Stainless microfluidic probe with 2D-array microapertures

Microfluidic probes (MFPs) have attracted attention as devices that enable local chemical treatment in a solution without introducing samples into channels constructed with physical walls. The inside of an MFP contains microchannels, with its microapertures connected to the microchannels at the end of the device, enabling local fluid control by injecting and aspirating fluid at each microaperture. At this time, the fluid is injected from one aperture and aspirated into the other aperture at an aspiration flow rate larger than the injection flow rate, thereby causing a fluid localization known as hydrodynamic flow confinement (HFC).^1,2 HFC applied to a sample overcomes the limitations of conventional microfluidic systems, which use channels constructed of physical walls, limiting the sample size and causing damage to the sample caused by the channel wall.³ In the field of tissue engineering and other biomedical applications, MFP technology is an emerging tool used for biopatterning,^4,5 stimulation experiments on biosamples such as cells and tissue slices,^6–8 and analysis of tumor cells circulating in blood.⁹ Aperture array structures may allow for microgel formation to generate three-dimensional biological structures.^10–12 In addition, MFPs with integrated temperature control and dielectrophoresis technology have been reported,^13,14 demonstrating the multi-functionalization of MFPs. Furthermore, by increasing the number of apertures to form an array, a complex treatment pattern and simultaneous local treatment in multiple areas have been proposed.^15–17 These multi-aperture arrays show the potential for manipulation of treatment regions, simultaneous processing in multiple regions, and formation of drug concentration gradients with their highly-flexible fluidic patterning. MFPs reported to date have been fabricated using silicon-glass or polydimethylsiloxane,¹⁸ but this method makes it difficult to increase the number of apertures in 2D because of the thick layers with the channel structures. Thus, the formation of an array with apertures of 50 µm spacing is limited to two rows,¹⁵ and no MFP with three or more rows to form 2D aperture arrays with narrow spacing has been reported, as far as we have investigated. Recently, resin-made MFPs using a 3D printer were developed,¹⁹ although the design of 3D microchannels is complex and requires long microchannels inside an MFP as the number of apertures is increased; it is also difficult to form an aperture array at narrow intervals and long microchannels because of the limited printing accuracy. The distance between apertures of the MFP is several hundred micrometers, and it is difficult to realize a high-density aperture array compared to conventional MFPs.

In this study, we focused on enhancing local processing by using a multi-aperture MFP and aimed at developing an MFP that can easily expand the 2D aperture array at narrow intervals. It allows us to arrange apertures flexibly in 2D while maintaining narrow aperture spacing, which leads to high-density biopatterning for sensors, space-resolved stimulation to cells and tissue slices, and formation of 3D gel structures for cell biology.

We propose a fabrication scheme based on the stacking and bonding of microfabricated stainless steel substrates to generate a 2D aperture array and developed a stainless MFP (stMFP) with a 6-row and 8-column aperture array with an aperture size of 100 × 150 µm at narrow intervals of 50 µm. We then evaluated the surface treatment performance of the MFP by biopatterning with a fluorescent antibody.

Increasing the number of apertures in two dimensions at narrow intervals requires stacking of microfabricated thin substrates to form multilayers. In conventional methods for fabricating an MFP,^15–17 polydimethylsiloxane, glass, and silicon substrates have been employed, although this method makes it difficult to form multilayer stacks of thin layers because of their physical properties and limitations in the fabrication process. To overcome these limitations, we used stainless steel substrates with high rigidity and chemical resistance to allow for facile bonding operations for thin substrates.²⁰ To form an aperture, a stainless-steel microchannel layer with a through-type channel structure and stainless-steel partition layers with a port structure were fabricated by photolithography and a wet etching process and then stacked and bonded [Fig. 1(a)]. By increasing the number of stacks, a scalable 2D aperture array was produced [Fig. 1(b)]. This stacking technique does not require a complicated 3D microchannel design and can form an aperture array in a scalable manner.

