WO2016049720A1

WO2016049720A1 - Microfluidic devices and use thereof

Info

Publication number: WO2016049720A1
Application number: PCT/BR2015/000102
Authority: WO
Inventors: Lucimara Gaziola DE LA TORRE; Aline Furtado OLIVEIRA; Reinaldo Gaspar BASTOS
Original assignee: Universidade Estadual De Campinas- Unicamp; Fundação Universidade Federal De São Carlos - Ufscar
Priority date: 2014-09-30
Filing date: 2015-07-02
Publication date: 2016-04-07
Also published as: BR102014024587A2

Abstract

The invention relates to structured microfluidic devices for generating a diffusive concentration gradient in biocompatible materials, which are easy to assemble and reusable. The invention describes the use of the device in any process requiring the ideal conditions of the substrate to be known, or the ideal conditions for cell performance, estimation of kinetic parameters and chemical response evaluation, such as chemotaxis.

Description

MICROFLUIDIC DEVICES AND THEIR USE

Field of the invention:

[1] The present invention fits into the field of microbiology and microfluidics to optimize the steps for selecting optimal conditions for microbial cell growth and thus improving the performance of the early stages in bioprocesses. More specifically, the invention relates to structured microfluidic devices for generating a diffusive concentration gradient in easily assembled and reusable biocompatible materials for general use in microbiology to assess cellular behavior.

Background of the invention:

[2] Currently, the sciences are looking for innovative technologies, allied to high productivity, lower costs and uptime.

[3] Within this context, microfluidics is a science that applies these principles and expands study alternatives in various areas of knowledge.

[4] In biotechnology, for example, microfluidics are involved in the development of systems capable of determining the optimal reaction conditions and assisting in the optimization of bioprocesses.

[5] This science operates on microfluoric devices with micrometer scale dimensions, requiring a small volume (10 ^{-6 '} to 10 ^{"" 1S} L) of samples and reagents.

[6] Thus, processes are fast in terms of mass and heat transfer and reactions take place under laminar flow regime, mimicking the icro-environment. in space precisely, enabling cell evaluations in real situations.

[7] Microfluidics developed from other sciences they sought. new techniques with fast and reliable results. The analytical was the most influential area, when the microfluidic potential was used in order to optimize the analytical methods. Microelectronics, in turn, collaborated in the development of the microfluidic platform, applying the photolithography technique in the construction of microfluidic devices.

[8] Subsequently, other supports and construction methods were: tested and microfabrication techniques boosted the production of microfluidic devices in various geometries and material types, configured according to the purpose of each research.

[9] Regarding geometry, microfluidic devices allow separation and manipulation of samples, generating mixing systems and concentration gradients.

[10] A. Gradient formation allows the observation of cellular behavior against different concentrations of a given substance, an important feature to understand cellular behavior and to determine parameters involved in bioprocesses.

[11] Among the many applications of microfluidics, animal cell studies stand out, where it is possible to monitor cell development in optimal situations, sequence DNA in microchip electrophoresis, and analyze gene expression with greater performance than techniques. animal cells.

[12] In bioprocesses, microfluidics are used to determine kinetic parameters in order to increase process productivity. In these studies, microfluidic devices are used to provide a promising environment for efficient cell growth.

[13] Microfluidic devices have the advantage of evaluating the biological behavior and kinetics of real-time reactions and simulating macroscale operations, which enables process optimization with greater efficiency in vitro than traditional techniques.

[14] Thus, microfluidics is an important tool for various biotechnology research, with emphasis on bioprocesses and the development of new technologies.

[15] The devices currently used in microfluidics have high versatility and concentration gradient formation capabilities that can contribute to studies in industrial microbiology, allowing the use of microfluidics to perform tests and analysis of early stages in bioprocesses, generating faster results. than current techniques. However, these devices have some critical points / technical problems / limitations, among which are: they are not fully reusable, they are not versatile regarding the use of aerobic or anaerobic system, greater difficulty in mounting the device, use of materials that may affect the reaction kinetics of cells and not so much consist of systems that [16] Some of the problems / limitations / critical points mentioned above are detailed in the documents described below.

