WO2018163101A1

WO2018163101A1 - Microfluidic sensor and method for obtaining such a sensor

Info

Publication number: WO2018163101A1
Application number: PCT/IB2018/051517
Authority: WO
Inventors: David Vincent BONZON; Marc Lany; Georges Henri MULLER; Philippe Renaud; Luc AEBERLI; Niklas VAN NEYGHEM; Thomas Braschler; Jonas CHOPPE
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2017-03-09
Filing date: 2018-03-08
Publication date: 2018-09-13
Also published as: EP3592695A1; WO2018163101A8

Abstract

The invention describes a method for producing hybrid microelectronic/microfluidic sensors at industrial scale. The method is characterized in that it comprises the following steps for obtaining said microfluidic channel: a) a first lamination step of a dry film resist onto a PCB panel; b) a photostructuration step of the dry film resist on the PCB panel; and c) a closure step of the photostructured dry film resist to obtain the microfluidic channel. The method adapts standard PCB manufacturing processes used at industrial level by repeating some of the passages thereof, in order to produce microfluidic channels built-in with the microelectronic components in the form of a photostructured dry film resist laminated on a previously obtained PCB panel. The microchannels are moreover simply integrated in the final sensors via standardized design rules and tools used in industrial PCB manufacturing. Microelectronic/microfluidic sensors obtainable by the presently invented method are also herein disclosed.

Description

Microfluidic Sensor and method for obtaining such a sensor

Technical Field

[0001 ] The invention relates to microfluidic devices compliant with standard PCB manufacturing procedures.

Background Art

[0002] Since its invention in the early 1900s, Printed Circuit Board (PCB) fabrication has become a robust standard in the electronic industry and led to a continuous development of this technology. At the end of the 20th century, PCB tracks resolution were reaching the 100 μιτι and fabrication process was sharing many similarities with microelectromechanical systems (MEMS) fabrication including photolithography and etching techniques. Consequently, researchers started to take advantage of PCB technologies to create microsystems. Nguyen et al. demonstrated a micropump based on a commercial PCB supplemented by piezo discs soldered onto the copper pads (Nguyen, N. T. et al. (2001 ) Sensors and Actuators A: Physical, 88(2), 104-1 1 1 ). Wego et al. demonstrated the possibility to create fluidic channel by directly using the PCB copper tracks with a laminated polymeric foil on the top (Wego, A., et al. (2001 ) Journal of Micromechanics and Microengineering, 1 1 (5), 528).

[0003] If purely electro-mechanical MEMS including pressure sensor, accelerometers and gyroscope are largely based on complementary metal-oxide-semiconductor (CMOS)-compliant process and largely benefited from the CMOS design and process standardization (Hierold, C. et al. (1996, February) In Micro Electro Mechanical Systems, 1996, MEMS'96, Proceedings. An Investigation of Micro Structures, Sensors, Actuators, Machines and Systems. IEEE, The Ninth Annual International Workshop on (pp. 174-179). IEEE; Pakula, L. S. et al., (2004) Journal of Micromechanics and Microengineering, 14(1 1 ), 1478), it is far from being the same for BioMEMS. BioMEMS is a growing family of microsystems targeting biological and medical applications (Bashir, R. (2004) Advanced drug delivery reviews, 56(1 1 ), 1565-1586). Amongst many applications, examples of BioMEMS are flow cytometer chips, liquid handling chips and electrode arrays. Flow cytometers are used in cell counting, analyzing and sorting with main applications in rapid analysis of a large cell population such as hematology (Givan, A. L. (2001 ) Methods in cell biology, 63, I 960; Gawad, S. et al. (2001 ) Lab on a Chip, 1 (1 ), 76-82). Electrode arrays are used for tissue electrical signal recording or stimulation. Rigid multi- electrode array (MEA) were first developed, on which cells were cultured (Gross, G. W. et al. (1977) Neuroscience Letters, 6(2), 101-105) and more recently, electrodes made on flexible probes steered this technology towards direct implantation (Boppart, S. A. et al. (1992) IEEE transactions on biomedical engineering, 39(1 ), 37-42; Cheung, K. C. et al. (2007) Biosensors and Bioelectronics, 22(8), 1783-1790). Even though the BioMEMS family was inspired from the initial MEMS in term of design, they have their own specificities and constraints. The microchannels they accommodate are generally in the range of tens of micrometers in width, such that they can be used with cells in suspension. Fluidic in- and outlets are much bigger than the electric pads used for wire bonding in CMOS chips. Polymeric flexible substrates often need to be used to interface cells and soft tissues. BioMEMS chips generally have larger dimensions in the order of square centimeters. On the other hand, BioMEMS can generally be produced with far lower resolutions than standard CMOS processes. Even if BioMEMS fabrication can benefit from well-established manufacturing technique such as injection molding, hot embossing and metal deposition methods (Greener, J. et al. (2006) Precision injection molding. Carl Hanser, Munich; Attia, U. M. et al. (2009) Microfluidics and Nanofluidics, 7(1 ), 1-28), the wide range of design and materials as well as their specific function makes it difficult to define standardized process for manufacturing. Current efforts go toward design (van Heeren, H., et al. (2015), 19: 1203. doi:10.1007/s10404-015-1639-4) and process standardization to facilitate BioMEMS commercialization (Volpatti, L. R., & Yetisen, A. K. (2014), Trends in biotechnology, 32(7), 347-350).

In the last decade PCB tracks resolution decreased toward 20 μιτι thanks to photoresist material improvement and new exposition techniques such as Laser Direct Writing. Microvia also emerged using Direct Laser Drill techniques. The use of flexible substrate such as polyimide becomes a standard. Consequently, PCBs are now sharing many similarities with BioMEMS; feature size are in the same order of magnitude (20 μιτι - 200 μιτι), their total surface can largely exceed the range of square millimeters and the substrate can be flexible and biocompatible using polymers such as polyimide. Researcher recognized those advances and started to make microfluidic chips by stacking of two or more standard PCBs and polymer layers (Wu, A., et al. (2010) Lab on a Chip, 10(4), 519-521 ). Recently, this technique was used by Vasilakis et al. (Applied Surface Science, 2016, 368, 69-75) to combine multiple PCBs and dry film resist layers to define at the same time electrodes and microfluidic channels.

