BRPI0703025B1 - Multilayer microdevice, Multilayer microdevice preparation process - Google Patents

Abstract

Multilayer microdevice, Multilayer microdevice preparation process. The present invention deals with a multilayer microdevice having integrated electrodes as well as their preparation process for use in chemical, clinical, biological, forensic, environmental, food, pharmacological analysis.

Description

(54) Title: MULTILAYER MICRODISPOSITIVE, MULTILAYER MICRODISPOSIT PREPARATION PROCESS (51) Int.CI .: B32B 27/36; G01N 27/447; G01N 27/453 (73) Holder (s): UNIVERSIDADE DE SÃO PAULO - USP. STATE UNIVERSITY OF CAMPINAS (72) Inventor (s): EMANUEL CARRILHO; JOSÉ ALBERTO FRACASSI DA SILVA; WENDELL KARLOS TOMAZELLI COLTRO

1/26 “MULTILAYER MICRODISPOSITIVE, MULTILAYER MICRODISPOSE PREPARATION PROCESS” [001] The present invention relates to a multilayer microdevice, as well as its preparation process, for use in chemical, clinical, biological, forensic, environmental, food analysis , pharmacological, among others.

State of the Art [002] Just as electronic microchips have revolutionized the worlds of computers and electronics, analytical microchips have revolutionized analytical chemistry in recent years [Wooley, A.T .; Lao, K .; Glazer,

A.N .; Mathies, R.A. Anal. Chem., V.70, p.684-688, 1998]. Initially, the main reason for miniaturization was to increase analytical efficiency rather than to reduce its size. However, with the change from the macro scale to the micro scale, other advantages were obtained such as reduced volume of reagents and samples (of the order of pL-nL), low manufacturing cost and analysis in a short time. This true revolution originated in the early 1990s, when Manz and colleagues proposed the introduction of the concept of microsystems for total analysis, or simply μTAS [Manz, A .; Graber, N .; Widmer, H.M. Sens. Actuators B, v.1, p.244-248, 1990].

[003] With the development of μTAS, it became possible to integrate several stages of conventional analytical processing such as, sample introduction, sample pretreatment, chemical reactions, chemical separation and detection in a single device [Wooley, A.T .; Lao, K .; Glazer, A.N .; Mathies, R.A. Anal. Chem., V.70, p.684-688, 1998; Manz, A .; Graber, N .; Widmer, H.M. Sens. Actuators B, v.1, p.244-248, 1990]. ΜTAS transform chemical information into electrical or optical signals, enabling easy automation. All of these factors, coupled with portability, have driven the explosive and massive development of micro-scale analytical systems in recent years [Reyes, D.R .; Iossifidis, D .; Auroux, P .; Manz, A. Anal. Chem., V.74, p.2623-2636, 2002]. These aspects have become relevant to the clinical and environmental field in which the term point-of-care has been used [Gulliksen, A .; Solli, L .; Karlsen, F .; Rogne, H .; Hovig, E .; Nordstrom, T. Sirevág, R. Anal. Chem, v.76, p.9-14, 2003; LAB-ON-A-CHIP: The Revolution in portable instrumentation. New York, John Wiley, 2001. p.7-135]. Due to the idea of inserting several steps normally developed in a laboratory on a chip, the

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2/26 μΤΆβ are also called lab-on-a-chip (LOC) [LAB-ON-A-CHIP: The Revolution in portable instrumentation. New York, John Wiley, 2001. p.7-135].

[004] The have been used intensively to solve a wide variety of bioanalytical problems. Microdevices for bacterial cell culture [Martin, K .; Henkel, T .; Baier, V .; Grodian, Ά .; Schon, T .; Roth, M .; Kohler, J.M .; Metze, J. Lab Chip, v.3, p.202-207, 2003; Taylor, Ά.Μ .; Rhee, S.W .; Tu, C.H .; Cribbs, D.H .; Cotman, C.W .; Jeon, N.L. Langmuir, v.19, p.15511556, 2003], clinical diagnoses [Li, P.H.; Wang, W.J .; Parameswaran, M. Analyst, v.128, p. 225-231, 2003; Christodoulides, N .; Tran, M .; Floriano, P.N .; Rodriguez, M .; Goodey, Ά .; Ali, M .; Neikirk, D .; Mcdevitt, J.T. Anal. Chem., V.74, p.3030-3036, 2002], environmental interests [Ohira, S.I .; Toda, K .; Ikebe, S.I .; Dasgupta, P.K. Anal. Chem., V.74, p. 5890-5896, 2002; Bromberg, Ά .; Mathies, R.A. Anal. Chem., V.75, p.1188-1195, 2003], immunoassays [Jiang, X.Y .; Ng, J.M.K .; Stroock, ΆΌ .; Dertinger, S.K.W .; Whitesides, G.M. J. Am. Chem. Soc., V.125, p.5294-5295, 2003; Sato, K .; Yamanaka, M .; Takahashi, H .; Tokeshi, M .; Kimura, H .; Kitamori, T. Electrophoresis, v.23, p.734-739, 2002], protein analysis [Jin, L.J .; Ferrance, J .; Sanders, J.C .; Landers, J.P. Lab Chip, v.3, p.11-18, 2003; Slentz,

B.E .; Penner, Ν.Ά .; Regnier, F.E. Anal. Chem., V.74, p.4835-4840, 2002], analysis and separation of DNΆ [Sanders, J.C .; Breadmore, M.C .; Kwok, Y.C .; Horsman, K.M .; Landers, J.P. Anal. Chem., V.75, p.986-994, 2003 .; Tian, H.J .; Landers, J.P. Anal. Biochem., V.309, p.212-223, 2002], polymerase chain reaction (PCR) [Sun, K .; Yamaguchi, Ά .; Ishida, Y .; Matsuo, S .; Misawa, H. Sens. Actuators, B, v.84, p.283-289, 2002] and also for DNΆ sequencing [Liu, S. Ά. Electrophoresis, v.24, p.3755-3761, 2003; Shi, Y.N .; ^ derson, R.C.

Electrophoresis, v.24, p.3371-3377, 2003; Blazej, R.G .; Paegel, B.M .; Mathies, R.A Genome Res., V.13, p.287-293, 2003] are some examples that demonstrate the potential of chemical microchips. Several analytical techniques can be developed on a micro-scale, especially gas chromatography (GC), high performance liquid chromatography (HPLC) and mainly capillary electrophoresis (CE) ^ uroux, P .; Iossifidis, D .; Reyes, D.R .; Manz, Ά. Anal. Chem., V.74, p.2637-2652, 2002];

[005] Ά EC is the main technique developed on a micro-scale due to the simplicity of its instrumentation and also due to the advantages regarding the transport of the fluid, which does not require additional components besides electrodes for application of electric field, thus eliminating the need to couple

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3/26 micro-pumps and micro-valves [Bilitewski, U .; Genrich, M .; Kadow, S .; Mersal, G. Anal. Bioanal. Chem., V.377, p.556-569, 2003]. Electrokinetic control can be used to transport the sample to a certain point where it can be chemically modified, diluted or mixed with another substance, detected and perhaps used to perform analyzes by coupling with other analytical techniques, such as mass spectrometry [Ermakov , SY; Jacobson, S.C .; Ramsey, J.M. Anal. Chem., V.72, p.3512-3517, 2000].

[006] μTAS, LOC's, microchips or analytical microdevices can be manufactured on a planar surface through lithographic processes such as x-ray lithography, soft lithography, electron beam lithography or UV lithography [Chen, Y .; Pépin, A. Electrophoresis, v.22, p.187-207, 2002]. These technologies were developed in the 1970s and 1980s and are used extensively in the microelectronics and semiconductor industries. Although lithographic processes are the most used methods in the preparation of μTAS, research by alternative methods, economically viable, is and will always be a permanent objective of the scientific community.