[17] US 8,216,526 B2 relates to a microfluidic device capable of generating multiple spatial chemical gradients simultaneously within a circular microfluidic chamber containing three inputs through which solutions of different concentrations are inserted. In this chamber a diffusive, non-convective concentration gradient is generated and applied to evaluate the bacterial chemotaxis process. Unlike the US patent document, the present invention describes a reusable device which has a diffusive concentration gradient in a two-level system. and using easily assembled materials that do not affect cellular performance.

[18] US 2004211054 describes microfluidic systems molded from polymeric material. The device presented in this document is composed of porous PDMS and a membrane with electrode assembly and is intended to generate electrical energy from fuel cells. In this case, it is an application of microfluidics in the electroanalytical area. Unlike the US document, the present invention consists of a diffusive gradient generation system for evaluating cellular behavior. The proposed devices are easy to assemble and have reversible sealing. Due to the properties of the materials used (hydrophilic and hydrophobic), they can be used to evaluate the behavior of different materials. Canadian patent document CA2830533, on the other hand, refers to a microfluidic device for conducting biological assays, which features irreversible sealing of PDMS in glass base, thus preventing complete sanitation of the system, containing micro tubes and pneumatic chamber in the microchannels. On the other hand, the proposed invention differs from the Canadian document mainly in that the sealing is reversible, which allows the reuse of said device. Additionally, the geometry of the proposed device allows, in addition to cell cultivation, to evaluate cell behavior against different concentrations of a given substrate, determine kinetic parameters and evaluate cell performance under the imposed conditions.

[19] In the paper "A robust diffusion-based gradient generator for dynamic cell assays" by Atencia et al., The authors describe a device for studying biological processes involving static or dynamic chemical gradients, such as chemotaxis. The device described in this article uses magnets for sealing, which can affect microbial cell performance. In contrast, the microfluidic devices provided by the invention are sealed under metal clamp pressure and have inlets made in the glass itself so as not to affect cell kinetics.

[20] In article ^no A simple and cost-effective method for fabricating integrated electronic-microfluidic devices PDMS layer laser-patterned "the feasibility of using PDMS glass-formed microfluidic systems as a reversible system was presented. microfluidics for the generation of concentration gradient and only analyzes the pressure supported by the system. The methodology used to construct the microchannels shows a structure made of glass-PDMS-glass, and the microchannels are built in PDMS (by photolithography).

[21] In the proposed invention, the constructive arrangement of the device comprises two levels in order to generate diffusive concentration gradient, which allows to evaluate free cells within the microchannel. In addition, the construction methodology differs in the materials and structures used.

[22] In view of the above, no prior art document containing microfluidic devices capable of generating diffusive gradient has been found, having biocompatible materials that do not affect the reaction kinetics of the cells, with ease in the assembly and cleaning process. a fully reusable device that is versatile in its use and yet allows for parallel testing.

[23] In view of this, the present invention provides three microfluidic devices which have been structured to generate a diffusive concentration gradient in biocompatible materials.

[24] Further, the invention describes the use of microfluidic devices to optimize the steps for selecting optimal conditions for microbial cell growth and thereby improving bioprocess production performance.

Brief Description of the Invention:

[25] The invention describes microfluidic devices. diffusive in biocompatible materials.

[26] More specifically, the invention refers to two devices which differ in the composition of materials, the three of which have a linear profile in the formation of diffusive concentration gradient.

[27] The term "chamber" as used herein indicates where cells are placed for diffuse concentration gradient analysis and is at the lower level of the devices. The term "microcamera" refers to chamber divisions (ranges) made virtually to perform cell counting. The term "channel" refers to the place from which solutions of different concentrations flow and has the shape of "y". The term "reversible sealing" refers to the fact that the device may be closed by any non-permanent means of attachment.

[28] The devices differ in the amount of layers at both the lower and upper levels and in the treatment of unlaminated PDMS to make it less hydrophobic.

[29] In a first aspect, the invention relates to a microfluidic device (Figure 1A), called a glass-to-glass microfluidic device, comprising:

- a lower level (i) containing the layers: smooth glass slide (l); Rolled, cut and cast PDMS containing at least one chamber (2), but preferably three chambers in which each chamber is a test, thus having a triplicate in a single system; and cover slip with holes (3);

- a higher level (ii) containing the layers: PDMS glass with holes (5).