Similarly to CMOS foundries that use a fully standardized process regardless of the design, the PCB production lines use standardized processes to produce different PCB designs. However, to be compliant with a PCB production line many constraints are imposed on the design and materials to be used. Many researchers already used existing PCB material such as photostructurable dry film resist to define their microfluidic network on substrate such as glass or silicon (Vulto, P. et al. (2005), Lab on a Chip, 5(2), 158-162) or used the same dry film resist as a master for soft lithography (Stephan, K. et al. (2007), Journal of Micromechanics and Microengineering, 17(10), N69). Alternatively, some microfluidic chips were also fabricated in PDMS by soft lithography but using directly the PCB and its copper traces as master (Li, C. W., et al. (2003), Analyst, 128(9), 1 137-1 142). Some other microfluidic chips were based on a PCB substrate on which a microfluidic layer made of SU-8 (Kontakis, K., et al.

(2009) , Microelectronic Engineering, 86(4), 1382-1384; Guo, J., et al. (2014), IEEE Sensors Journal, 14(7), 21 12-21 17) or PDMS (Marshall, L. A., et al. (2012), Analytical chemistry, 84(21 ), 9640-9645) was subsequently added. Finally, microfluidic chips where made directly by the stacking of two or more standard PCBs and polymer layers (Wu, A., et al.

(2010) , Lab on a Chip, 10(4), 519-521 ). [0006] However, despite the great amount of solutions proposed in this field and the efficiency of the obtained devices, the methods for obtaining the same are either cumbersome, expensive or requires a drift from the standard processes used in the PCB manufacturing chain, thus requiring adaptation of the production protocols used in industry which is not always feasible, cheap or easily affordable at industrial level. Most of the prior art hybrid microfluidic-microelectronic devices have been developed to solve specific technical issues or as proof-of-concept, and therefore the manufacturing method has been adapted each time, and on a laboratory-scale with tailored tools and conditions. What is lacking in the art is a universal method for producing such hybrid devices (e.g., BioMEMS) which is as much compliant as possible to industrial processes actually in place, so to optimize well-known operations, machines and expertise in the field.

Summary of invention

[0007] The present invention aims at solving the limitations of the prior art approaches and solutions in terms of manufacturing of PCB-based microfluidic devices. It is based on a simple and elegant intuition adapting the standard PCB fabrication line to produce a BioMEMS, suitable for small to large scale production.

[0008] One aim of the present invention was to put in place a manufacturing process to fabricate hybrid devices adapted to be used as sensors, including a fluidic network and microelectronic components, by exploiting a certified, complete PCB production line and its standardized industrial PCB process flow.

[0009] Another aim of the invention was to fabricate said sensors possibly at high industrial scale and on PCB panels.

[0010] Still another aim of the invention was to exploit standard designing tools typically used in the PCB design, and compliant with designing rules thereof, for defining microfluidic channels, so to easily produce built-in microchannels with PCB industrial machinery. All these aims have been accomplished through the present invention, as disclosed in the following description and in the appended claims. [001 1 ] The key idea behind the inventive concept of the invention was to repeat some of the passages of a standard, industrial PCB fabrication process in order to exploit well-defined, known steps to implement a fluidic network in the form of a photostructured dry film resist laminated on a previously obtained PCB panel in a one-step, easy and repeatable manufacturing procedure.

[0012] In this context, it must be highlighted that usually, for mass production, PCB manufacturers combine several individual boards into a single panel, as schematically depicted in Figure 1 , by using rational design rules. This process, called panelization, is a must-have with the consideration of PCB manufacturing efficiency. On the one hand, panelization leads to the improvement of PCB manufacturing efficiency so that lead time can be greatly reduced. At the same time, for small PCBs with irregular shapes, panelization is the most effective manufacturing way. For PCB assembly, panelization is crucial because it can downsize the cost of labor and it's convenient to control products' quality. In general, more boards packed into a set of panels means more efficient (less costly) manufacturing (and, generally speaking, the more boards that fit on a panel the lower the per- board price). An example of general panelization rules can be retrieved for instance at http://electronicdesign.com/boards/pcb-designers-need-know- these-panelization-guidelines.

[0013] Moreover, and importantly, for the sake of automation and repeatability, the machinery and processes used in an industrial setting are setup to handle uniformly-sized panels. In this context, the panels and the printed circuits thereon are also further characterized by the presence of fiducial marks providing common measurable points for all steps in the assembly process so that all automated assembly equipment can accurately locate the circuit pattern. Fiducial marks are generally categorized in the following types: global fiducials, used to locate the position of all features on an individual printed circuit board; local fiducials used to locate the position of an individual land pattern or component that may require more precise location; image fiducials, global fiducial marks on a multiple printed circuit board fabrication panel that are located within the perimeter of an end-product printed circuit board; and panel fiducials, global fiducial marks on a multiple printed circuit board fabrication panel that are located outside the perimeters of the end-product printed circuit boards. Design rules have been standardized by the Surface Mount Equipment Manufacturers Association, and are supported by IPC - Association Connecting Electronics Industries.

[0014] PCB panelization can be envisaged, for example, as a panelization with break-away but without spacing between each single printed circuit, with break-away and spacing between each single printed circuit, without break-away or panelization with stamp holes (small holes connecting single boards that look like stamp saw tooth). The large panel is then broken up or "depaneled" as a certain step in the process - depending on the product, it may happen for example right after in-circuit test (ICT), after soldering of through-hole elements, or even right before the final case-up of the assembly. There are six main depaneling cutting techniques currently in use: hand break, pizza cutter / V-cut, punch, router, saw and laser.