To fabricate the stMFP, 1-mm diameter through holes were designed on 50-μm thick stainless steel substrates as partition layers, and channel structures were designed as microchannel layers with a minimum width of 150 µm at the end of 100-μm-thick stainless steel substrates [Fig. 2(a)]. Microchannel layers with 1-mm diameter through holes were also prepared to connect the microchannels to the ports on the side of the device. In this configuration, the ports were connected to the apertures individually through isolated microchannels.

In this study, seven partition layers and six microchannel layers were fabricated by photolithography and wet etching of stainless steel substrates (SUS304), and an stMFP with a 6-row and 8-column aperture array was developed by stacking and diffusion-bonding [Figs. 2(b) and 2(c)] through the processing service provided by KYOSEI Co., Ltd (Tokyo, Japan). The end surface of the stMFP was flattened by polishing machines (Metaserv 2000, BETA, BUEHLER, Lake Bluff, IL, USA). The stMFP has independent apertures with an average size of 94.5 × 151.8 µm for each design value of 100 × 150 µm [Fig. 2(d), Table I], and the apertures correspond to the 48 inlet ports on the side of the device (details are shows in the supplementary material). This configuration allows for the selection of fluid injection/aspiration apertures individually. In addition, stainless steel has high chemical resistance, enabling the removal of organic stain from the device with an organic solvent and strong alkaline solution, as well as reusing the stMFP after experiments involving biosamples. In this fabrication scheme, increasing the number of stacking layers and microchannels in a single layer increases the number of apertures in the row and column directions, respectively. Particularly, scalability in the column direction, which is limited in conventional methods, can be achieved because of the bonding property of stainless steel. The stMFP has 48 microapertures controlled individually and allows 2D patterning on the samples with a single device.

An MFP injects fluid from one aperture and aspirates the fluid at the other aperture to form HFC, enabling application of a chemical solution to a localized sample area. The HFC is used for biopatterning on a substrate in cell biology and biosensor applications.⁹ Thus, we demonstrated biopatterning using the stMFP and evaluated the treatment area on the substrate surface as the pattern area. In biopatterning, a fluorescent antibody is used as the injection solution and localized on the chemically modified glass substrate to cause the antibody to adhere to the glass (Fig. 3). By observing the fluorescence of the antibody attached to the glass substrate under an optical microscope, the treatment area can be estimated.

Biopatterning was performed by localizing a 0.25 mg/ml IgG fluorescent antibody [anti-mouse IgG (whole molecule)-TRITC antibody produced in the goat IgG fraction of the antiserum, buffered aqueous solution, SIGMA] on poly-L-lysine-coated slide glass (S7441, Matsunami Glass Ind., Osaka, Japan). The stMFP was supported by stainless steel (SUS304) jigs with screw holes corresponding to each inlet port on the side of the device and a jig made by a 3D printer [Fig. 4(a)], and the position was controlled by an xyz stage and goniometer, which allow the stMFP to be translocated in an open-to-air chamber where the spacer is installed. Syringe pumps (legato111, legato180P, legato220, and legato270P, KD Scientific, Holliston, MA, USA) connected through tubes were used to inject and aspirate the solution, and 1-mL syringes (SS-01T, TERUMO, Tokyo, Japan) were used for injection whereas 10-mL syringes (SS-10SZ, TERUMO) were used for aspiration. A polytetrafluoroethylene (PTFE) tube (AWG-30, Chukoh Chemical Industries, Ltd., Tokyo, Japan) with silicone tubes (high-strength silicone tube 0.5 × 3 mm, Sanyo Furue Science Co. Ltd., Saitama, Japan) attached to the tip was used to connect the syringe needle (NN-2232R, TERUMO) to the tube connection. A flangeless fitting (XP-283 or XP-286, IDEX Health and Science, Oak Harbor, WA, USA) was attached to the tip of the peak tube (1532L, IDEX) and connected to the screw hole of the stainless steel jig. To maintain the distance between the sample and the end of the stMFP constant, Kapton tape (653F No. 25, Teraoka Seisakusho Co. Ltd., Tokyo, Japan) with a film thickness of 55 µm was attached to the poly-L-lysine-coated slide glass as the spacer that does not block stMFP movement along x and y directions. The MFP did not close the top of the chamber; the chamber is open to air. Biopatterning was conducted in a Petri dish filled with pure water, and the fluorescence of the glass substrate after biopatterning was observed with a deconvolution microscope (DV-KT-EX, Seki Technotron Corp., Tokyo, Japan).