[007] One of the alternative paths is the production of polymeric devices using LIGA technology whose acronym, from German, stands for Lithography, Electroforming and Molding (Lithography, Galvo und Abformung) [Manz, A .; Graber, N .; Widmer, H.M. Sens. Actuators B, v.1, p.244-248, 1990]. The LIGA technology was originally conceived based on x-ray lithography (LIGA-RX), but recently, thanks to advances in the materials area, a technological variant based on lithography with ultra-violet radiation (LIGAUV) has become possible. This technology makes it possible to manufacture microstructures with a high aspect ratio (for example, a wall 200 μm high by only 2 μm wide) [Manz, A .; Graber, N .; Widmer, H.M. Sens. Actuators B, v.1, p.244-248, 1990].

[008] Another very attractive method for the rapid production of microdevices was recently proposed by do Lago and collaborators [do Lago,

C.L .; Silva, H.D.T .; Neves, C.A .; Brito-Neto, J.G.A .; Fracassi da Silva, J.A. Anal. Chem., V.75, p. 3853-3858, 2003]. In this process, the image patterns are printed on a polyester film using a laser printer to selectively deposit a toner layer, which defines the dimensions of the microfluidic channels. After printing, these patterns are laminated against a sheet of

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4/26 transparency or against a specular image of the pattern, forming channels with single or double layer of toner, respectively.

[009] Although the miniaturization of separation devices has been developing rapidly in recent years, the development of effective detection systems is at a late stage [Wang, J. Talanta, v.56, p.223-231, 2002 ; Schwarz, M.A .; Hauser, P.C. Lab Chip, v.1, p.1-6, 2001]. Since the μTAS concept was proposed in the early 1990s, the most widely used detection method, especially in the first decade, was laser-induced fluorescence (LIF) [Seiler, K .; Harrison, D.J .; Manz, A. Anal. Chem., V.65, p.1481-1488, 1993]. This detection mode is perfectly suited to be used in bioanalytical applications. Another detection method that has recently received considerable attention in connection (primarily) with proteomic analysis is mass spectrometry (MS) [Ramsey, R.S .; Ramsey, J.M. Anal. Chem., V.69, p.1174-1178, 1997; Bings, N.H .; Wang, Skinner, C.D; Colyer, C.L .; Thibault, P .; Harrison, D.J. Anal. Chem., V.71, p.3292-3296, 1999]. Although they offer high sensitivity and a high degree of information, LIF and MS require complete control of off-chip instrumentation and have a high instrumental cost, aspects that compromise the benefits of miniaturization and portability.

[010] If LIF and MS present complex and costly instrumentation, electrochemical techniques have attracted, in recent years, considerable interest directed to microsystems [Lacher, N.A .; Garrison, K.E .; Martin, R.S .; Lunte, S.M. Electrophoresis, v.22, p. 2526-2536, 2001; Vandaveer IV, W.R .; Pasas, S.A .; Martin, R.S .; Lunte, S.M. Electrophoresis, v.23, p.3667-3677, 2002]. Electrochemistry offers great promise for microsystems, with aspects that include remarkable sensitivity, the very miniaturization of the detector and instrumentation control, regardless of the optical path length or sample turbidity, low cost, simplicity and high compatibility with microfabrication techniques [Wang, J. Talanta, v.56, p.223-231, 2002]. Since the metallic deposition processes are already well established, the implementation of detection methods using electrodes integrated on the same platform has become quite attractive. The integration of electrodes for amperometric detection [Scott Martin, R .; Gawron, A.J., Lunte, S.M .; Henry, C.S. Anal. Chem., V.72, p.3196-3201, 2000; Nacher, N.A .; Lunte, S.M .; Scott Martin, R. Anal. Chem., V.76, p.2482-2886, 2004] and conductometric [Laugere, F .; Guijt, R.M .; Bastemeijer, J .; Steen, G.V.D .; Berthold, A .; Baltussen, E .; Sarro, P .; van Dedem, G.W.K .; Vellekoop, M. Anal.

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Chem., V.75, p.306-310, 2003; Guijt, R.M .; Baltussen, E .; van der Steen, G .; Frank, H .; Billiet, H .; Schalkhammer, T .; Laugere, F .; Vellekoop, M .; Berthold, A .; Sarro, L .; van Dedem, G.W.K. Electrophoresis, v.22, p.2537-2541, 2001] in electrophoresis microdevices have recently been proposed. Among the electrochemical detection modes, amperometric detection provides the best detectability. However, due to its robustness, simplicity and low cost, the use of conductometric detection in microsystems has increased a lot in recent years [Vandaveer IV, W.R .; Pasas, S.A .; Martin, R.S .; Lunte, S.M. Electrophoresis, v.23, p.3667-3677, 2002].

[011] Conductometric detection is, in principle, an electrochemical method of universal detection. The conductance of a solution (L) is dependent on the electrode area (A), its distance (/), the concentrations (c) of all charged species and their molar conductivity (λ), according to equation 1:

L = A Σλ, (Eq. 1) [012] Conductometric detection can be conducted in two different modes: (i) contact mode and; (ii) non-contact mode. In the conventional mode (with contact), the electrodes are placed directly in contact with the solution, a fact that can generate some problems in the presence of a high electric field, as is the case with capillary electrophoresis. Among the possible problems, there is the formation of bubbles caused by electrolysis and also the degradation of the electrode. In addition, the presence of an electric field requires adequate protection of the detector's electronic circuit to eliminate possible electrical interference. In non-contact mode, the electrodes are positioned on the outside of the microchannel (without contact with the solution), preventing problems related to contact mode.

[013] The electrodes essentially form capacitors with the electrolyte, as long as the channel wall is a dielectric. These capacitors are characterized by the impedance, X _c , also known as capacitive reactance, which is inversely dependent on the capacitance, C, which is calculated by equation 2:

x _c = G (Eq. 2) ω (.

where C represents capacitance, and ω is given by (2π χ frequency).

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6/26 [014] When applying a sinusoidal signal to one of the electrodes, an alternating current can be capacitively coupled to the working electrolyte and captured at the second measurement electrode. According to equation 2, the higher the frequency, the lower the reactance or impedance that a capacitor offers to the charge flow, allowing the conductivity variations of the solution inside the microchannel to be observed even with the electrodes positioned on the outside of the microchannel.

[015] To use this detection mode in miniaturized systems, it is essential that the electrodes are positioned as close as possible to the microfluidic channel, in order to guarantee a good capacitive coupling. As shown in equation 3, the capacitance, C, is dependent on the distance, d, between the two conductive plates (electrodes) of the parallel capacitor, where d is equivalent to the thickness of the insulating layer (dielectric).

C = yJ-A (Eq. 3) d

s _o = vacuum permittivity (8.854 χ 10 ¹² F m ^-1 ) s _r = medium dielectric constant A = electrode surface area

Replacing equation 3 in equation 2:

Xc = -? - (Eq. 4) ωε ₀ ε _Γ Ά [016] Equation 4 clearly shows that X _c is directly dependent on the thickness of the insulating layer, and that higher frequency signals should be used for more insulating layers thin (or less thick).