[0015] The method of the invention is particularly intended for the production of hybrid microfluidic/microelectronic device sensors at large industrial scale and directly at the PCB panel ' level, so to adapt standard processes (e.g. panelization, fiducials etc.), materials and machinery used in PCB mass production to a new kind of products such as BioMEMS without altering the commonly used practice, but only by repeating certain steps mastered by PCB fabricators. Moreover, since medical device companies are constantly challenged with a stringent and continuously evolving regulatory environment, a further aim of the invention was to develop a method allowing to obtain microelectronic/microfluidic sensors for use in e.g. clinics or diagnostics that are produced by already certified manufacturers in terms of Good Manufacturing Practices (GMP), ISO standards and the like.

[0016] In some implemented embodiments, two BioMEMS chips were produced: a flow cytometer with a design adapted to PCB production constraints, and an electrode array placed on a flexible probe tip. To demonstrate its compliance with a standard PCB fabrication line, the fabrication was outsourced at a PCB workshop operating a standard PCB production line. The flow cytometer chip was used to evaluate the PCB fabrication capability in term of resolution for microfluidic channel and metallic tracks. Its operation was demonstrated with polystyrene beads.

[0017] Accordingly, one object of the present invention relates to a method for producing a microfluidic layer comprising at least a microfluidic channel on a Printed Circuit Board (PCB) panel, the method comprising the following steps:

[0018] a) a first lamination step of a dry film resist onto said PCB panel;

[0019] b) a photostructuration step of the dry film resist on the PCB panel; and [0020] c) a closure step of the photostructured dry film resist to obtain the microfluidic layer.

[0021 ] In one embodiment, the method further comprises a first step of producing a PCB panel comprising a PCB or a plurality thereof.

[0022] In one embodiment, the PCB or plurality thereof are produced via lamination and photostructuration of a dry film resist disposed onto a metallized substrate.

[0023] In preferred embodiments, the method is characterized in that the photostructuration step of b) is determined by a Gerber file.

[0024] In preferred embodiments, the method is characterized in that the closure step of c) is a lamination step.

[0025] In preferred embodiments, the method is characterized in that the lamination step of c) is a lamination step of a dry film resist.

[0026] In preferred embodiments, the method is characterized in that the lamination step of c) is performed on the PCB panel.

[0027] In preferred embodiments, the method is characterized in that steps a), b) and c) are performed before the depaneling of the PCB panel.

[0028] In one embodiment, the method is characterized in that steps a) and b) are sequentially repeated so to obtain a stack of dry film resists.

[0029] In one embodiment, the method is characterized in that the microfluidic layer comprises an array of microfluidic channels. [0030] In one embodiment, the method is characterized in that the microfluidic channel is an elongated channel, a reservoir, a well or combinations of the foregoing.

[0031 ] In one embodiment, the method is characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some vias present in the PCB panel.

[0032] In one embodiment, the method is characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some electric components or electrodes present in the PCB panel.

[0033] In one embodiment, the method is characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some electric components or electrodes present in the PCB panel through at least some vias present in the PCB panel.

[0034] In one embodiment, the method is characterized in that the dry film resist is selected from a list comprising Riston, Ordyl or Kolon.

[0035] In one embodiment, the method is characterized in that the photostructuration step is performed by UV light insolation and development.

[0036] Another object of the present invention relates to a Printed Circuit Board (PCB) panel comprising a microfluidic layer obtainable through the above method.

[0037] Another object of the present invention relates to the use of the method for manufacturing a microfluidic sensor or a plurality thereof comprising a

Printed Circuit Board (PCB) and a microfluidic layer.

[0038] Still another object of the present invention relates to a sensor comprising a Printed Circuit Board (PCB) and at least a microfluidic channel obtainable through the above method.

Brief description of drawings

[0039] In the Figures:

[0040] Figure 1 depicts a schematic overview of standard PCB panel features; [0041 ] Figure 2 depicts one embodiment of the fabrication process used for manufacturing a sensor according to the invention. In the depicted embodiment, steps 1 to 1 1 are common to standard PCB fabrication, whereas step 12 and 13 are repeated from steps 4 and 5 to create microfluidic channels;

[0042] Figure 3 shows an optical micrograph of test patterns allowing to identify the smallest feature obtained for dry film resist channels (left) and metal tracks (right) in a standard PCB fabrication line;

[0043] Figure 4 shows a) Design and b), c) SEM of the fabricated electrode array probe;

[0044] Figure 5 shows a) Graph showing fibroblasts growth after 5 days incubation with 7 different combinations of material used for PCB fabrication and the control without PCB. b) Graph showing the copper concentration in the well for the 7 combination of materials after the 5 days incubation, c) Optical micrograph of the fibroblast culture in the control well after 5 days incubation, d) Optical micrograph of the fibroblast culture in the well containing the PCB made only of polyimide after 5 days incubation;

[0045] Figure 6 shows a) SEM and b) design of the flow cytometer chip with liquid electrodes, c) Impedance magnitude signal over time of 20 μιτι beads suspended in PBS and flowing in the 40 μιτι microfluidic channel of the fabricated flow cytometer. Each impedance peak represents the passage of one 20 μιτι bead in the sensing area;

[0046] Figure 7 shows a Gerber file used for designing a sensor according to the invention. It includes a layer called "microfluidic layer" used to define the pattern of microchannels to be included in the sensor, that is implemented as all other PCB layers in the manufacturing process;

[0047] Figure 8 shows a partially depaneled panel of hybrid microelectronic/microfluidic sensors produced according to the method of the invention;

[0048] Figure 9 depicts one embodiment of a Coulter counter that can be produced with the process of the present invention. Description of embodiments

[0049] The present disclosure may be more readily understood by reference to the following detailed description presented in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0050] As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term "comprising", those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of."