[30] Where:

- the cutout of the laminated PDMS (2) surrounds the walls of the microchannels and the smooth glass blade (1) defines the bottom of the microchannels (chambers);

the holes in the laminate (3) are preferably drilled by chemical corrosion and fit into the ends of the chambers;

- Laminated channel PDMS (4) allows the flow of solutions of different concentrations;

- sealing is performed by adhering the laminated PDMS to the glass;

- the holes in the glass slide (5) may optionally contain fittings for solution inlet hoses (9);

The materials have transparency, which allows to view the biological material present in the chambers through an inverted microscope, preferably glass and laminated PDMS;

- The device is reusable.

[31] In a second aspect, the microfluidic device (FIGS. 1B and 1C), referred to as the glass-PMDS microfluidic device (or Glass ^ -mPMDS when the chemically modified PDMS) comprises:

- a lower level (i) containing the layers: microchannel glass slide (chambers) (6); and PDMS with Y-shaped channel and hollow holes (7);

- An upper level (ii) containing the glass sheet layer with holes (5) and may comprise fittings for solution inlet hoses (9). - the chambers are present in the low relief glass sheet (6) and were preferably made by chemical corrosion;

- the channel is in low relief, in Y shape, on the upper surface of the PDMS (7) and allows the flow of solutions of different concentrations;

the holes in the PDMS (7) are within the channel region and fit the ends of the microchannels (chambers) of the glass slide (6);

Optionally PDMS (2) may be chemically modified, for example with polyethylene glycol divinyl ether (DEV-PEG), to make it less hydrophobic, and is referred to as mPDMS (8) after modification;

sealing is by any movable fastening means, preferably by pressing metal clips;

The materials have transparency, which allows to view the biological material present in the chambers through an inverted microscope, preferably glass and the PDMS-the device is reusable.

[33] The invention describes the use of the device for any process that needs to know optimal substrate conditions or ideal conditions for cellular performance, estimation of kinetic parameters and evaluating chemical responses such as chemotaxis.

[34] Still more specifically, the invention describes the use of microfluidic devices to optimize the steps for selecting optimal conditions for microbial cell growth and thereby improving bioprocess production performance. Microbial cells can be selected. or aerobic and cells that do or do not undergo chemotaxis.

[35] In the glass-glass device model (Figure IA) a laminated PDMS was used to form the microchannels (upper and lower level) and further promote adhesion between the glass bases. In the glass-PDMS (Figure 1B) and glass-mPDMS (Figure 1C) models (mPDMS is chemically modified PDMS to make the surface hydrophilic or less hydrophobic), the lower microchannels (where there is diffusive gradient and cell presence) were constructed. by wet corrosion on the glass (by the conventional method) and the upper microchannels (where the solutions of different concentrations flow) were made by the PDMS that forms the channel.

[36] To seal the glass-PDMS and glass-mPDMS models, a metal clip was used on both sides to avoid internal pressure difference in the device. And since the microchannel where the gradient is generated is built into the glass, this ensures that there is no deformation of it. Furthermore, the proposed invention optionally features fixed connectors for inlet and outlet streams on the upper blade.

Brief description of the figures:

[37] For a complete and complete view of the object of this invention, reference figures are given as follows.

[38] Figures 1A-C schematically represent the proposed microfluidic devices, wherein (a) is the glass-glass device, (b) is the glass-PDMS device and (c) is the glass-mPDMS device. of the laminated PDMS glass-to-glass microfluidic adhesion sealing device, wherein (a) is the laminated PDMS smooth glass laminate and the apertured glass laminate, (b) is the inoculation of the cell suspension within the cultivation, (c) is the adhesion of the Y-channel laminated PDMS on the upper blade, (d) is the upper and lower adhesion of the device and (e) is the assembled glass-glass microfluidic device.

[40] Figures 3A-C show the linear gradient profile along the glass-to-glass microfluidic microchannel (chamber), where (a) is the linear gradient formation with rhodamine B and mosaic image captured after 30 min with magnification. 20x, (b) is the linear profile by fluorescence intensity and (c) is the nearly linear gradient profile by color intensity in the glass-glass device.