[017] Conductively coupled non-contact conductometric detection (C ⁴ D) was applied to electromigration techniques in the early 1980s for the isotachophoretic determination (ITP) of organic and inorganic anions [Gas, B .; Demjanenko, M .; Vacík, JJ Chromatogr., V. 192, p. 253-258, 1980; Vacík, J .; Zuska, J .; Muselasová, IJ Chromatogr., V. 320, p.233-239, 1985]. In 1998, Zemann and colleagues [Zemann, AJ; Schnell, E .; Volgger, D .; Bonn, GK Anal.Chem., 1998, v.70, p.563-567] and Fracassi da Silva and do Lago [Fracassi da Silva, JA; do Lago, CL Anal.Chem., v.70, p. 4339-4343, 1998] independently proposed the use of C ⁴ D as a detection method for capillary electrophoresis (CE). As of this moment, the number of publications

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7/26 related to instrumentation and different applications with CE-C ⁴ D has increased considerably. Further details can be found in recently published review articles [Kubán, P .; Hauser, PC; Electroanalysis, v.16, p.20092021, 2004; Kubán, P .; Hauser, PC; Electrophoresis, v.25, p. 3387-3397, 2004; Kubán, P .; Hauser, PC; Electrophoresis, v.25, p. 3398-3405, 2004; Brito-Neto, JG; Fracassi da Silva, JA; Blanes, L .; do Lago, CL Electroanalysis, v. 17, p.1198-1206, 2005; Brito-Neto, JG; Fracassi da Silva, JA; Blanes, L .; do Lago, CL Electroanalysis, v.17, p.1207-1214, 2005].

[018] The use of C ⁴ D in electrophoresis microdevices was first proposed by Guijt and colleagues in 2001 [Guijt, RM; Baltussen, E .; van der Steen, G .; Frank, H .; Billiet, H .; Schalkhammer, T .; Laugere, F .; Vellekoop, M .; Berthold, A .; Sarro, L .; van Dedem, GWK Electrophoresis, v.22, p.2537-2541, 2001]. In this first work, aluminum electrodes were incorporated directly into the separation channel. To avoid contact with the solution, the electrodes were coated with a thin layer of SiC. Lichtenberg and collaborators [Lichtenberg, J .; Rooiji, NF; Verpoorte, E. Electrophoresis, v.23, p.3769-3776, 2002] proposed a similar prototype, designing the platinum electrodes close to the separation microchannel, however, isolated with a thin glass wall (10-15 μm). The microsystems proposed by Guijt and Lichtenberg, although manufactured by highly sophisticated technologies, did not show good analytical results when compared with similar systems. In addition, the steps of the processes involved are quite expensive and also complex. A very simplified system was proposed by Pumera and collaborators [Pumera, M .; Wang, J .; Opekar, F .; Jelínek, I .; Feldman, J .; Lowe, H .; Hardt, S. Anal. Chem., V.74, p.1968-1971, 2002]. In this system, two electrodes were prepared from aluminum foils and fixed on top of the PMMA microchips. The distance between the electrodes and the microchannel was defined by the thickness of the PMMA plate (cover) (—175 μm). Wang et al. [Wang, J .; Chen, G .; Muck Jr, A. Anal. Chem., V.75, p.4475-4480, 2003; Wang, J .; Chen, G .; Muck Jr, A .; Collins, GE Electrophoresis, v.24, p.3728-3732, 2003] used a similar process to manufacture a mobile C ⁴ D system, allowing a quick positioning of the electrodes in different positions on the separation channel. The construction of electrodes for C ⁴ D using Cu tapes was also used by different groups [do Lago, CL; Silva, HDT; Neves, CA; Brito-Neto, JGA; Fracassi da Silva, JA Anal. Chem., V.75, p. 3853-3858, 2003; Tanyanyiwa, J .;

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Hauser, P.C. Anal. Chem., V.74, p.6378-6382, 2002; Tanyanyiwa, J .; Abad-Villar, E.M .; Fernández-Abedul, T.F .; Costa-Garcia, A .; Hoffmann, W .; Guber, A.E .; Gerlach, A .; Hauser, P.C. Analyst, v.128, p.1019-1024, 2003]. Although it is a practical method, the repeatability of the electrode configuration is highly dependent on the user's technical skill. Therefore, the preparation of electrodes with the same dimensions (length and width), their fixation always in the same position on the separation channel, evenly spaced by the same distance, is completely compromised. This has consequences for reliability, repeatability, reproducibility and consequently for serial manufacturing.

[019] Tanyanyiwa and collaborators [Tanyanyiwa, J .; Hauser, PC Anal. Chem., V.74, p.6378-6382, 2002; Tanyanyiwa, J .; Abad-Villar, EM; FernándezAbedul, TF; Costa-Garcia, A .; Hoffmann, W .; Guber, AE; Gerlach, A .; Hauser, PC Analyst, v.128, p.1019-1024, 2003] demonstrated that the sensitivity of C ⁴ D can be improved with the use of a high amplitude excitation signal (HV, 100-500 V). US 2005109621 deals with the use of C ⁴ D with HV in electrophoresis. This document refers to the development of C ⁴ D analytical instrumentation with HV for conventional electrophoresis and also for the micro-scale system. For the micro-scale electrophoresis system, the electrodes were prepared using Cu adhesive tapes. It should also be noted that the microchips (glass and PMMA) used in the work of the group by Hauser et al. were obtained commercially.

[020] Kubán & Hauser showed the effects of the geometry of the detection cell and the operational parameters on the use of C ⁴ D in miniaturized systems were presented [Kubán, P .; Hauser, PC Lab Chip, v.5, p.407415, 2005].

Goals

The objectives of the present invention were:

- simplicity and speed in preparing the devices;

- possibility of repeatability in the preparation of the electrodes, which does not occur in the electrodes prepared manually, enabling their mass production or on an industrial scale;

- low cost, enabling the use of these integrated microchips as disposable analysis systems;

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- low consumption of reagents and samples and, consequently, low production of environmental waste.

Short Description [021] The present invention deals with a multilayer microdevice comprising a surface (i) containing microchannels, a surface (ii) with integrated detection electrodes and reservoirs. The invention also contemplates the process of preparing a multilayer microdevice.

Detailed Description [022] The present invention deals with a multilayer microdevice comprising a surface (i) containing microchannels, a surface (ii) with integrated detection electrodes and reservoirs.

[023] According to the invention, the material that constitutes the microchannels produced by the direct printing method can be chosen from toner, toner in different colors, toner with magnetized nanoparticulate materials, thermoplastic nanoparticulate materials, polymeric and ferromagnetic composites; preferably, toner is used, which can be from printers or photocopiers. Microchannels can be arranged to create a three-dimensional matrix of channels, which can allow a wide variety of analytical operations such as dilutions, reactions, concentration, among others.

[024] The material that constitutes the surfaces (i) and (ii) can be chosen from glass, polyester, PMMA, PC, quartz, silicon, preferably, polyester is used. The microchannels obtained have a height ranging from 5 to 15 μm; preferably, 6 and 12 μιη are produced by a process containing a single layer of toner (STL) or a double layer of toner (DTL).

[025] As for the integrated electrodes, these can be presented in different geometries and positions, and can be used for detection with or without contact. According to the invention, each microdevice may have two or more electrodes.

[026] The electrodes are made of conductive material and, optionally, material that allows better adhesion of the layer of conductive material on the substrate. The conductive material can be chosen from aluminum, gold, platinum, palladium, carbon, copper, titanium, chrome, nickel and silver. Aluminum is preferably used because of its low cost. However, the materials that allow better adhesion of the conductive material on the substrate are titanium and chromium; preferably, titanium is used.

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10/26 [027] According to the invention, the reservoirs present in the microdevices are positioned on the surface (ii) containing the electrodes, and the material used in the reservoirs can be chosen from PVC, polyethylene, or any other ductile, malleable material and that is inert. The basic device requires a minimum quantity of two reservoirs for its operation, preferably four being used for demonstration of injection and separation on the same platform. The microdispotivo can accommodate a varied number of reservoirs, which, in addition to the electrolytes for analysis, can contain specific reagents for separation or detection.

[028] The invention also contemplates the process of integrating electrodes into the multilayer microdevice, which comprises the following steps:

(a) surface preparation (i) containing microchannels;

(b) surface preparation (ii) containing electrodes;

(c) integration of surfaces (i) and (ii);

(d) insertion of reservoirs in the microdevice obtained in steps (a) (c).