[0051 ] A "microfluidic device", "microfluidic chip" or "microfluidic platform" is generally speaking any apparatus which is conceived to work with fluids at a micro/nanometer scale. Microfluidics is generally the science that deals with the flow of liquids inside channels of micrometer size. At least one dimension of the channel is of the order of a micrometer or tens of micrometers in order to consider it microfluidics. Microfluidics can be considered both as a science (study of the behaviour of fluids in micro- channels) and a technology (manufacturing of microfluidics devices for applications such as lab-on-a-chip). These technologies are based on the manipulation of liquid flow through microfabricated channels. Actuation of liquid flow is implemented either by external pressure sources, external mechanical pumps, integrated mechanical micropumps, hydrostatic pressures or by combinations of capillary forces and electrokinetic mechanisms. [0052] The microfluidic technology has found many applications such as in medicine with the laboratories on a chip because they allow the integration of many medical tests on a single chip, in cell biology research because the micro-channels have a similar size as the cells and allow such manipulation of single cells and rapid change of drugs, in protein crystallization because microfluidic devices allow the generation on a single chip of a large number of crystallization conditions (temperature, pH, humidity...) and also many other areas such as drug screening, sugar testers, chemical microreactor or micro fuel cells.

[0053] In the frame of the present invention, a microfluidic device can be easily adapted to work with fluid volumes spanning from millilitres down to femtoliters, and the dimensions can be adapted accordingly to have channels within the millimetre scale.

[0054] The method of the invention is particularly suitable for the development of microfluidic sensors. Particularly, due to the exploitation of standard Printed Circuit Board manufacturing process, the invented method allows for the creation of small, cheap and ready-to-use hybrid microelectronic/microfluidic sensors, in which electronic sensing/stimulating capabilities provided from the PCB components can be coupled with the handling of small volumes of liquid samples. A typical example of such a sensor could be a miniaturized, portable Coulter counter.

[0055] In the '50s, Wallace Coulter described the Coulter counter principle and means to detect particles in a fluid (US2656508A). This principle allows for characterizing dielectric particles in a fluid in term of number and size. The basic components necessary to build a Coulter counter are: i) two chambers filled with conductive saline medium, ii) an orifice separating the two chambers and iii) one electrode immersed in each chamber. When an electrical current is applied between the two electrodes, most of the electrical resistance or impedance is in the orifice. If a particle passes through the orifice, it displaces an equivalent volume of saline resulting in an increase in impedance. [0056] A Coulter counter played an important role in the development of the first ever cell sorter, and was involved in the early days of the development of flow cytometry. Cells, being poorly conductive particles, alter the effective cross-section of the conductive microchannel. If these particles are less conductive than the surrounding liquid medium, the electrical resistance across the channel increases, causing the electric current passing across the channel to briefly decrease. By monitoring such pulses in electric current, the number of particles for a given volume of fluid can be counted. The size of the electric current change is related to the size of the particle, enabling a particle size distribution to be measured, which can be correlated to mobility, surface charge, and concentration of the particles.

[0057] The most successful and important application of the Coulter Principle is in the characterization of human blood cells, used to diagnose a variety of diseases, that is the standard method for obtaining red blood cell counts (RBCs) and white blood cell counts (WBCs) as well as several other common parameters. When combined with other technologies such as fluorescence tagging and light scattering, the Coulter Principle can help produce a detailed profile of patients' blood cells. In addition to clinical counting of blood cells (cell diameters of -6-10 micrometres, typically), the Coulter principle has established itself as the most reliable laboratory method for counting a wide variety of cells, ranging from bacteria (< 1 micrometre in size) to plant cell aggregates (>~1200 micrometres).

[0058] The Coulter Principle has proved useful for applications well beyond cellular studies. The fact that it individually measures particles, is independent of any optical properties, is extremely sensitive, and is very reproducible has appeal to a wide variety of fields. Coulter Counters have a wide variety of applications including paint, ceramics, glass, molten metals and food manufacture. They are also routinely employed for quality control. As it will be more apparent in the following description, the method of the present invention would permit inter alia to fabricate miniaturized, portable microfluidic sensors exploiting the Coulter principle, such as those described for instance in US8608891 or in US 2010/0006441 in a liquid electrode configuration, in a quick and cheap one-step process not requiring any microfabrication or assembly step.

[0059] A method for manufacturing a microfluidic layer on a PCB panel and, in particular embodiments, for producing microfluidic sensors according to the invention is schematically depicted in Figure 2. This is a general description of a typical, standard PCB manufacturing process; however, some of the described steps can be absent or interchanged in particular embodiments according to the invention, without departing from the general inventive concept. One of the key challenges of the invention, as repeatedly stated elsewhere, was to find a simple and not expensive means to create in one step microelectronic/microfluidic devices, reliable enough to produce efficient sensors such as BioM EMS (even at large scale), and easily adaptable to standard processes/machinery commonly used in PCB manufacturing plants.

[0060] Typically, a polymeric flat substrate 100, having an upper surface, a bottom surface and a thickness spanning from about 5 μιτι to about 5mm, typically between 10 μιτι and 1 .6mm, is metallized via a lamination step with a thin metal film (or lamina) 101 (about 1 to 70 μιτι in thickness) on either or both sides of the polymeric substrate 100 (Figure 2, item 1 ) and used as a starting material for the process. It has to be noted that the thickness of the substrate 100 and that of the film 101 is usually determined by the application requirements. The term "lamination" refers to the technique of manufacturing a material layer by layer (or lamina by lamina), so that the obtained composite material achieves improved properties from the use of differing materials. A laminate is a permanently assembled object by heat, pressure, welding or adhesives.

[0061 ] The polymeric flat substrate 100 is preferably composed of dielectric polymeric materials such as FR4 (glass reinforced epoxy), Kapton (polyimide) or Liquid crystal polymers, while the thin metal film 101 is generally speaking composed of highly conductive metals such as copper or noble metals such as gold or platinum. In common industrial practice, PCB manufacturers receive a starting material composed of an already metal-laminated support ready to be used; for example, a basic building block in this process can be an FR-4 panel with a thin layer of copper foil laminated to one or both sides. In multi-layer boards, multiple layers of material are laminated together.