[41] Figures 4A-C show the linear profile of the gradient along the PDMS microfluidic microchannel, where (a) is the formation of the linear dye gradient as a function of contact with the upper level currents, (b) is the color intensity gradient profile along the microchannel and (c) is the nearly linear gradient profile in the lower microchannels of the glass-PMDS device.

[42] Figure 5 schematically depicts the chamber in which the diffusive concentration gradient is generated in microfluidic device.

[43] Figures 6A and 6B correspond to the image of growth of Saccharomyces cerevisiae ATCC 7754 along the microchannel (chamber) and time, at times 0 and 1 hour, with initial concentration of 1.02 x 10 ³ cel / pL. microfluidic root canal, where (a) are the growth profiles of the Saccharomyces cerevisiae ATCC 7754 yeast along the chambers (position) and time and (b) is the schematic diagram of the chamber and the cell count divisions, called the microchamber. .

[45] Figures 8A and 8B correspond to the growth curves of S. cerevisiae ATCC 7754 in (a) microfluidic device and (b) conventional batch cultivation.

[46] Figure 9 graphs the estimated kinetic parameters for cell growth kinetics for S. cerevíseae described by Monod.

Detailed Description of the Invention:

[47] The invention describes structured microfluidic devices for generating a diffusive concentration gradient in easy-to-assemble, reusable, biocompatible materials for cellular behavior assessment.

[48] The proposed microfluidic devices are configured at two levels (lower and upper) and their geometries were based on the study by Atenda et al., 2012 already available in the state of the art.

[49] However, the new microfluidic devices described differ in the composition of materials used, are reusable and easy to assemble, use effective and simple sealing methods and connections and do not affect microbial cells by magnetism.

[50] The microfluidic devices of the present invention are:

(i) glass-glass (Figure IA),

⁽ ii ⁾ glass-PMDS (polydimethylsiloxane) (Figure 1B), and (Figure 1C), wherein mPDMS corresponds to a chemically modified PDMS to prevent cell adhesion by the intrinsic characteristic of cellular hydrophobicity to the also hydrophobic polymer.

[51] The materials used, glass and PDMS, are both reusable without loss in system configuration and effectiveness, provided no organic solvent that can deform the PDMS is employed.

[52] Table 1 briefly shows the materials that make up each of the devices of this invention. Their structure is shown in Figures 1A-C.

Table 1: Summary of the Three Device Models

proposed microfluidics.

Proposed microfluidic device materials

Glass-glass Glass-PMDS Glass-mPMDS

Blade of

Flat Glass Blade (1) Glass with Glass with

cameras (6) cameras (6) i

mPDMS with

PDMS with

holes and

Laminated PDMS (2) Holes and Channel

Y-channel in Y (7)

(8)

Blade of

Glass coverslip with glass with

holes (3) inputs and inputs and outputs (5) outputs (5) ii

Laminated format PDMS

Y-channel ()

Glass slide with [53] In the proposed microfluidic devices, the lower level has microchannels suitable for the cultivation of microbial cells. Such microchannels are called chambers and in them the formation of the diffusive concentration gradient occurs.

(54] At the upper level there are two solutions of different concentrations, in laminar regime, which access the lower level through holes located at the ends of the chambers, thus generating the diffusive gradient at the lower level.

[55] The microfluidic devices proposed here are best described below.

Glass-glazing device (Figure IA):

[56] The glass-to-glass device is a fully transparent hydrophilic system that allows the evaluation and visualization of free cell behavior under an optical microscope.

[57] In addition, glass is a tough material and does not allow gas to pass through, making studies with anaerobic or facultative cells possible.

[58] In the glass-glass device, the lower level (i) is formed of plain glass slide (or coverslip) (l). Above this slide, laminated PDMS (2) is placed, which bypasses the walls of the microchannels and promotes adhesion between the glass base and the upper layer. The geometries made in the laminated PDMS were created by ablation in a CO ₂ laser machine of the glass-glass device and made by glass coverslip (3) with 0.5 mm holes, which perfectly fit the lower microchannels (chambers) and that give [59] The upper level is formed by laminated PDMS in Y-channel geometry (4), through which solutions flow. The holes are made by wet corrosion process and the PDMS by soft lithography. And to close the device, a glass slide (5) containing two inlets and one outlet is used, further comprising the fitting of the hoses (9) through which the syringe pump solutions will be pumped.