[029] The microchannels prepared in step (a) can be obtained by corrosion, molding, pressing, printing or lithography or processes derived from them. The material used in the formation of microchannels can be chosen toner, toner in different colors, toner with magnetized nanoparticulate materials, thermoplastic nanoparticulate materials, polymeric and ferromagnetic composites, preferably, toner. Microchannels can be arranged to create a three-dimensional matrix of channels, which can allow a wide variety of analytical operations such as dilutions, reactions, concentration, among others. The material that constitutes the surfaces (i) and (ii) can be chosen from glass, polyester, PMMA, PC, quartz, silicon; preferably, polyester.

[030] Electrodes can be obtained by lithography, printing, corrosion, electroforming, chemical evaporation or thermal deposition. The electrodes can also be manufactured using toner masks. The lithography process comprises the following steps:

i. covering the substrate with photosensitive material;

ii. photogravure of the desired image;

iii. revelation;

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11/26 iv. deposition of a layer containing conductive material to form the electrodes;

v. removal of photosensitive material.

[031] According to the invention, the lithography process optionally comprises a stage prior to stage (i) of cleaning the substrate; besides presenting, optionally, intermediate steps to reduce the thermal stress of the photosensitive material.

[032] The integrated electrodes can have different geometries and consist of conductive material and, optionally, material that allows better adhesion of the layer of conductive material on the substrate. The conductive material can be chosen from aluminum, gold, platinum, palladium, carbon, copper, silver, chrome, nickel or titanium; preferably, aluminum is used. The material that allows better adhesion of the conductive material on the substrate can be chosen between chromium and titanium, preferably titanium is used.

[033] Step (c) of the process of integrating electrodes into the multilayer microdevice can occur by thermal lamination process, using a document laminator or thermal press, and the surface containing the electrodes can be positioned according to the type of desired detection, with or without contact with the microchannels.

[034] The invention also contemplates multilayer microdevice obtained by the process previously described.

[035] The following are examples to better elucidate the invention, however, they should not, however, be taken for limiting purposes of the invention.

Example 1: Manufacture of Microchannels [036] The fluid microchannels were manufactured according to the direct printing process, according to PI 0105260-8. In addition to the advantages related to cost and time of manufacture, the potential of this process for the manufacture of microdevices for electrophoretic separations with amperometric detection [Coltro, W.K.T., Fracassi da Silva, J.A .; Silva, H.D.T .; Richter, E.M .; Furlan, R .; Angnes, L .; do Lago, C.L .; Mazo, L.H .; Carrilho, E. Electrophoresis, v.25, p.3832-3839, 2004; He, F.Y .; Liu, A.L .; Yuan, J.H .; Coltro, W.K.T .; Carrilho, E .; Xia, X.H .; Anal. Bioanal. Chem., V.382, p.192-197, 2005; Liu, A.L .; He, F.Y .; Hu, Y.L .; Xia, X.H. Talanta, v.68, p.1303-1308, 2006] or microdevices for mixing solutions [Liu, A.L .; He, F.Y .; Wang, K .; Zhou, T .; Lu, Y .; Xia, X.H. Lab Chip, v.5, p.974-978, 2005] was recently addressed in the literature. The process outline

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12/26 direct printing is shown in Figure 1, which shows the preparation of the structures with a simple layer of toner (SCT), however, it is also possible to design structures with a double layer of toner (DCT). The steps of the microfabrication process for structures with SCT based on direct printing are: (I) polyester film; (II) polyester film with a toner layer (a) deposited;

(III) alignment between the device scheme printed on the polyester film with polyester film without perforated toner (for access to the reservoirs); (IV) lamination stage and; (V) microdevice finished with glued reservoirs (b) (PVC tubes or the base of 100 μL pipettor tips).

[037] The first stage of the manufacturing process, not shown in the Figure, is related to the preparation of the device design. The device configuration was designed, on a 1: 1 scale. The pattern was printed on a polyester film (I) (model CG 3300, 3M, Sumaré-SP) using a Laser printer (model LaserJet 2200d, Hewlett Packard). It is important to note, at this point, that the use of polyester films properly manufactured for use in laser printers is extremely important for the success in the manufacture and consequently instrumental operation of this microdevice.

[038] Printing was performed in the optimized mode, with a resolution of 600 dpi operating in vector mode. During printing, a toner layer (II) (model Q4096A, Hewlett Packard) was deposited by the printer in order to form the microchannels. The thickness of the toner layer (~ 6 μm), represented by the black color, defines the height of the microchannels obtained and the white regions represent the reservoirs and microchannels formed.

[039] The step of sealing the microchannels manufactured by the direct printing process will be presented below, as further details, in which the step of integration with the microfabricated electrodes will be addressed. Additionally, for illustrative purposes and for a better understanding of the user, this step is also inserted (generically) in Figure 1. As the objective is the construction of structures with an SCT, the sealing was performed against a standard polyester film (clean or without toner). However, to access the microchannels, holes were made directly on the polyester film (without toner) with the aid of an adapted paper punch (III). The use of guides (or marks) facilitates the step of drilling the appropriate places for access to the microchannels. The printed image pattern and the perforated polyester film were aligned (III) and laminated (IV) together.

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13/26 [040] After lamination, PVC tubes 5 mm long and 7 mm outside diameter were bonded (V) to the microdevices, with epoxy resin, in order to form reservoirs for the solutions, thus ending the process of microfabrication. In addition to PVC tubes, polyethylene tubes or the base of 100 μL pipettor tips can be used for the same purpose. The step of bonding the reservoirs must be done with care in order to avoid clogging the end of the microchannels with the resin used.

[041] Figure 2 shows two images of the projected microdevice. Figure 2a shows the scheme of the injection and separation microchannels. The separation microchannel is delimited by points 1 and 4. In turn, the injection microchannel is delimited by points 2 and 3. The geometry of the injection microchannel is extremely important for any quantitative analysis, as will be discussed below. The geometry used for this (detection) system consisted of a double-T, as highlighted in Figure 2a. Figure 2b shows the microchannel scheme with the positioning of the electrodes (e1 and e2 - in detail) on the separation microchannel (isolated from contact with the microchannel through the thickness of the polyester film).

Example 2: Manufacture of Electrodes by lithography [042] The microelectrodes were manufactured combining a photolithographic step and a metallic deposition step, using polyester films as substrates.

[043] The microfabrication process of the electrodes is shown in Figure 3, where: the blank bar represents the polyester film; in gray, the photoresist layer; and in black, the metallic material deposited.

[044] The polyester film, due to its flexibility, was fixed on a glass plate to give greater rigidity facilitating the preparation of the electrodes. The photolithographic process is limited to the stages of choosing and cleaning the substrate, covering with a photosensitive material (photoresist), photogravure of the desired image through the incidence of UV radiation, revelation of the photogravure pattern and corrosion of the material. As this process was used only for the manufacture of planar microelectrodes, the corrosion step was not necessary in this step.

[045] Initially, the substrate (polyester film) was subjected to a cleaning step (I). The substrate was then coated (II) with a layer of

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14/26 positive photoresist (Shipley S1811 - Shipley, Marlborough, MA, USA). The configuration of the electrodes, contained in a mask, was transferred with exposure to UV radiation to the positive photoresist (III) and, after the development stage (IV), a metallic layer (Ti / Al) was deposited via sputtering (V). The sputtering technique can optionally be replaced by chemical vapor deposition. The titanium layer (20 nm) was deposited in order to guarantee a better adhesion of the aluminum layer (150 nm) with the substrate. To avoid problems with metal adherence, before metallization the substrates were subjected to an oxygen plasma for 4 minutes in order to eliminate any organic material. When removing the photoresist with acetone, the electrodes were easily obtained (VI), as long as they have adhered perfectly to the substrate surface. The removal of the photoresist layer and consequent obtaining of the electrodes is called lift-off. Aluminum (material used in the construction of the electrodes) was chosen due to its low cost compared to other materials. However, materials such as gold (Au), platinum (Pt), palladium (Pd), carbon (C), copper (Cu), silver (Ag), nickel (Ni) or even chromium (Cr) can be deposited through the same procedure. The manufacture of Au, Pt, C, Ni, Ag and Cr electrodes on polyester films is attractive not only for applications in separation microchips, but also as disposable biosensors. This technology could significantly reduce the cost of the prototypes used to control blood glucose levels and other clinical diagnoses, for example,

Cleaning the substrates

The substrate cleaning step initially consisted of organic degreasing (acetone, methanol and 2-propanol) followed by washing with a piranha H ₂ SO ₄ / H ₂ O _{2 solution} (2: 1) for 30 minutes. This step was carried out in order to remove organic residues and promote a better adhesion of the photoresist to the substrate. After each step using an acid solution, the samples were washed, in abundance, with deionized water and dried with ultra-pure nitrogen.