Biopatterning using one injection aperture and another adjacent aspiration aperture was conducted to confirm that the stMFP could form the HFC and to evaluate the minimum surface treatment area. The injection flow rate (Qi) was fixed at 1.00 µl/min, and the aspiration flow rate (Qa) was controlled to change the flow ratio of Qa to Qi (Qa/Qi). The fluorescence image of antibodies patterned on a substrate was analyzed with ImageJ ver1.52a software (NIH, Bethesda, MD, USA), and the surface treatment area under different flow ratio conditions was evaluated.

Biopatterning using neighboring two apertures showed that the treatment area was reduced by increasing the flow ratio (Qa/Qi). This occurred because of an increase in the flow velocities in the region surrounding the aspiration, which translates into a reduction in the section size of the confinement,² indicating that the stMFP successfully formed the HFC. At a flow rate ratio of 6.50, the minimum treatment area was 3.3 × 10³ µm², with the treatment area reaching up to 7.6 × 10⁴ µm² [Fig. 5(a)].

Next, patterning using eight aspiration apertures in one row and 1–4 injection apertures in the nearby row was conducted to evaluate the treatment area under different aperture arrangements. The flow rate at each opening was fixed at Qi = 1.00 µl/min and Qa = 5.00 µl/min, and biopatterning was conducted for 2 min with different numbers of injection apertures between 1 and 4.

Biopatterning under different aperture arrangement conditions showed that the treatment area was expanded in the row direction by increasing the number of injection apertures [Fig. 5(b)]. This patterning also showed that the size and shape of the surface treatment area can be controlled by selecting the aperture arrangement and surface treatment using multiple apertures can form a wide-range treatment area without scanning of an MFP. In addition, a complicated treatment area can be formed simultaneously by controlling the aperture arrangement and flow rate, improving the efficiency of biopatterning. These characteristics will be useful in cell biology applications, including the formation of cell culture patterns and chemical stimulation of cells.^21–23 

The advantage of the stMFP is that the injection and aspiration of fluid are controlled with arrayed apertures individually. Thus, we demonstrated that stMFP treatments with different solutions could pattern two types of biomolecules simultaneously on a substrate. IgG fluorescent antibodies (goat anti-mouse IgG solution [TRITC 0.25 mg/ml] and goat anti-mouse IgG solution [FITC 0.25 mg/ml]) with different fluorescence colors were used as injection solutions. First, in the aperture arrangement composed of the injection aperture row and aspiration aperture rows, the IgG solutions of TRITC and FITC were alternately arranged in the injection aperture row [Fig. 6(a)]. By conducting biopatterning at an injection flow rate of 10 µl/min at each injection aperture and aspiration flow rate of 12.5 µl/min at each aspiration aperture for 2 min, simultaneous treatment of multiple solutions over a wide range could be performed. The left and right ends of the aspiration aperture row were set to stabilize the flow field.¹⁰ 