[60] The sealing of the glass-glass device is performed by adhering the laminated PDMS, which gives good adhesion and no leakage throughout the test time even at higher flow rates. The device is reversible sealing and step mounted which are shown in Figure 2A-E.

Glass-PDMS (Figure 1B) and Glass-mPDMS (Figure 1)

1C):

[61] The reusable glass-PDMS and glass-rnPDMS devices are more easily constructed and offer many possibilities for applications with free cells, adherent cells, chemotaxis and non-chemotaxis cells, as well as anaerobic or aerobic systems.

[62] Both models are configured in the same way and also feature geometry based on the work of Atencia et al., 2012.

[63] The lower level (i) of these devices is formed by glass slide containing microchannels (6) for cell culture (chambers), which were made by wet corrosion, which gives durability, strength and larger hydrophilic area characteristics. ; and a PDMS part configuring the Y-shaped channel and containing 0.5 mm holes (7 ⁾ which as input to the lower level. The PDMS part was made by soft lithography.

[64] In the glass-PDMS device, native PDMS was used, ie without chemical modification. Due to the hydrophobicidity of PDMS, cells, also hydrophobic, may adhere to the polymer, and as an alternative to this behavior, the use of a cell suspension surfactant may be used to prevent adhesion.

[65] PDMS can also be chemically modified (mPDMS) with polyethylene glycol divinyl ether (DEV-PEG) and other methods available in the scientific literature to provide a less hydrophobic surface, hereinafter modified PMDS ( 8).

[66] In order to compose the upper level (ii) and close both devices, a glass slide (5) containing two inlets and one outlet is provided, including the fitting of the hoses (9) through which the pumping will be pumped. solutions by syringe pump.

[67] The glass-PDMS or mPDMS device was sealed with metal clamp pressure, which provided efficient sealing, allowing stable flow of upper level currents and lower level gradient generation capability.

[68] Therefore, the device was able to form a linear and purely diffusive concentration gradient in the lower chambers, proving to be applicable for cell studies.

[69] Therefore, the present invention further provides the use of microfluidic devices to optimize the steps of microbial and thus improve production performance in bioprocesses.

Operational Characterizations of Devices:

- Flow limits for constant flow generation:

[70] After assembly of the devices, flow determination was defined as a function of constant flow generation at the upper level and thus forming the diffusive concentration gradient at the lower level.

[71] Thus, flow limits were determined by observing the formation of flow oscillations at the upper microchannel interface, through which currents of different concentrations flow.

- Formation of diffusive concentration gradient:

[12] In order to evaluate the generation capacity of the diffusive concentration gradient of molecules for the glass-glass device, rhodamine B solution was used in one stream and distilled water in the other. The cell culture chamber was filled with distilled water and the linear gradient generation capacity and time were evaluated.

[73] In the Glass-PDMS and Glass-mPDMS devices, liquid and hydrophilic food coloring was used in the lower chambers of each device and, in the inlet streams, 40 and 0 g / L glucose solution, with water as solvent.

[74] The diffusion concentration gradient profile was assessed for color intensity within the lower microchannel (cell culture chamber).

- Results obtained: [75] The flow rate is constant at the upper level, but the concentrations of the two input streams are distinct. This procedure is imposed at the upper level to ensure the formation of a diffusive concentration gradient in the lower chambers.

[76] Thus, the flow limits that generate a stable laminar flow flow were experimentally determined because the oscillation of currents (in the upper channel) interferes with the formation of linear concentration gradient (generated in the lower chamber).

[77] Thus, flow behavior as a function of solution flow rates was analyzed, ranging from 5 to 60 pL / min, observing whether the current profile was oscillatory or stable.

[78] Flow remained macroscopically stable (without oscillations) at a flow rate of 15 to 25 pL / min.

- Formation of diffusive concentration gradient:

[79] From the determinations of the operating parameters, the diffusive concentration gradient formation capacity of the three proposed microfluidic devices was analyzed.