Spinning

The substrates were coated with photosensitive material using a spinner. The spinner is a mechanical device used to spread viscous solutions in a uniform manner resulting in an ultraPetition film 870170085429, from 11/06/2017, p. 24/47

15/26 thin. This device operates at a high speed, is coupled to a vacuum system and the thickness of the films is determined according to a material calibration curve. The spinning time depends on the characteristic of the radiation sensitive material and also on the desired thickness. In the experiments performed, the S1811 positive photoresist was used, which has a low optical transparency that makes it ideally suited for thin films. The spinning step was performed at 4000 rpm for 30 s, obtaining a thickness of approximately 1 μm.

Photogravure

After the scattering of the photoresist, the pre-bake stage was performed before the photogravure stage. The sample was placed on a hot plate at 65 ° C for 5 min and then on another hot plate at 95 ° C for 15 min. The sample was placed back on the hot plate at 65 ° C for another 5 min. This procedure helps to reduce thermal stress. The pre-bake time is directly related to the elimination of the solvent contained in the photoresist. Once removed from the heating plate, the samples were left to rest for 10 min before being exposed to UV radiation. The exposure time is directly related to the thickness and the pre-bake time, to which the photoresist was exposed. In the experiments carried out, the Karl Suss MJB 3 UV300 Aligner was used. For a power of 10 mW / cm ^2, an exposure time of 9 s was used.

Revelation

As S1811 is a thermo-activated resin, after the exposure process, the sample was subjected to a heat treatment on a heating plate at 95 ° C, for 15 min. The samples were left to rest for 10 min before starting the development step. The development step is necessary to remove the photoresist that received radiation, as being a positive photoresist, the regions irradiated with UV radiation become soluble in organic solvent and can be removed easily. The non-irradiated areas remain intact (polymerized).

[046] The AZ 351 developer was used, which was placed in a beaker under magnetic stirring at room temperature. The sample was fixed on a Petition holder 870170085429, of 11/06/2017, p. 25/47

16/26 rotating sample to improve process uniformity. The development time used was 20 s. After development, the substrate containing the electrode configuration was then washed with deionized water and dried with N ₂ .

[047] In order to reduce the intrinsic stress of the S1811 at the end of the photogravure process, another post-bake step was performed (95 ° C for 15 min). After this stage, the photoengraving process of the electrode configuration was completed.

[048] The use of this process for the construction of planar electrodes makes it possible to project and evaluate different geometries, or even to project an electrode array. These factors represent additional advantages over the use of electrodes prepared with Cu adhesive tapes. Figure 4 shows examples of different geometries.

Example 3: Manufacture of the Electrodes by direct printing [049] The lithographic process for the manufacture of the electrodes can be optionally substituted by the direct printing process, similarly to the manufacturing process of the microchannels. In this case, the printed diagram defines the area of the electrodes that are formed with deposition via sputtering. After metal deposition, the toner is easily removed using acetone or acetonitrile. This alternative allows the manufacture of masks for the manufacture of electrodes in a matter of minutes without the need for the entire infrastructure for the photolithographic steps. In comparative terms, the production of the masks through the conventional process (photolithography) requires between 2 and 3 hours, while the direct printing process requires only 5-10 minutes.

Example 4: Integration of electrodes / microchannels [050] As already mentioned, the sealing of microchannels made of polyester-toner was done through a thermal lamination step, using a document laminator from Gazela model AC91 230. In general, the Sealing processes of glass microchips, for example, involve high temperatures (500-700 ^o C), are slow, expensive and require a completely clean environment depending on the type of material that is used. Additionally, the use of

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17/26 high temperatures can damage the conductive material during its integration, rendering the device unusable.

[051] When sealing via thermal lamination, the substrate containing the printed microchannels and the substrate containing the microfabricated electrodes were aligned and laminated together at 120 ° C and 40 cm min ^-1 , as shown in Figure 1. At this temperature, the melting occurs between the toner layer and the polyester film, promoting an effective sealing of the microchannels, without damaging the electrodes.

[052] During the lamination step, the face of the polyester film containing the electrodes was carefully placed so as to be out of contact with the microchannel, that is, the electrodes were isolated from the microchannel by the very thickness of the polyester film (~ 100 pm). The lamination does not cause any damage to the adhered electrodes. One of the great advantages of using thermal lamination to integrate microelectrodes with microchannels made of polyester-toner is that there is the possibility of using these electrodes for other electrochemical detection modes. Electrodes for amperometric detection and their derivations, conductometric with contact, voltammetric or potentiometric can be perfectly manufactured on the polyester film and integrated with the microchannels. As in this case, the aforementioned detection modes require contact with the solution, just position the electrode face in contact with the microchannel, before lamination.

[053] The idealization of these microchips with integrated electrodes makes it possible to mass produce these prototypes, without losing precision in the positioning of the electrodes. In addition, the projection of different geometries as well as the manufacture of electrode array in series enriches, as shown in Figure 4, the consolidation of this process. The cost of these integrated microchips was calculated based on the market values of polyester films, toner cartridges and aluminum targets. It is estimated that each prototype will cost a maximum of R $ 0.20 (twenty cents of reais). For comparative purposes, integrated microchips, made of glass, cost around € 500 (five hundred euros) each

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18/26 unit (according to a quotation made recently with Micronit Microcfluidics).

[054] In addition to the simplicity, speed and low cost of the manufactured prototypes, another great advantage is that many devices can be manufactured in parallel at all stages involved. In the projection of microchannels, as long as a letter or A4 size sheet is used, dozens of microstructures can be printed on a single sheet. Similarly, dozens of substrates can be submitted in parallel for metallic deposition (via sputtering), depending on the size of the deposition chamber. In the lamination stage, the last stage involved in the manufacture of these prototypes, depending on the size of the laminator, several microchips can also be laminated in parallel.

[055] These advantages stimulate mass production, reduce unit cost and also reduce manufacturing time. In addition, the invention becomes industrializable and its construction is not dependent on the user's technical ability to position the electrodes manually, or handcrafted.

[056] Figure 5 represents a simplified scheme of the microfabrication process used in the present invention, where (A) microchannel fabrication through the direct printing process; (B) preparation of the electrodes on the polyester film (upper side); (C) alignment of the perforated polyester film (containing the electrodes) and the polyester film with the printed microchannels before the lamination step; 1: polyester films; 2: microchannels printed on the polyester film; 3: microfabricated electrodes on the polyester film; and (a) polyester film; (b) printed microchannels; (c) polyester film; (d) microfabricated electrodes; (e) perforated polyester film; (f) printed microchannels.

[057] Figures 6A and 6B show a schematic of the microdevice made of polyester-toner, where: I, microchannel for introducing the sample; II, analytical separation microchannel. 1, 2, 3 and 4 represent the sample, buffer, sample disposal and buffer reservoirs, respectively. The electrodes for connection to the

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19/26 signal generator and the detector circuit are represented by 5 and 6, respectively.