[0062] In certain embodiments, at this stage the metalized panel undergoes the process of panelization whereby a number of PCBs are grouped for manufacturing onto a larger board (the panel). This can consist of a single design, but sometimes multiple designs are mixed on a single panel. The panelization is the rational design put in place by the manufacturer or the client thereof in order to group all the designed PCBs into a single panel, mainly for the sake of cost efficiency.

[0063] As it will be evident, the panelization process directly derives from the design(s) of the one or preferably more PCBs present in a panel. To facilitate this design process, a standard electronics industry file format, called Gerber, is used to communicate design information to manufacturing for many types of printed circuit boards. This file format, a hybrid machine control language and image, is a core component of the electronics manufacturing supply chain, and describes the printed circuit board layers images: copper layers, solder mask, legend, etc. PCBs are designed on a specialized electronic design automation (EDA) or a computer-aided design (CAD) system. The CAD systems output PCB fabrication data to allow manufacturing. This data typically contains a Gerber file for each image layer (copper layers, solder mask, legend or silk...). During bare board fabrication Gerber is the standard input format for photoplotters and all other fabrication equipment needing image data, such as legend printers, direct imagers or automated optical inspection (AOI) machines or for viewing reference data in different departments. Gerber files can also contains a 'stencil' layer for solder paste and the central locations of components to allow the PCB assembler to create the stencil and place and bond the components.

[0064] As will be more apparent later on in the description, the fabrication of microfluidic sensors according the present invention, and particularly the microfluidic path(s) thereon, is highly facilitated thanks to the possibility of exploiting the high precision and universality of standard industrial design software/machinery. In particular, the Gerber file format and other computer-aided design tools permit with an extreme ease and reproducibility to create in a single file bunch, and without departing to standard tools used in industry, all the needed image layers for creating both the electronic and the fluidic elements of a hybrid sensor.

[0065] The metalized panel is then usually pierced to obtain holes. Holes through a PCB are typically drilled with small-diameter drill bits made e.g. of solid coated tungsten carbide. The drilling is performed by automated drilling machines with placement controlled by computer-generated files called drill tape or drill file. The drill file describes the location and size of each drilled hole (Figure 2, item 2). Holes may be made conductive, by e.g. electroplating or inserting metal eyelets (hollow), to electrically and thermally connect board layers. Some conductive holes are intended for the insertion of through-hole-component leads. Others, typically smaller and used to connect board layers, are called vias 103 (Figure 2, item 3). For purposes which will become more apparent with the following description, in the context of a sensor such as a Coulter counter, vias 103 can act as both electrical and fluidic connection between the microfluidic channel/array and the external environment. However, this step is not mandatory in the manufacturing of microfluidic sensors according to the invention, and is described herein for non-limiting, clarity purposes.

[0066] As depicted in Figure 2, item 4, the holed panel is laminated with a so called dry film resist or photoresist 104. A dry film photoresist is applied using dry lamination, where photoresist 104 is evenly rolled across the surface of the support 100 with a controlled degree of pressure and temperature.

[0067] A "photoresist" is a photosensitive material used in the microelectronics industry to form a patterned coating on a substrate surface. In general, a dry film photoresist has a top layer as a separation sheet, composed of Polyethylene film (PET), the bottom layer is a support or protective film, composed of polyester (PE), and the middle layer a photosensitive layer. Its thickness depends on the application and ranges from few microns to 150μιτι. [0068] A pattern is transferred from a photomask to the substrate using a process called photolithography. In this process, a photoresist 104 is coated on the metal laminated substrate 100 and exposed to light through a mask (not depicted) to perform a photostructuration of the resist 104 (Figure 2, item 5). A photochemical reaction occurs in these exposed regions of the resist 104 which is then easily dissolved in a developer solution.

[0069] Generally speaking, the components of photosensitive layer are monomers, photo initiators, polymer binder, and some additional additives such as adhesion promoters and dyes. Monomers are the main components of a dry film. A monomer is initially solvable in developer and becomes unsolvable after UV light exposure and heat treatment. Photo initiators generate free radicals under the UV light exposure. When a dry film resist 104 is exposed under UV light, photo initiators absorb UV energy and generate free radicals. The monomers, initially spread uniformly in a dry film, start to polymerize under the stimulation of these free radicals. After that, polymers cross link to etch other and become unsolvable in developer solution

[0070] The resist pattern depends on the photomask pattern and the polarity of resist 104. Positive photoresist responds to the light in such a way as to make the exposed regions dissolve more quickly during the development process. In other words, the unexposed regions of the resist will remain unchanged (as shown in (Figure 2, item 5). Negative photoresists respond to light in opposite manner such that the unexposed regions of the resist will dissolve in the developer solution, while the exposed regions remain behind. In the frame of the present disclosure, particularly advantageous dry film resists are Riston®, Ordyl® or Kolon®.

[0071 ] In the subsequent steps, optionally a tin mask 105 is deposed on the exposed metal layer 101 present on the support 100, and the remaining dry film resist 104 is stripped (Figure 2, items 6 and 7). Finally, the thin metal layer 101 not protected by the tin mask 105 is etched (Figure 2, item 8), and the tin mask 105 itself is then etched to leave patterned metal contacts 101a deriving from the metallic thin layer 101 on the polymeric substrate 100 (Figure 2, item 9). Additionally, a solder mask 107 can be deposed and structured for the purpose of insulating some of the metal contacts 101a (Figure 2, item 10). Optionally, the metal contacts 101a can undergo a further metallization with e.g. gold 106 (Figure 2, item 1 1 ), typically for biocompatibility purposes.