[80] In order to evaluate the diffusive concentration gradient generation capacity in the glass-glass device, when measuring the rhodamine fluorescence intensity (Figure 3A), stable and linear gradient formation was observed after approximately 30 minutes ( see Figure 3B).

[81] Also, gradient formation was also observed using lower microchannel dye and imposing different glucose concentrations in the currents (0 and 40 The microchannel was linear for both fluorescence intensity assessment and dye use (Figure 3C).

[82] The linear gradient profile indicated the absence of convection within the microchannels, confirming the diffusive concentration gradient. Moreover, the concentration gradient, indicated by fluorescence intensity, remained stable after the generation of the linear gradient.

[83] For glass-PDMS device models. and glass-mPDMS, dye concentration gradient formation was observed after a time as a function of color variation in the lower chamber (see Figure 4A). This color gradient is a result of the flow of glucose matter and dye in the lower chamber, since there was a glucose mass flow from the highest concentration to the lowest concentration region.

[84] The region with the lowest dye color is the region where the glucose concentration flows at 40 g / L. In this case, a qualitative analysis indicates that the dye diffusivity in this region will be lower, as greater resistance to mass transfer is imposed by the presence of glucose.

[85] At the other end of the chamber, where coloration is most intense, indicating higher dye concentration, is the region where there is no glucose. In this case, the dye diffusivity in the medium will be higher, creating a liquid dye flow throughout the chamber.

[86] Thus, the resulting ^' total dye flow (NA) occurs from highest to lowest concentration of that the transport resistance of matter is lower at the lowest concentration imposed (Figure 4B). The color gradient began to form after 5 min, becoming stable and linear at approximately 35 min (while dye is present) (Figure 4C).

Application of the glass-glass device to evaluate microbial cellular behavior:

[87] Saccharomyces cerevisiae ATCC 7754 yeast cell tests to analyze microbial cellular behavior. In view of the micrometer scale of the microfluidic system, the concentration of S. cerevisiae used was 0.5x10 ² cel / pL.

[88] The microfluidic system was mounted in laminar flow and the cell suspension was placed in the culture chambers. To impose the difference in glucose concentration, two degassed solutions of YPD medium were used, varying only the glucose concentration, being one medium absent from glucose.

[89] Microbial growth was observed using confocal microscope with temperature control system at 30 ° C. Microchannel images were captured hourly, 10h and anaerobically.

[90] - Evaluation of S. cerevisiae cells;

[91] From the images acquired from the microfluidic system over time, direct cell counting was performed using ImageJ software for both counting and drawing the microcameras.

[92] For direct counts, 0.5 mm intervals were determined (for the 0 to 40 g / l gradient of microcameras (Figure 30).

[93] Microchannel endpoints have been excluded because there is no gradient generation in these regions, as they are the gateway to the different-glucose solution streams to the microchannel (see Figure 5).

- Batch cultivation:

[94] For batch cultivation, assays were performed using YPD medium in a volume of 100 mL in shaken Erlemmeyer flasks, varying only the glucose concentration from 0 (control) to 40 g / l glucose.

[95] Cell concentration was determined by absorbance reading (600 nm) every 1 h with the aid of a calibration curve obtained by counting in a Neubauer chamber.

Determination of kinetic parameters:

[96] To determine Sacchoromyces cerevisiae ATCC 7754 specific growth rate (μ _χ ), the slope of the linear region of the semi-logarithmic growth curve was considered using Equation 1:

i d

μ X X dt (Equation 1)

[97] whereas growth kinetics as Monod equation, the maximum specific growth rate (p _m ax) and saturation constant (K _s), which indicates affinity of the microorganism. substrate (S) can be determined according to Equation 2:

t * max-S

μ _s + S (Equation 2) in diffusive concentration gradient environment:

[98] The glass-to-glass microfluidic device was suitable for the cultivation of Saccharomyces cerevisiae ATCC 7754, as it observed an increase in cell number over time, allowing investigation of kinetic parameters and comparison with conventional culture cultivation assays. batch and literature data.

[99] In addition, the operation of the device was evaluated for different concentration ranges as well as the use of microchannel subdivisions of different sizes for cell counts.

