueous liquid – the droplet poured on such surfaces quickly slipped from the hydrophobic film and flowed toward the hydrophilic channel where it was confined and retained. The average value of α for the thin-film-coated glass was 99°, whereas the confined droplet exhibited a contact angle depending on the amount of the liquid dispensed on the surface. This was due to the confining hydrophobic film action – the film repelled the droplet and pushed it toward the hydrophobic compartments of different sizes and shapes. The average light transmission in the visible wavelength range (450-700 nm) through the ablated section of the glass slides fluctuated between a minimum of 74% and 85% having no particular trend depending on the laser speed and power parameters (Figure 4a). However, the transmission was proportional to the pattern hatch distance (Figure 4b). At 200 μm HD, the mean transparency in the visible wavelength range was 74%, and it increased to ~ 84% at an HD of 400 μm, as well as, the improvement of 10% also resulted in a less blurry picture when seen through these regions. The transparency was reduced by increasing pattern density. The transparency losses were also induced by laser processing itself as melting, in-place 11 ACS Paragon Plus Environment


redeposition, and formation of debris resulted in defects that scatter the light at a large angle resulting in a low definition of an image. However, such losses of definition are also expected in defect-free surfaces having uniform channel profile due to light scattering from their edges. Therefore, for a transparent substrate containing grooves and channels, a post-annealing process can be adapted to reduce the defects for which the image definition would be primarily limited by the scattering process. These results imply that the transparency was reduced with high solid fraction Ø and higher surface roughness r due to material loss and defects.

Figure 4. Optical transmission analysis of the ablated glass substrates in the wavelength range of 450-700 nm. (a) Average transmission for glass substrate ablated at a constant hatch distance of 400 μm and different laser scanning speeds and powers. (b) Optical transmission for glass


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substrates with different hatch distances, and a constant speed and power of 400 mm.s-1 and 7.8 W, respectively.

The patterned array can be used in biological assays to avoid the cell overlapping. The plant cells’ distribution analyzed via a fluorescence microscope revealed the structure and count of cells with high resolution (Figure 5). The fluorescence microscopy images showed the difference of cell distributions on plain SLS substrates and laser-processed surfaces. The plain SLS substrates had a random cells distribution at different areas of the slide with varying ranges of countable cell distribution of 49-128 cells.cm-2 and high density where cells aggregated (Figure 5a, d). This limited the process of assaying cells to detect their shapes and sizes in a reproducible process. The ablated glass substrates with microchannels showed an even distribution of ~27-36 and ~15-24 cells.cm-² (Figure 5e-h, i-l) for HDs of 400 μm and 200 μm, respectively. No cell overlapping was observed due to surface tension effect which allowed the spread of cells in aqueous medium and resulting in the separation of cells43. This followed the hemi-wicking state shown in Figure 2b and Supporting Information Video 1, 2. Hence, increasing the pattern density resulted in decreased cell population and improved the cell distribution efficiency. This showed the effectiveness of the modified glass in terms of distributing a cluster of cells within a given area. Therefore, cell separation, in this application case, allowed studying the cell division stages in the male organ of Arabidopsis flower bud. This high contrast cells distribution have significant applications in tissue engineering and a regenerative medicine 44. Laser-patterned microchannels on glass slides offer self-segregating properties for biological cells due to the spreading feature of aqueous media on patterned surfaces. The microscopic fluid dynamics at the modified surfaces indicated that the spreading of droplet occurred at a higher rate due to increased molecular flow caused by the modified liquid-superhydrophilic solid interface, especially along the engraved linings


45.

The kinetic


energy of flow in the fast-spreading liquid droplet broke the cell-cell coalescence and efficiently dispersed them in the media.

Figure 5. Fluorescence microscopy of cells and their distribution analysis. (a-c) Cells dispersed over a plain soda-lime glass slide at different locations. (d) Cell population at a different position of untreated plain glass slides. (e-g) Cell dispersed over a CO2 laser-patterned glass substrate with periodic 2D microchannels at an HD of 400 μm. Scale bar = 50 μm. (h) Cell population at a different position of laser treated glass slides (HD = 400 μm). (i-k) Cell dispersed over a CO2 laser-patterned glass substrate with periodic 2D microchannels at an HD of 200 μm. Scale bar = 50 μm. (l) Cell population at a different position of laser treated glass slides (HD = 200 μm). Increasing the pattern density resulted in decreased cell population and improved the distribution efficiency.


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Material and Methods A continuous wave CO2 laser (λ = 10.6 μm, Pmax ≈ 60 W, LS3040, HPC Ltd., West Yorkshire) was used to fabricate HHAs on precleaned soda lime glass slide (SLS) samples (76×26×1 mm3, ThermoFisher Scientific) (Figure 1). The laser beam was focused by a meniscus lens (diameter = 20 mm, focal length = 50 mm) giving a minimum beam spot diameter of ~ 100 μm. Supporting Information Table S3 shows the parameters to create different patterns by varying the hatch distance (HD), scan gap (G), scanning speed (S), and power (P). Throughout the texturing process, the z-axis distance was maintained at the focal length. The geometry of the patterns was drawn using computer-aided design software LaserCut 6.1 (Leetro, Chengdu, China). Microscopic images of the patterned glass substrates were captured using an optical microscope (Axio Scope.A1, Zeiss, Jena, Germany). The laser processed glass slides were also examined by Alicona microscope (G5 InfiniteFocus, Alicona, Kent, UK) to measure the roughness and microstructured profile. Contact angle (α) was measured for all modified surfaces by applying 2.00 ± 0.06 μL drops at 24 °C at a relative humidity level of 23-25% (Supporting Information Figure S3, S4 and Table S4). To investigate the wetting dynamics, videos were recorded for spreading droplets and top view pictures of the wetted glass were captured to measure the liquid spread area and wicking distance. Images were analyzed using ImageJ software (Wayne Rasband, National Institute of Health, USA). Optical transmission measurements of CO2-laser treated glass substrates in the wavelength range of 450-700 nm were performed using a spectrophotometer (USB2000+, Ocean Optics, Oxford, UK) integrated with an optical microscope (Axio Scope.A1). SLS glass slides were spin-coated (100 rpm, 30 s) with a hydrophobic transparent layer based on water (95 vol%), isopropyl alcohol (4 vol%), and non-ionic surfactant (

Laser Inscription of Microfluidic Devices for Biological Assays - ACS

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