[0072] Having described the generalities of a standard PCB manufacturing process, the inventive concept behind the invention allows to produce e.g. a hybrid microelectronic/microfluidic sensor by simply adapting said process in order to create microfluidic channels built-in with the PCB component on a panel. In particular, by repeating steps 4 and 5 as depicted in Figure 2, one or several time, one or more built-in fluidic path(s) 108 can be easily and automatically produced (Figure 2, item 12). In the frame of the present disclosure, all the structural elements composing the microfluidic channels/paths are referred to as a single entity called "microfluidic layer". Accordingly, vias and through-holes are not included into the definition of microfluidic layer.

[0073] To do so, therefore, a first lamination step of a dry film resist 104 is performed on the PCB panel, followed by a photostructuration of the same (Figure 2, item 12). At this step, the microfluidic channel(s) 108 is (are) characterized by the fact that at least the walls thereof are defined by a dry film resist 104. This process is hugely facilitated by the possibility of designing the microfluidic channel 108 or even arrays thereof via computer-aided design tools, and convert the design into Gerber files as they was additional PCB layers. In this way, standardization and compliance to industrial workflow is guaranteed, as well as the reproducibility of the obtainable sensors at industrial scale. As a consequence, the present method is further characterized by the fact that the photostructuration steps for producing a microfluidic channel or arrays thereof in a sensor according to the invention is determined at the Gerber file level, that is, the photostructuration pattern of the microfluidic portion of the sensor is dictated by the design provided by a Gerber file for the microfluidic layer, and implemented via the machinery using a Gerber file usually used in PCB manufacturing such as a photoplotter. This is schematically depicted in Figure 7. [0074] Finally, a closure step of the photostructured dry film resist 104 is performed to close the channel(s) 108, thus creating the floor thereof (Figure 2, item 13). This closure step can be performed in several ways; for instance, a transparent panel, made for instance of glass or transparent polymeric materials, can be used, allowing for transparency of the resulting sensors. However, in a preferred embodiment, the closure step can be advantageously a lamination step, and particularly a lamination of a dry film resist, such as those used in the production of the PCB components of the sensors. In this way, the entire embedded microfluidic part of the sensors would be produced through a classical PCB process, thus reducing costs and time. For the same reasons, preferably the entire process is performed before the depaneling of the PCB panel into the single sensors. The result of the entire process is shown in Figure 8.

[0075] Thanks to the possibility of rationally designing in a simple manner the microfluidic channels 108 of the microfluidic layer, and implementing them in an industrial workflow, very complicated designs can be envisaged. For instance, in some embodiments several layers of microfluidic channels 108 or arrays thereof can be stacked one on the others by simply repeating the lamination and photostructuration steps several times, and the channels 108 can be set out as elongated channels, reservoirs, wells and the like, thus granting an extreme adaptation to the sensing needed. Moreover, the photostructuration step for producing the microfluidic paths 108 can be adapted to fluidically connect the same with at least some vias 103 present in the PCB panel, and/or with at least some electric components or electrodes present in the PCB panel, and/or with at least some electric components or electrodes present in the PCB panel through at least some vias 103 present in the PCB panel. In this context, the manufacturing process of the invention would facilitate the production of a miniaturized, portable flow cytometer such as a Coulter counter based on a PCB workflow, as the one schematically depicted in Figure 9, or a liquid electrode flow cytometer as depicted in Figure 7. [0076] EXAMPLES

[0077] In an implemented embodiment, the fabrication of a hybrid microelectronic/microfluidic sensor consisted of a standard PCB process in which two steps are repeated in order to add the fluidic layer to a PCB on which metallic tracks have been patterned to be used as electrodes. The repeated steps for microfluidic channel definition are based on similar equipment and parameters than other conventional PCB fabrication steps. To demonstrate its compliance with a standard PCB fabrication line, the fabrication was outsourced at a PCB workshop.

[0078] The standard PCB process used in this work is the same as shown in Figure 2, and starts with a 100 μιτι Kapton film (100) covered by 18μιτι of copper (101 ) on both side (step 1 ) (Pyralux, DuPont, US). Holes and vias 103 are drilled by CNC machining (step 2) or laser etching for microvias, and metallized with 18 μιτι copper using electroless deposition (step 3). Dry film resists 104 (KOLON Pk 1640, Lifestyle Innovator, KR) are laminated on both sides (step 4) and structured by photolithography followed by the resist 104 development (step 5). In the depicted PCB process, tin 105 is preferably deposited as mask (step 6) through which the dry film resist 104 and copper 101 are etched (steps 7 and 8). The tin mask 105 is then etched (step 9). Optionally, a photosensitive epoxy based insulating mask 107 (Elpemer, Lackwerke Peters, DE), later called solder mask, is deposited by serigraphy and subsequently photostructured (step 10). Also optionally, a layer 106 of ~5 μιτι nickel and subsequently ~ 50 nm gold is chemically deposited (step 1 1 ) to cover the copper traces and pads. At this stage, a conventional PCB is complete with a substrate for mechanical stability in addition to metallic traces, via and pads as well as a solder mask for the electrical insulation. In order to fabricate the microfluidic layer of the BioMEMS chip on the same fabrication line, another layer of dry film resist 104 is laminated and photostructured on a PCBs panel with similar equipment and parameters than used for the steps 4 and 5. These repeated steps defines the fluidic channels 108. The same step 5 is repeated once again but without photostructuration to deposit a layer of dry film resist 104 to close the microfluidic channel 108.

[0079] Results

[0080] Limits and resolution

[0081 ] Figure 3 shows the PCB process resolution for microfluidic channel openings in the dry film resist (KOLON Pk 1640, Lifestyle Innovator, KR) and metallic tracks in the copper layer. Fabricated features were systematically 10-20% bigger than the design dimension for features smaller than 100 μιτι and this effect was more important for smaller features. Out of three chips, the minimal channel opening that was completely developed over different batches was measured to be on average 45μιτι with a standard deviation of Ο.δμιτι with a design dimension given at 40μιτι. Smaller dimensions down to 20μιτι were usually not completely developed forming a u-shaped channel, but still usable to perfuse liquid with limited access to underlying electrodes. Similarly and out of three chips, the smallest metallic tracks width, with a spacing of similar dimensions between them, was on average 43μιτι with a standard deviation of 1.8μιτι for a given design dimension of 50μιτι. In this later case, fabricated feature were systematically 10-20% smaller than the design dimension.