Cell growth profile in a diffusive concentration gradient microfluidic device:

[100] S. cerevisiae ATCC 7754 yeast was inoculated into the device. In the upper chamber, the flow of YPD medium was maintained in both entries, with YPD medium without glucose and another YPD medium with 40 g / L glucose.

[101] Cell behavior was monitored for 10 h and yeast growth was observed on the imposed substrate gradient (Figure 6A and B).

[102] In an assay using lower cell concentration, images of cells along the concentration gradient chamber could be obtained under an automated microscope. From these images, the direct cell count (Figure 7A) was performed, correlating with the position in the microchannel and consequently with the glucose concentration (Figure 7B).

[103] It can be seen from Figure 7B that after 3 h of cell growth follow-up, there was a higher number of This behavior was maintained until 10 h, the evaluated period (Figure 7A).

[104] In this concentration range, the observed result was as expected, as the optimal glucose concentration for cell growth should not be greater than 25% (w / v), as in the early growth phase yeast It uses glucose for its metabolism and a high content of this sugar raises the osmotic pressure of the medium.

[105] Thus, it can be noted that yeast cells showed the same behavior as expected in a conventional fermentation system, proving the feasibility of evaluating microbial growth in this glucose concentration range and consequently determining the kinetic parameters in microfluidic system.

- Growth kinetics and comparison with conventional batch cultivation:

[106] Once cell growth is observed in the microfluidic device and taking the number of cells for each gradient microchamber over 10 h, microbial growth curves can be constructed for each glucose concentration range obtained and the velocity determined. specific growth rate under these conditions (Figure 8A).

[107] To compare the results obtained with the microfluidic device, a series of batch cultures were performed using concentrations from 0 (control) to 40 g / L glucose, having the same conditions as those used in the microfluidic device and cell concentration. of micro ^¬ organism. In this way, one can construct the curves of batch (Figure 8B).

[108] In the microfluidic system, a growth profile with higher cell numbers was observed under conditions of 23 to 30 g / L glucose, with behavior similar to batch cultivation.

[109] However, the glucose concentration is constant in microfluidics and therefore a volume of 100 rTIL YPD medium was used for batch cultivation, with the same initial cell concentration employed in microfluidics in order to try to reproduce the same. condition. Thus, in batch cultivation, a smaller number of cells with larger oscillations was observed over time.

[110] Nevertheless, after obtaining the microbial growth curves in both systems, the maximum specific growth rates of yeast in the conventional batch and microfluidic system were determined, as noted in Table 2 below.

Table 7: Comparison between S. cerevísiae ATCC 7754 specific growth rate in microfluidic system with concentration gradient and batch fermentation.

System cultivation Conventional batch microfluidic cultivation

μ _Χ (Μ) (h ^"1 ) V> x (B) (h ^{" 1} )

Glucose (g / L) Glucose (g / L)

(Ux ± SD) (Ux ± SD)

30 0.17 ± 0.12 10 0.21 ± 0.23

10 0.20 ± 0.17 20 0.24 ± 0.16

16 0.24 ± 0.19 30 0.21 ± 0.01

23 0.24 ± 0.1, 40 0.19 ± 0.05 36 0.20 ± 0.07

[111] From Table 2, it is observed that there was no statistically significant difference between the averages of specific growth rates, since experiments on microfluidics and conventional batching were conducted at low cell concentrations and any value within the range. The glucose concentration studied corresponded to an amount sufficient to support microbial growth.

- Monod kinetics in microfluidics:

[112] Microfluidic conditions and the achievement of specific cell growth rates in this system allow the expansion of growth kinetics investigations, thereby investigating the parameters of cell growth kinetics described by the Monod equation.

[113] In addition, it is possible to evaluate device operation at lower limiting substrate concentrations and at smaller microcamera intervals for counts to compare the effect of the gradient itself within the microcamera.

[114] Knowing that glucose concentration is constant in the microchannel, this allows the determination of Monod kinetic growth parameters, not only in the early times, as in conventional systems, since substrate variation influences the results.

[115] Thus, at a 0 to 3 g / L glucose gradient and with interval counting microcameras limiting substrate (S) function (glucose) (Figure 9).

