FIELD OF THE INVENTION

The present invention relates to microfluidic systems. More particularly, the invention relates to a microfluidic apparatus and a corresponding method for creating a concentration gradient, in particular a lateral chemical gradient.

BACKGROUND OF THE INVENTION

Several important physiological processes are mediated by chemical gradients in the environment. A well-known example is the process of chemotaxis, i.e., the directed cell migration of cells in a concentration gradient of a soluble factor (chemoattractant). Chemotaxis is critical for the body's immune response, and is involved in a wide range of processes, including cancer metastasis, angiogenesis, bone formation, and wound healing. The study of chemotaxis is also relevant from the clinical point of view, since many diseases (e.g. atherosclerosis, cancer, AIDS, etc.) are related to changes in cell motility. Recently a link between monocyte mobility and atherosclerotic risk has been suggested. More specifically, patients with high risk factors for Coronary Artery Disease (CAD), such as hypercholesterolemia, diabetes and smoking, demonstrate a drastic reduction in monocyte mobility. This gives a potential for CAD risk stratification based upon monocyte chemotaxis. Chemical concentration gradients are also used to study other processes besides cell migration, such as axon guidance, cell polarization, stem cell differentiation or cytotoxicity assays.

Traditionally, cell migration has been studied in trans-well assays such as the Boyden chamber, or in planar assays such as the Zigmond chamber or the Dunn chamber. In these assays, a concentration gradient is created by diffusion across a membrane filter or in a shallow channel connecting two reservoir chambers, which are filled respectively with pure buffer and a concentrated solution of chemoattractant. These devices provide basic information on the migratory response of cells, but allow very little control on the generated gradient. As they are based on diffusion, a considerable time is required to establish a concentration gradient across the detection region. The shape of the gradient is defined by the diffusion dynamics and cannot be adjusted externally. Moreover, the gradient decays in time and can vary considerably during the time required for the assay, which is typically in the order of Ih.

Microfluidic devices have been recently recognized as powerful and versatile tools for generating chemical gradients. These devices greatly improve gradient stability and control compared to conventional assays such as the Boyden, Zigmond and Dunn chambers. This has opened the door to a new type of bioassay in which the response of single cells is analyzed with unprecedented detail. Microfluidic methods have the promise to reduce the time needed for migration assays from hours to minutes. The simplest microfluidic device for generating a chemical gradient is a y-type channel structure (see, for example, F. Lin, E. C. Butcher, Lab Chip 6, 1462-1469 (2006)). In this device, a concentrated solution of chemoattractant is injected from one inlet and pure buffer is injected from the other inlet. A concentration gradient is formed in the channel when chemoattractant molecules diffuse across the interface between the streams. In contrast to the conventional devices described above, the gradient is stable over time and can be maintained as long as fresh buffer and chemoattractant solutions are supplied. The shape of the gradient is typically sigmoidal, but can be varied to some extent by adjusting the relative flows of solutions. The direction of the gradient can also be inverted by changing the inlet configuration. The y-channel structure has however important disadvantages, which arise from the fact that the gradient formation still relies on diffusion. The extension of the gradient is determined by the diffusion length L _d = yJ4Dt , where D is the diffusivity (~1.7 x

10 ^"6 Cm ² S ¹ for 10 kDa molecules) and t is the residence time of the chemoattractant in the system, i.e., t = x/ U where x is the position along the channel (as measured from the inlets) and f/is the flow velocity. For characteristic values of x = lOOμm and U= lOOμm/s, the diffusion length L _d is in the order of 30μm. This means that a sharp gradient is present in the middle region of the channel or, in other words, that only a limited fraction of the channel width can be used in the assay. A smoother gradient can be obtained by increasing the residence time (i.e., either by increasing the channel length or decreasing the flow velocity) so that Ld becomes comparable to the width W of the channel. Due to the square root dependence, however, in order to increase the extension of the gradient by a factor of 10 the channel has to be 100 times longer (or the flow speed 100 times smaller). Long channels are not suitable for miniaturization, while low flow velocities introduce considerable time-delays when the gradient is applied, removed, or inverted.