Example 5: Conductometric Detection without Contact [058] The scheme [Fracassi da Silva, J.A .; Guzman, N .; do Lago, C.L. J. Chromatogr. A, v.942, p.249-255, 2002] of operation of the non-contact conductometric detector is illustrated in the block diagram of Figure 7. An alternating high frequency signal is applied to one of the planar microelectrodes using a signal generator. When applying this signal, a current is generated, which is transmitted by the current / voltage converter (i / e) reflecting all possible paths taken by the signal. Once the geometric parameters of the detection cell are kept constant, the current variation obtained directly reflects variations in the composition of the solution inside the microchannel. Thus, the lower the resistance of the solution, that is, the greater the conductance, the greater the current recorded.

[059] After the current signal is transformed into voltage, using the i / e converter, the signal is rectified in half-wave. The resulting signal is filtered through an RC (low-pass) filter and finally goes through a baseline amplification and compensation step (Vos). The resulting DC signal (Vs) is applied directly to the serial interface input, used for data acquisition in the operating system, preferably Linux (Slacker distribution).

[060] The current measured by the electronic circuit reflects the total impedance of the system. The impedance, in turn, is the combination of the capacitive reactances of the capillary (X _w ), inter-electrodes (X _o ) and the resistance of the solution (R _s ), and is best expressed in terms of the inverse of the complex impedance: a complex admittance (Y):

Y = G + jB =

R_, Λ ² + Xw (Xo + Xw) + J

R _s + Xw

Xo (Rs ² + Xw ² ) (Eq. 5) [061] The real part of the admittance is called conductance (G), and should not be confused with the conductance of the solution inside the channel. The imaginary part is called susceptibility (B). The admittance module, which is proportional to the measured current, is given by:

\ R ² X _o ² + R ² + Xw (Xo + Xw) f

Xo (Rs ² + Xw ² ) (Eq. 6)

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20/26 [062] When working with fixed values of operating frequency, maintaining the geometry of the oscillometric cell and ignoring the capacitance variations (or more correctly of the dielectric constant) influenced by the temperature, the variations in the Y admittance will be due to the variations in the conductivity in the solution. Signal generator [063] The excitation signal for the C ⁴ D circuit was generated from an MFG 4202 function generator (Minipa, São Paulo, SP). The digital generator is capable of generating sine, square and triangular waveforms in the frequency range of 0.2 Hz to 2 MHz, with amplitude adjustment (maximum voltage of 20 V _pp ) and wave symmetry, digital displays with 5 digits for frequency and 3 digits for amplitude and 50 Ω output impedance.

Electronic circuit [064] The detector circuit, shown in Figures 8 and 9, was assembled in two modules, as shown in Figure 7. In the first module (Figure 8), the detection cell, the current / voltage conversion stage ( i / e), the rectifier and the RC filter were placed on a single plate using surface mounted devices (SMD). A shield (aluminum box and PDMS) was placed around it to minimize the capture of noise. In the second module (Figure 9), the final stages of amplification and compensation of the baseline were placed, as well as the power supply of the circuit.

Example 6: Data acquisition system [065] The data acquisition system was developed using an RS232 serial port as an interface. All development was done on the Linux operating system and the software was programmed through Borland Kylix 3 using C and C ++ languages. The software developed was named MICSENS. The entire system is controlled using a Pentium 233 MHz microcomputer, 32 Mb of RAM and 4.0 Gb HD. The system developed for data acquisition of the C ⁴ D was also programmed, with the aid of a microcontroller, to control the electrical potentials applied by the high voltage source. Thus, the potentials for introducing and separating the sample into the microchannels were applied in an automated manner.

Example 7: Applicability [066] The applicability of these microdevices with integrated electrodes, and completely disposable, was evaluated with electrophoretic separations from a mixture of inorganic cations (K +, Na + and Li +). Although different geometries

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21/26 have been projected, only the results of one of them will be presented. The microsystem used consisted of two electrodes (750 μm wide) for detection, configured in the anti-parallel direction, spaced by 750 μm and positioned 5 mm from the buffer disposal reservoir.

[067] Initially, the channels were filled with solution (MES / His 20 mmol L ' ¹ , pH 6.0) through the capillarity phenomenon. After filling, a 1.0 kV potential was applied momentarily to the separation microchannel until the baseline stabilized. Then, the detection conditions were optimized (output signal depending on the frequency and amplitude of the applied sine wave) before starting the electrophoretic experiments. Figure 10 presents some dc signal curves as a function of the frequency and amplitude of the sine wave, parameters that can be optimized in each system.

[068] The absolute value of the baseline (V _dc ) was monitored, for approximately 60 s, with the channel filled with the running electrolyte. The experiments were carried out in a frequency range of 100 to 900 kHz and with an amplitude range of 1-10 V _pp . Figure 10a shows the baseline signal as a function of the operating frequency for different amplitudes. The baseline signal as a function of the amplitude of the sine wave, for different frequencies, is shown in Figure 10b.

[069] As can be seen in Figure 10a, for each amplitude value there is a maximum response indicating its best operating frequency. This point is shifted to lower frequencies when the amplitude of the sine wave is increased. When the response signal is observed as a function of amplitude (Figure 10b), a linear dependence for low amplitudes is observed. For the curve constructed at 400 kHz, linearity is observed up to approximately 8 Vpp. This linear dependency is reduced for higher frequencies. In summary, both figures show that for a wave of amplitude equal to 10 Vpp, the frequency of 400 kHz is the best choice. Therefore, for the other experiments, these conditions were chosen as standard conditions. In these conditions, electrophoretic experiments began.

[070] For electrophoretic experiments, there is a need for electrokinetic control in microchannels. Figure 11 shows an outline of the operating system. Figure 11A shows the configuration of the microdevices. The configuration consists, basically, of two microchannels for sample introduction (I) and analytical separation (II). In the injection microchannel,

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22/26 configured as a double-T, points 1 and 3 indicate the sample and sample disposal reservoirs, respectively. On the other hand, points 2 and 4, in the separation channel indicate the reservoirs for the running buffer and the disposal buffer, respectively. Point (III), highlighted, represents the region where the detection electrodes are positioned.

[071] Electrophoretic separations were performed after stabilization of the baseline. The sample, added to reservoir 1 (see Figure 11B), was introduced electrokinetically under the application of a potential difference of 1.0 kV / 10 s between reservoirs 1 and 3. After this time (10 s) the entire microchannel of injection is filled with sample (see Figure 11C). The sample plug, defined by the double-T geometry, was moved in the direction of the detector when applying the potential difference between reservoirs 2 and 4 (see Figure 11D). In this way, the analytes are separated according to their electrophoretic mobility and detected with C ⁴ D. The volume corresponding to the injection plug, defined geometrically by the double-T, is equal to 270 pL in this geometry). In Figure 11D, the points indicated by e1 and e2 indicate the electrodes that are connected to the signal generator and the electronic circuit, respectively.

[072] For a better visualization of the electrokinetic control in the microchannels, Figure 12 shows a sequence of images captured, in real time, in order to present the electrokinetic phenomenon in the steps of filling the injection channel and introducing the sample plug in the microchannel. separation. These images were captured with the aid of an optical microscope equipped with a CCD (charge coupled device) camera. For these experiments, a 20 mmol L ^-1 solution of potassium phosphate, pH 7.0, was used as an electrolyte solution. To view the images, 20 mg of methylene blue dye were dissolved in 1 mL of the electrolyte solution. Images A, B and C were captured after 1, 5 and 10 s, respectively, under the application of an electrical potential in the injection channel. Images D, E and F were captured after 1, 3 and 5 s, respectively, by applying electrical potential in the separation channel. Electrokinetic control was performed as shown in Figure 11. The applied potential, in both stages, was equal to 1.0 kV.