[0082] Electrodes array probe fabrication

[0083] A flexible MEA was designed in which the electrodes are patterned on a needle shape flexible substrate in view of its insertion in a biological tissue. The probe is 20 mm long and 1.2 mm in width and include six distal microelectrodes (200 μιτι in diameter) connected to proximal connection pads with 70 μιτι width conductive traces, as presented in Figure 4a). Figure 4b) shows the probe resulting from the standard PCB fabrication process with 18 μιτι thick copper tracks and electrodes patterned on top of a 100 μιτι thick standard Kapton layer. Figure 4c) shows the final probe once the solder mask is deposited and structured to define the electrodes and provide electrical insulation of the tracks.

[0084] Cell toxicity assay with the electrodes array probe [0085] An assay for cell compatibility with the different material used in the standard PCB fabrication process was performed. The electrode array probes shown in Figure 4 were fabricated in different batches with adapted designs to exhibit only certain features and materials of the final probe. The first batch consists of the polyimide substrate only (PI). The second presents copper tracks patterned on the polyimide substrate (Pl-Cu). The third has nickel-gold deposited on the copper tracks (Pl-Cu-Au). The fourth has the solder mask as final step (PI-Cu-Au-SO). The following combination of materials were also produced: polyimide with copper tracks and solder mask but without gold deposition (PI-Cu-SO) and polyimide with the dry film resist only (PI-RI) as well as polyimide with solder mask only (PI-SO). All batches went through the complete PCB fabrication process and were consequently in contact with all the chemicals used for the complete process except the nickel-gold deposition that was performed on one panel only. PCBs of each batch were placed in one well each with 1 ml. of cell culture media (DM EM + 10% FBS) and seeded with 20Ό00 cell/mL human foreskin fibroblasts, passage 10. Every combination was triplicated. Three wells were prepared as control with fibroblasts only.

[0086] Figure 5a) shows the culture growth after 5 days of incubation at 37 °C using Alamar Blue (Invitrogen, DAL1025, diluted 1 :10 in DMEM/FBS + 33mM HEPES pH 7.4) and an automatic plate reader (Spectramax Paradigm, Molecular Devices, fluorescence with kinetics mode, reads every 30s, excitation 535nm, emission 595nm). The cell numbers were determined by comparison of the slope of the fluorescence signal from 10 to 30 minutes of incubation at 37°C to controls with known amounts of cells seeded 4h prior to the Alamar blue test. The cell growth was then defined as the number of cells observed after 5 days divided by the number of cells seeded (fold increase). The control well showed a cell growth of 2.5 fold. In the wells containing the PCB made of polyimide only (PI), the cell density increase was even larger and reached 2.8 fold. This may be related to the extra surface offered by the probe in the well but essentially shows that the cell growth rate is not affected by the polyimide substrate that has passed through the whole PCB fabrication process. Figure 5c) and d) also shows that the cell morphology of the two fibroblast culture do not significantly differ. Similarly, Figure 5a) shows that the PCB batch made of polyimide with the solder mask only (PI-SO) does not affect the cell growth with an increase of 2.7 fold. Copper is cytotoxic and could be leached from the substrate. We therefore quantified the concentration of Cu2+ ions in the cell culture media by use of a modified Bicinchonic acid assay (BCA, detection of Cu2+ via formation of a bicinchonic acid- Cu+ complex in the presence of adipic dihydrazide as a rapid reducing agent). When bare copper is in contact with cell solution (Pl-Cu), almost all of the cells died and nearly 2mM of free Cu2+ is measured (Figure 5b). Copper leaching can be substantially reduced by covering the tracks, but not the active areas, by a solder mask, and indeed, the cell growth is close to normal, with 2.2 fold increase over the 5 days of culture, which is only 20% lower than the control. Finally, adding a nickel-gold layer on top of copper (Pl-Cu-Au) avoids microscopically evident electrode corrosion, and strongly decreases concentration of copper in solution after 5 days as shown in Figure 5b). Further, the combination of nickel-gold coating with a solder mask coverage for non-active areas allows to restore cell growth to slightly below control level (1 .7 fold).

[0087] PCB-made flow cytometer and beads detection

[0088] As a proof of concept for a BioMEMS fabricated in a PCB production line, a flow cytometer fabricated and presented in Figure 6a) and b), uses a liquid electrodes configuration, which consists of metal electrodes placed in recessed cavities orthogonal to the main channel and filled with liquid. This allows to constrict the electric current along the recessed channel to the sensitive area in the main channel where it is used to detect particles. By providing a resolution that is limited to the microfluidic fabrication, this minimizes the alignment problems between the metallic tracks and the fluidic channel. The main channel, in which particles flow, has a width of 40μιτι, whereas the perpendicular side channel that defines the liquid electrode is 28μιτι in width and connects larger cavities in which the copper electrodes are provided. Despite an imprecise alignment of the copper electrodes with the side channel can be observed, this only has a minor influence on the current constriction and electric field created in the main channel.

[0089] To examine the functionality of the flow cytometer chip, 20 μιτι polystyrene beads dispersed in PBS were flown along the microfluidic main channel by applying a pressure of -2mbar at the outlet. The impedance was measured using the lock-in demodulation principle and performed with an integrated lock-in (SI-QSD, Sensima, CH). On one electrode, an excitation frequency of 49 kHz with amplitude of 0.5 Vpp is applied, while on the second electrode, the current is amplified using a transimpedance amplifier (OPA2350, Tl, USA). This amplified current is measured and the impedance is determined using the acquisition, demodulation and filtering capability of the integrated lock-in. The impedance signal is then recorded with a computer.