[116] From the experimental data, the saturation constant (K _s ) and the maximum specific growth velocity (τώχ), described by the Monod equation (Equation 2), can be estimated.

[117] Nonlinear least-squares fit yielded 0.32 ± 0.01 h ^_i and 0.27 ± 0.04 g / L, respectively, for _m á and K _s (Figure 9 ).

[118] Through this growth curve and the results for the kinetic parameters, described by the Monod equation (Equation 2), it is observed that the kinetic data obtained in microfluidics are consistent with literature data determined by conventional fermentation.

[119] Thus, the feasibility of this diffusive gradient-forming microfluidic device is noted for investigating the kinetic parameters of the microorganism and thereby increasing the productivity of a bioprocess reaction.

[120] Given the tests and results obtained, the microfluidic devices proposed here were able to generate a gradient with linear and stable profile.

[121] The geometry and compositions of the three devices of the present invention were efficiently sealed, allowing stable flow of upper level currents and lower level gradient generation capability.

[122] The three devices showed linear and purely diffusive concentration gradient formation in the chambers (lower microchannels) and were applicable. [123] The glass-glass device, as it has hydrophilic surfaces and is absent from gas exchange, was chosen for free cell studies under anaerobic conditions and is applicable for microbial cultivation, having similar kinetic parameters to conventional cultivation, which may contribute in bioprocesses.

[124] Furthermore, devices with diffusive concentration gradient generation allowed the determination of Monod kinetics with values consistent with those in the literature, and the results were obtained more practically when compared to conventional techniques.

[125] Given this, the proposed devices have been shown to be efficient for determining kinetic and viable parameters for bioprocess optimization.

Claims

1. Microfluidic device characterized in that it is structured to generate a diffusive concentration gradient comprising:

- a lower level (i) containing the layers: smooth glass slide (l); Rolled, cut and cast PDMS containing at least one chamber (2); and Hole lamina (3);

an upper level (ii) containing the layers: Y-shaped hollow channel laminated PDMS (4); and glass slide with holes (5).

Device according to Claim 1, characterized in that the cut-off of laminated PDMS (2) surrounds the microchannel walls and the smooth glass slide (1) defines the bottom of the microchannels (chambers).

Device according to Claim 1, characterized in that the holes in the coverslip (3) are preferably drilled by chemical corrosion and fit to the ends of the chambers.

Device according to Claim 1, characterized in that the rolled-channel laminated PDMS (4) allows the flow of solutions of different concentrations.

Device according to claim 1, characterized in that the sealing is performed by adhering the laminated PDMS to the glass.

Device according to claim 1, characterized in that the holes in the glass slide (5) may optionally comprise solution inlet hose connections (9).

7. Microfluidic device comprising: - a lower level (i) containing the layers: microchannel glass slide (chambers) (6); and PDMS with Y-shaped channel and hollow holes (7);

- An upper level (ii) containing the layer of glass slide with holes (5);

Device according to Claim 7, characterized in that the chambers are present on the glass sheet (6) in low relief and preferably made by chemical corrosion.

Device according to Claim 7, characterized in that the channel is undercut, Y-shaped, on the upper surface of the PDMS (7) and allows solutions of different concentrations to flow.

Device according to Claim 7, characterized in that the holes in the PDMS (7) are within the channel region and fit the ends of the microchannels (chambers) of the glass slide (6).

Device according to Claim 7, characterized in that the PDMS (7) can be optionally chemically modified (8).

Device according to Claim 7, characterized in that the sealing is carried out by any movable fastening means, preferably by clips.

Device according to claim 7, characterized in that the holes in the glass slide (9) may optionally comprise connections for solution inlet hoses.

Use of the device as defined in claims 1 to 6, characterized in that for any process it is necessary to know the ideal substrate conditions or ideal conditions for cellular performance, estimation of kinetic parameters and evaluation of chemical responses such as chemotaxis.

Use of the device as defined in claims 7 to 13, characterized in that for any process it is necessary to know the ideal substrate conditions or the ideal conditions for cell performance, estimation of kinetic parameters and to evaluate chemical responses such as chemotaxis.