[073] With these pre-determined parameters, the electrophoretic separation of the mixture of inorganic cations (K ⁺ , Na ⁺ and Li ⁺ ) was performed as electrolyte, under the application of an electric field equal to 250 V cm ^-1 , using the MES / His buffer 20 mmol L ^-1 , pH 6.0, 1.0 kV / 10 s injection, 250 V cm ^-1 electric field,

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23/26 operating frequency of 400 kHz, 10 V _pp , and one of the resulting electropherograms is shown in Figure 13. Despite presenting a low production cost per unit, more than 50 electropherograms were registered using a single prototype. In the electropherogram shown, the equimolar mixture of cations was prepared in a concentration equal to 25 μmol L ^-1 . It can be seen that the three analytes were efficiently separated in approximately 75 s. For the electropherogram shown, the resolution between the K + and Na + peaks was 1.7 and between the Na + and Li + peaks it was 1.4.

[074] Although in conventional electrophoresis the introduction of the sample is performed by electrokinetic and / or hydrodynamic actions, in miniaturized systems this step is done mainly by electrokinetic processes [Chen, G .; Wang, J. Analyst, v. 129, p.507-511, 2004; Fang, Q. Anal. Bioanal. Chem., V.378, p.49-51, 2004], although there are already some works with hydrodynamic injection [Backofen, U .; Matysik, F-M .; Lunte, C.E. Anal. Chem., V.74, p.40544059, 2002; Solignac, D .; Gijs, M.A.M. Anal. Chem., V.75, p.1652-1657, 2003]. The sample introduction system is one of the most important steps in the analytical processing developed in microfluidic devices. Such importance is due to the fact that the characteristics of the sample plug, formed by the intersection of the channels, will determine the quality of the analytical separation. In addition, for a quantitative study there is a need for good repeatability of injections. This is a critical point and problems related to the repeatability of electrokinetic injection have been observed [de Jesus, D.P .; BritoNeto, J.G., do Lago, C.L .; In: 13th Annual Meeting of Analytical Chemistry. Book of Abstracts. Niterói, 2005. ref.H-057].

[075] Different alternatives were studied in order to assess what was the best methodology for performing the electrokinetic injection stage in microchips. The alternative that provided satisfactory results consisted of a cleaning step (clean-up) with the running buffer itself (MES / His 20 mmol L ' ¹ , pH 6.0) between the subsequent injections. In other words, after each electrophoretic separation performed, the injection channel was cleaned with a buffer solution. The cleaning step was monitored with the C ⁴ D signal and also by the electrophoretic current in the microchannels. With this procedure, a series of 10 injections was performed with the cleaning step and another series of 10 injections was performed without the cleaning step to compare the results. The analyte used in both series was K + at a concentration of 75 μmol L ^-1 , which has high mobility

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24/26 electrophoretic and is easily detected with C ⁴ D. The injection plug, defined by the geometry of the microchannels, was only 270 pL (V = 150 χ 300 χ 6 μm ³ ).

[076] The results of both series are shown in Figure 14. As can be seen in Figure 14A, the series of injections without the cleaning step shows a decrease in the peak height for the first injections. The relative standard deviation (RSD) for the presented sequence was approximately 30%. This decrease is related to the formation of a sample stacking in the injection channel resulting in regions with different concentrations of the analyte, and as long as the plug is not completely mobilized by the channel, the signal decreases to its basal level.

[077] From the data related to the simulations, it was clear that if the injections were performed under the same conditions, the results would be different. With the addition of the cleaning step, between the subsequent injections, the same decrease in peak height was not observed (Figure 14B). The clean-up does not eliminate the presence of the phenomenon, it only allows the sample plug to be inserted into the microchannels always under the same conditions. The RSD for this new sequence was less than 5%.

[078] The success obtained in the repeatability of the electrokinetic injection in the microchannels made it possible to carry out a first quantitative test in the completely disposable homemade microchips. Figure 13 shows a typical electropherogram for a mixture of K +, Na + and Li + at a concentration of 25 μmol L ^-1 each and Figure 15 shows a sequence of electropherograms obtained in different concentrations (Fig. 15A, where: (a) 25, (b) 50, (c) 75 and (d) 100 μmol L ^-1 ) and also the analytical curves for the analyzed cations (Fig. 15B). The correlation coefficients for K +, Na + and Li + were 0.9985, 0.9976 and 0.9926, respectively. The sensitivity obtained was 9.55 (K +), 8.51 (Na +) and (Li +) mV / pmol L ^-1 . The detection limit (LD) estimate, considering the signal / noise ratio equal to 3, was equal to 3, 4 and 7 μmol L ^-1 for K +, Na + and Li +, respectively.

[079] The LD's obtained for the three cations in our system are higher than those obtained in the C ⁴ D system with HV [Tanyanyiwa, J .; Hauser, PC Anal.Chem., V.74, p.6378-6382, 2002; Tanyanyiwa, J .; Abad-Villar, EM; Fernández-Abedul, TF; Costa-Garcia, A .; Hoffmann, W .; Guber, AE; Gerlach, A .; Hauser, PC Analyst, v.128, p.1019-1024, 2003], and are also greater than those presented by Pumera and collaborators [Pumera, M .; Wang, J .; Opekar, F .; Jelínek, I .; Feldman, J .; Lowe, H .; Hardt, S. Anal. Chem., V.74, p.1968-1971,

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2002]. However, the LD's of our system are comparable to the LD's found for a system with optimized electrode geometry [Kubán, P .; Hauser, P.C. Lab Chip, v.5, p.407-415, 2005] and even smaller than those found with microfabricated electrodes [Laugere, F .; Guijt, R.M .; Bastemeijer, J .; Steen, G.V.D .; Berthold, A .; Baltussen, E .; Sarro, P .; van Dedem, G.W.K .; Vellekoop, M. Anal. Chem., V.75, p.306-310, 2003; Guijt, R.M .; Baltussen, E .; van der Steen, G .; Frank, H .; Billiet, H .; Schalkhammer, T .; Laugere, F .; Vellekoop, M .; Berthold, A .; Sarro, L .; van Dedem, G.W.K. Electrophoresis, v.22, p.2537-2541, 200; Lichtenberg, J .; Rooiji, N.F .; Verpoorte, E. Electrophoresis, v.23, p.37693776, 2002]. This analytical study is extremely important in order to establish a reliable method to carry out validated and certified analyzes.

[080] For a total of 10 injections performed sequentially, the mean migration time for K +, Na + and Li + cations was 37 ± 1, 56 ± 1 and 72 ± 2 s, respectively. The efficiency of the obtained separation was calculated in number of plates. The values obtained for Li +, Na + and K + were approximately 18800, 7800 and 11250 dishes / m, respectively. These values were calculated according to equation 7, considering t _m , the migration time from the analyte to the detector ew, the width of the peak base:

N = 1óf 3 ^} (Eq. 7) [081] The theoretical value for efficiency, or the expected efficiency for separation, is equal to 2.30 * 10 ⁵ plates / m. The theoretical value was calculated according to equation 8, with L _ef and L _pUg representing, respectively, the effective length of the separation channel (3.4 cm) and the length of the sample plug defined in the double-T geometry (300 μm):

N _T = 16 and f < ^L plug J (Eq. 8) [082] Dividing the values referring to the experimental efficiency obtained and the theoretical value, the values found were 8.2, 3.4 and 4.9%. These values are much higher than the values obtained for the amperometric separation in the polyester-toner microdevices [Coltro, WKT; Fracassi da Silva, JA; Silva, HDT; Richter, EM; Furlan, R .; Angnes, L .; do Lago, CL; Mazo, LH, Carrilho, E. Electrophoresis, v.25, p.3832-3838, 2004]. The difference (greater than 10%) is due to the fact that the oscillometric detection is performed in the separation channel, that is,

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26/26 about the channel (on channel). Especially in the case of amperometric detection, the working electrode was positioned at the end of the separation channel (end-channel). In the on-channel detection mode, there is no bandwidth expansion referring to the channel outlet, as observed in the DA.