[0090] Figure 6c) shows the impedance magnitude traces over time obtained when 20 μιτι beads flows in the 40 μιτι microfluidic channel of the flow cytometer. Measured peaks have on average a 60 Ω magnitude and a 300 ms dwell time representing a 20 μιτι polystyrene beads passage in the sensing area.

[0091 ] This work uses a state-of-the-art PCB fabrication facility with a resolution of 45μιτι for both metallic tracks and fluidic channels made of dry film resist. Although this resolution is far from the processing capability of the CMOS industry, it is good enough for many BioMEMS applications. In these applications, features size are dictated by the object to be analyzed, which is typically a biological cell with dimensions in the range of 20 μιτι or a biological tissue that is even larger. In this context, the PCB resolution is well suited for a range of simple BioMEMS applications. Nowadays, some PCB manufacturers announce a resolution capability for metallic tracks down to 20 μιτι, two folds smaller that resolution presented in the present study.

[0092] In the implemented embodiments, a 40μιτι thick dry film resist was used to define the fluidic channel. When higher channel are required, it is possible to laminate more than one layer of dry film resist. [0093] A constraint of using the standard PCB fabrication line to produce BioMEMS is the restricted choice of materials. The polyimide substrate that is standard in PCB fabrication is a biocompatible material and already used in different implants. The present work confirmed that a polyimide substrate that went through the full PCB fabrication process and its related chemicals keeps its cell compatibility. However, the most common material for metallic trace in a standard PCB process is the copper which is toxic for the cells and can consequently by used only when the performed BioMEMS analysis is an end-point measurement. The choice of material becomes critical when BioMEMS are in contact with a living tissue. The use of a solder mask which presents a good compatibility with cells and covers copper tracks significantly reduce copper cytotoxic effects. The problems of biocompatibility of the metal tracks in PCBs can however be solved by using alternative PCB manufacturing methods that use noble metal only for metallic tracks and biocompatible material such as polyimide or liquid crystal polymer (LCP) for substrates and thus making PCB good candidates even in the case of direct implants.

[0094] For every BioMEMS application that can accommodate those constraints, advantages are numerous. First, PCBs are processed in large panels and the production is driven by the electronic market. PCB fabrication costs are typically below 1 cent per millimeter square for small batches of 100 pieces or more. This cost is certainly interesting compared with the one of MEMS foundries. Moreover, only a small number of components needs to be produced to reach this level of price compared with injection molding. The PCB industry for BioM EMS fabrication also has an interesting prototyping possibility with potential for mid-scale production as well as large scale production.

[0095] Finally another interesting advantage that BioMEMS production could inherit from PCB industry is their standardization in the design to production pipeline. Similarly, in the CMOS industry, an IC designer focuses on the design and rely on a standard and well defined process. As long as the design is CMOS process compliant and thanks to the standardization of this technology, the steps from the designing phase to the production are simplified and no process development has to be undertaken. A similar advantage appeared in the work of the inventors for the two chips produced on the PCB production line. The chip design was adapted to be compatible with the standard PCB process and consequently no effort had to be conducted on the process development. The production of the chip is then fully defined by the standard Gerber files and can be performed by different PCB manufacturers without process specific instructions except the repeat of the dry film resist deposition and structuration for the microfluidic layer, which is already an existing step in the line. This is certainly an advantage when the end goal of a research project is to reach industrial application. Along with the simplicity of production management, comes the advantage to outsource the production to partners being already certified for their production line and processes.

Claims

1 . A method for producing a microfluidic layer comprising at least a microfluidic channel on a Printed Circuit Board (PCB) panel, the method comprising the following steps:

a) a first lamination step of a dry film resist onto said PCB panel;

b) a photostructuration step of the dry film resist on the PCB panel; and c) a closure step of the photostructured dry film resist to obtain the microfluidic layer.

2. The method of claim 1 , further comprising a first step consisting in the production of a PCB panel comprising a PCB or a plurality thereof.

3. The method of claim 2, wherein the PCB or plurality thereof are produced via lamination and photostructuration of a dry film resist disposed onto a metallized substrate.

4. The method of any previous claim, characterized in that the photostructuration step of b) is determined by a Gerber file.

5. The method of any previous claim, characterized in that the closure step of c) is a lamination step.

6. The method of claim 5, characterized in that the lamination step of c) is a lamination step of a dry film resist.

7. The method of claims 5 or 6, characterized in that the lamination step of c) is performed on the PCB panel.

8. The method of any previous claim, characterized in that steps a), b) and c) are performed before the depaneling of the PCB panel.

9. The method of any previous claim, characterized in that steps a) and b) are sequentially repeated so to obtain a stack of dry film resists.

10. The method of any previous claim, characterized in that the microfluidic layer comprises an array of microfluidic channels.

1 1 . The method of any previous claim, characterized in that the microfluidic channel is an elongated channel, a reservoir, a well or combinations of the foregoing.

12. The method of any previous claim, characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some vias present in the PCB panel.

13. The method of any previous claim, characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some electric components or electrodes present in the PCB panel.

14. The method of any previous claim, characterized in that the photostructuration step is adapted to fluidically connect the microfluidic layer with at least some electric components or electrodes present in the PCB panel through at least some vias present in the PCB panel.

15. The method of any previous claim, characterized in that the dry film resist is selected from a list comprising Riston, Ordyl or Kolon.

16. The method of any previous claim, characterized in that the photostructuration step is performed by UV light insolation and development.

17. A Printed Circuit Board (PCB) panel comprising a microfluidic layer obtainable through the method of claims 1 to 16.

18. Use of the method of claims 1 to 16 for manufacturing a microfluidic sensor or a plurality thereof comprising a Printed Circuit Board (PCB) and a microfluidic layer.

19. A sensor comprising a Printed Circuit Board (PCB) and a microfluidic layer obtainable through the method of claims 1 to 16.