Furthermore, the condition L _d ~ W also implies that the gradient will evolve rapidly along the channel. In the case of chemotaxis assays, cells in different sections of the channel will experience different conditions. In the prior art this is avoided by limiting the measurement area to a single microscope field (in the order of 100 x 100 μm), over which the gradient does not change considerably. This however means that only a small fraction of the channel (and consequently only a small fraction of cells injected in the device) is analyzed. To increase the efficiency of the assay, it would be beneficial to make use of a larger fraction of the channel. This involves sequential imaging of high power microscope fields, a task which can be easily accomplished with conventional microscopes equipped with an automated translation stage. A prerequisite for this is, however, a gradient which does not change significantly along the channel.

To address the issues of the y-channel structure, micro fluidics devices have been designed in which gradient generation relies on flow patterns rather than diffusion.

These devices are typically based on complicated networks of micro fluidic channels, either in a branched configuration or by "tapping" from different positions in channels connecting two reservoirs. In these devices, the gradient shape can be controlled precisely and is defined by the inputs and the structure of the micro fludic network. The gradient is essentially independent of the flow rate, and can be maintained over several millimeters along the detection chamber under laminar flow conditions.

The microfluidic network approach has however other drawbacks. The network needed to create the gradient occupies a considerable fraction of the device. The size of the network is essentially proportional to the resolution of the gradient. As a result, the potential of the device for miniaturization and high throughput experimentation is limited. Moreover, the narrow microfluidic channels forming the network increase the chance of leakage and are easily blocked by entrapped air bubbles, debris, etc.

WO 2003/011443 A2 discloses a laminar mixing apparatus and methods wherein a microfluidic device having groove features on the channel walls is used to create a homogeneous mixture.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an apparatus and method that combines the high degree of gradient control and stability of flow-based devices, with the simplicity and ruggedness of a simple y-type channel structure.

It is an aim of particular embodiments of the present invention to provide an apparatus and method that are capable of creating a lateral chemical gradient, e.g. a growth factor gradient, in a microchannel.

According to a first aspect of the present invention there is provided a microfluidic device for creating a concentration gradient comprising: a microfluidic channel, the microfluidic channel including two or more inlets for introducing fluid solutions, two or more channel segments, wherein at least one segment includes a ridged element including a plurality of ridges in and/or on an interior channel wall, characterized in that the ridged element is adapted to create a localized secondary flow effect and a local mixing effect in that segment of the channel for the formation of said concentration gradient over the width of the channel. The microfluidic apparatus and method allows precise (spatial and temporal) control over the chemical gradient. An important property is that the gradient is independent of the flow speed through the channel. Stable gradients can therefore be established in seconds using high flow speeds and do not decay over time, contrary to methods relying on diffusion. The gradient is defined by the geometry of the grooved structures and can be maintained over several millimeters along the channel. The device is also much simpler than existing flow-based gradient generators based on complicated networks of microfluidic channels, and is less prone to blockage and leaking. An important application of the device is the study of chemotactic properties of cells (in particular of monocytes) in a growth factor gradient, which is important for diagnosis and risk stratification of CAD. The apparatus can be manufactured relatively cheaply.

Advantageous embodiments of the device of the present invention, in particular of the ridged elements within the channel segments, are defined in the dependent claims.