Patterning using different solutions showed that six processing areas (6.1 × 10⁴ µm² on average) were formed independently in one large processing area (3.8 × 10⁵ µm²) without mixing of the solutions [Fig. 6(b)]. This suggests that multiple solutions can be applied simultaneously to the substrate. Such a configuration can be applied to cells on a substrate and tissue slice⁷ for simultaneous and local chemical treatment in cell biology. Finally, to demonstrate 2D patterning by the stMFP, the apertures were arranged in four rows and four columns [Fig. 7(a)]. In addition, in this biopatterning, TRITC and FITC IgG fluorescent antibodies were arranged alternately in the row and column direction so that the solution injected from each injection aperture was separated, and the aspirating aperture rows were set on the left and right sides [Fig. 7(a)]. Biopatterning was conducted at an injection flow rate of 10 µl/min at each injection aperture and at an aspiration flow rate of 17.5 µl/min at each aspiration aperture for 2 min. For patterning with the 2D aperture array, surface treatment areas of two types of fluorescent antibodies were formed without gaps in one wide processing area (4.5 × 10⁵ µm²), with 2D treatment areas generated in the row and column directions [Figs. 7(b) and 7(c)]. The stMFP is capable of high-density multiple-solution treatment through 2D treatment area formation and treatment with a solution with a high degree of freedom such as in a complicated processing pattern. The advantage of the stMFP is configurability: various patterns such as those shown in Figs. 5–7 can be realized with a single device by selecting apertures for aspiration and injection from 48 apertures. In addition, injection and aspiration pumps are used, where the injection and aspiration flow rate of each aperture are uniform by putting multiple syringes as a set on the pumps. In principle, bio-pattering can be achieved with two pumps (i.e., an aspiration and an injection pump). To control the flow rates of 48 apertures individually and dynamically should broaden the potential of 2D flow patterning, although further integration of such a fluidic control system is a technical challenge.

These 2D patterning methods can be used in applications such as patterning of different cells to analyze cell–cell interactions or the formation of concentration gradients in studies of chemotaxis of cells or bacteria.²⁴ In cell biological applications, the flow generated by an MFP induces shear stress to cells. The stress depends on the gap between the MFP and the sample and the flow rate.² Thus, controlling the experimental setting should be considered to minimize the effect of shear stress while forming the desired surface treatment area.

The stMFP is reusable; the stain inside the stMFP is removed by washing it with a strong alkaline solution (ex. sodium hydroxide solution) or organic solvent (ex. acetone, toluene, isopropyl alcohol, etc.) because of its high chemical resistance. In addition, the stMFP can easily increase the numerical aperture in two dimensions at narrow intervals compared to conventional MFPs;¹⁸ however, burrs and errors exist in the apertures formed by under-etching during the wet etching process. The apertures of the stMFP have a maximum aperture size of 1.6 × 10⁴ µm², which are ∼1.5 times larger than the minimum one of 1.1 × 10⁴ µm². The difference in the aperture size potentially changes the flow velocity field, which may affect the spread of the injection solution. The unsymmetrical patterns shown in Figs. 5–7 are considered to be due to the difference in the aperture sizes derived from the fabrication error. Laser microprocessing²⁵ would be a solution to fabricate apertures of uniform size.

We developed an stMFP with 48 apertures in a 6-row and 8-column aperture array by stacking seven partition layers and six microchannel layers for multifunctional and simultaneous 2D flow patterning. To evaluate the treatment capability of the stMFP, biopatterning on glass substrates was conducted, confirming that the HFC was formed on the stMFP and that the minimum patterning area was 3.0 × 10³ µm². In addition, by conducting simultaneous processing using multiple apertures and a solution, the size and shape of the treatment area were controlled, and a high-density pattern of two types of molecules was formed in the wide-range treatment area. Biopatterning using a 4 × 4 aperture arrangement showed that the stMFP enabled the generation of a 2D treatment pattern and simultaneous treatment of multiple areas with a high degree of freedom. The stMFP can be used in various applications, such as cell biology, and shows various advantages such as multifunctionalization and reusability.

See the supplementary material for the design of stainless substrates of the stMFP.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

This work was supported by PRESTO (Grant No. JPMJPR14FB) of the Japan Science and Technology Agency (JST) and JSPS KAKENHI (Grant Nos. 15K13324, 16H06077, 18K18837, 18K06175, and 20K21900) of the Ministry of the Education, Culture, Sports, Science, and Technology (MEXT). The work was partly conducted at the Kagawa University Nano-Processing Facility, supported by the “Nanotechnology Platform Program” of the MEXT.