[083] A direct comparison between the results obtained with our microchips and those published by the Wang and Hauser groups is difficult due to a number of factors. Both groups use commercial microchips made of glass or PMMA, which have excellent definition and resolution of the channels. On the other hand, our microchips are manufactured without a “clean environment” and the definition of the channels is completely dependent on the resolution of the laser printer used. For this reason, the width and height of the microchannels represent the limitations of this process. The width considered “limit” is equal to 150 μm. Regarding the height, it is possible to build microchannels with 6 or 12 μm. In the process presented in this patent application, the manufacture of 6 μm high microchannels was adopted, in which a simple layer of toner was deposited on the polyester film. The manufacture of 12 μm high microchannels can be manufactured using a double layer of toner. In addition to the disadvantages related to the definition and resolution of microchannels, we believe that microchips made of polyester-toner have a high potential as portable and disposable analysis microchips for monitoring the environment, clinical diagnostics, chemical, forensic, defense analyzes national, food, fuel, among others.

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Claims (25)

1. Multilayer microdevice comprising a surface containing toner microchannels, electrodes for detection, characterized by the fact that it presents microchannels with preferential height between 6 and 12 μm arranged on the surface (i) in order to create a three-dimensional matrix of channels and allow a variety analytical operations, at least two electrodes manufactured by lithography or toner mask and integrated into the surface (ii) and at least 2 detection tanks with different geometries and positions, which are made of conductive material, preferably aluminum, in order to enable its adhesion on the substrate, chosen from titanium or chromium, preferably titanium. 2. Multilayer microdevice according to claim 1, characterized in that the material that constitutes the microchannels can be chosen from toner, toner in different colors, toner with magnetized nanoparticulate materials, thermoplastic nanoparticulate materials, polymeric and ferromagnetic composites. 3. Multilayer microdevice according to claim 2, characterized in that the toner can be from printers or photocopiers. 4. Multilayer microdevice according to claim 1, characterized in that the material that constitutes the surfaces (i) and (ii) can be chosen from glass, polyester, PMMA, PC, quartz, silicon. 5. Multilayer microdevice according to claim 4, characterized in that the material that constitutes the surfaces (i) and (ii) is polyester. 6. Multilayer microdevice according to claim 1, characterized in that the preferred conductive material is chosen from aluminum, gold, platinum, palladium, carbon copper, titanium, chromium, nickel or silver. 7. Multilayer microdevice according to claim 1 characterized by the fact that the integrated electrodes can be used for detection with or without contact with the microchannels. 8. Multilayer microdevice according to claim 1, characterized by the fact that they preferably have four reservoirs. 9. Multilayer microdevice according to claim 1, characterized in that the material that constitutes the reservoirs can be chosen from PVC, polyethylene or any other ductile, malleable and inert material. 10. Process for preparing a multilayer microdevice, as defined in claims 1 to 9, characterized by comprising the following steps: Petition 870180034489, of 04/27/2018, p. 20/22 2/3 (a) surface preparation (i) containing microchannels, preferably by direct printing; (b) surface preparation (ii) containing integrated electrodes, manufactured by lithography or toner mask; (c) integration of surfaces (i) and (ii); (d) insertion of reservoirs in the microdevice obtained in steps (a) - (c). 11. Process for preparing a multilayer microdevice according to claim 10, characterized in that the microchannels prepared in step (a) can be obtained by means of corrosion, molding, pressing, printing or lithography or processes derived therefrom. 12. Multilayer microdevice preparation process according to claim 10 characterized by the fact that the material used in the formation of microchannels can be chosen from toner, toner in different colors, toner with magnetized nanoparticulate materials, thermoplastic nanoparticulate materials, polymeric and ferromagnetic composites preferably toner. 13. Process for preparing a multilayer microdevice according to claim 10, characterized by the fact that the microchannels can be arranged to create a three-dimensional matrix of channels, which can allow a wide variety of analytical operations such as dilutions, reactions, concentration, among others. Process for preparing a multilayer microdevice according to claim 10, characterized in that the material that constitutes the surfaces (i) and (ii) can be chosen from glass, polyester, PMMA, PC, quartz, silicon; preferably, polyester. 15. Multilayer microdevice preparation process according to claim 10, characterized in that the electrodes can be manufactured using toner masks. 16. Process for preparing a multilayer microdevice according to claim 10, characterized in that the lithography process comprises the following steps: i. covering the substrate with photosensitive material; ii. photogravure of the desired image; iii. revelation; iv. deposition of a layer containing conductive material to form the electrodes; v. removal of photosensitive material. Petition 870180034489, of 04/27/2018, p. 21/22 3/3 17. Process for preparing a multilayer microdevice according to claim 16, characterized in that the lithography process comprises, optionally, a step prior to step (i) of cleaning the substrate. 18. Multilayer microdevice preparation process according to claim 16, characterized by the fact that the lithography process optionally comprises intermediate steps to reduce the thermal stress of the photosensitive material. 19. Process for preparing a multilayer microdevice according to claim 10, characterized by the fact that the electrodes can have different geometries and positions. 20. Process for preparing a multilayer microdevice according to claim 10, characterized in that the electrodes consist of conductive material chosen from aluminum, gold, platinum, palladium, carbon, copper, titanium, silver, nickel or chromium. 21. Process for preparing a multilayer microdevice according to claim 20, characterized in that the conductive material is aluminum. 22. Process for preparing a multilayer microdevice according to claim 10, characterized by the fact that the electrodes are optionally constituted of material that allows better adhesion of the layer of conductive material on the substrate. 23. Process for preparing a multilayer microdevice according to claim 22 characterized by the fact that the material that allows better adhesion of the layer of conductive material on the substrate can be chosen from titanium or chrome; preferably, titanium is used. 24. Process for preparing a multilayer microdevice according to claim 10, characterized in that step (c) can take place by thermal lamination process. 25. Process for preparing a multilayer microdevice according to claim 10, characterized in that the surface (ii) can be positioned with or without contact with the microchannels. Petition 870180034489, of 04/27/2018, p. 22/22 1/13 Channels , X X * FIGURE 2 Petition 870180034489, of 04/27/2018, p. 7/22 2/13 ΙΗ ι Η ι ΙΗ FIGURE 3 Petition 870180034489, of 04/27/2018, p. 8/22 3/13 Petition 870180034489, of 04/27/2018, p. 9/22 4/13 Petition 870180034489, of 04/27/2018, p. 10/22 5/13 FIGURE 6 Petition 870180034489, of 04/27/2018, p. 11/22 6/13 Petition 870180034489, of 04/27/2018, p. 12/22 7/13 FIGURE 8 Petition 870180034489, of 04/27/2018, p. 13/22 100 nF PT1 Y ♦ —ο Λ D / A ‘/ 2 OPA2604AP 300 kO 10 kO PTAO ΐ 100 nF 10 kO l · 10 kn Serial interface ‘/ 2 OPA2604AP 28 kO +12 V YOU PI 1 kíl BZX79C4V7 8/13 220 kil 10 kil FIGURE 9 Petition 870180034489, of 04/27/2018, p. 14/22 9/13 Frequency (kHz) 2.0 V ▲ 4.0 V • 10 V V (V) pp '' FIGURE 10 Petition 870180034489, of 04/27/2018, p. 15/22 10/13 Petition 870180034489, of 04/27/2018, p. 16/22 11/13 FIGURE 12 Petition 870180034489, of 04/27/2018, p. 17/22 12/13 Peak Height (V) Sequential Injections Sequential Injections FIGURE 14 Petition 870180034489, of 04/27/2018, p. 18/22 13/13 FIGURE 15 Petition 870180034489, of 04/27/2018, p. 19/22