In a preferred embodiment, each of the two or more channel segments includes a dissimilar ridged element for creating localized secondary flow effects and local mixing effects in respective segments of the channel. The ridged element may be included in a channel floor. The ridged elements may include at least two parallel ridges separated by a groove that is substantially even with a channel floor. With respect to a width of the channel perpendicular to fluid flow direction, the ridged element may be in a middle portion of a channel floor only, particularly for a first segment. These arrangements have the advantage of providing precise control over the specific location of the secondary flow effects and local mixing effects within the fluids, leading to a predictable gradient across the width of the channel. Each segment may include a plurality of zones oriented parallel to fluid flow, e.g., nine zones, for the optimal arrangement of the ridged elements and the local flow and mixing effects. In at least a first segment type, there may be no ridged element in the three zones adjacent each side wall of the channel. In another arrangement, only a middle three zones include the ridged elements, and at least the first segment type includes the ridged element in only two of the three middle zones. Alternatively, the ridged element in the first segment may include a laterally staggered arrangement. Second and subsequent segments may include two rows of generally parallel ridged elements arranged side-by-side. The two generally parallel rows of ridged elements in each subsequent segment may be spaced farther apart than in a preceding segment. These arrangements provide the advantage of identifying the portions of the fluid stream which are subject to the secondary flow effects and local mixing effects, and include an orderly manner to methodically create a predictable gradient. The height of the ridged element above the floor of the channel may be about 20-40% of the total height of the channel. The ridges of the ridged element may be angled with respect to the fluid flow direction. The angle may be between 30° to 60°. The first half of the ridged element in each segment may be angled clockwise from the fluid flow direction, and a second half of the ridged element in each segment may be angled counterclockwise from the fluid flow direction. In this manner, the exact physical embodiment of the ridged elements may be selected to match the properties of the fluids to create the desired gradient. Various fluid chemicals may be introduced through the inlets, e.g., a concentrated growth factor solution and a pure buffer solution. The various fluid chemicals may be chemoattractants, but can also be toxins. The dissimilar fluids will create a sharp gradient, while fluids having fewer differences will form a more gentle gradient. Careful selection of the fluids provides the advantage of a gradient that is exactly appropriate for the testing to be accomplished. The microfluidic device may include a perpendicular grooved/ridged element as a cell capture area, located after a cells inlet and before a channel exit, wherein cells are captured between a plurality of perpendicular ridges in a plurality of perpendicular grooves. Captured cells may be observed in chemotaxis studies. According to a second aspect of the present invention there is provided a method of creating a concentration gradient with a micro fluidic device comprising the steps of: constructing a micro fluidic channel, the microfluidic channel including two or more fluid inlets and two or more channel segments, wherein at least one segment includes a ridged element including a plurality of ridges in and/or on an interior channel wall; and introducing fluid solutions into the inlets and through the channel; and creating a localized secondary flow effect and a local mixing effect in the fluid solutions via the ridged element, wherein the concentration gradient is formed.

A microfluidic device according to the present invention includes numerous advantages. These advantages include the elimination of complicated fluidic structures.

Further, the creation of the gradient in the microfluidic device is independent of the velocity of flow through the channel. The creation of a stable gradient does not rely strictly upon diffusion and closely mimics a coronary artery. Also, the invention may integrate valves to reverse the gradient. It shall be understood that the claimed method has similar and/or identical preferred embodiments as the apparatus and as defined in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. In the following drawings Figs. IA and IB show a plan view of a prior art microfluidic device for producing a chemical gradient;

Fig. 2 shows a plan view of an embodiment of a microfluidic device for producing a chemical gradient in accordance with an embodiment of the present invention; Figs. 3-5 illustrate plan views of embodiments of segments for a microfluidic device for producing a chemical gradient in accordance with an embodiment of the present invention;

Fig. 6 illustrates a plan view of individual ridges for a microfluidic device for producing a chemical gradient in accordance with an embodiment of the present invention; Figs. 7 A-E illustrates exemplary concentration profiles of two fluids at specific points along the channel of a microfluidic device in accordance with an embodiment of the present invention, Fig. 8 illustrates a plan view of a microfluidic device for producing a chemical gradient including a cell capture area in accordance with an embodiment of the present invention, and

Fig. 9 illustrates a perspective view of the perpendicular groove and ridge arrangement of a cell capture area for a microfluidic device for producing a chemical gradient in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Figs. IA and IB illustrate a prior art microfluidic device which relies upon a complex flow pattern to create a gradient generation. These devices are typically based on complicated networks of microfluidic channels, either in a branched configuration or by "tapping" from different positions in channels connecting two reservoirs. In these devices, the gradient shape can be controlled precisely and is defined by the inputs and the structure of the microfluidic network. Fig. IA shows a schematic block diagram of a complex prior art device. In this example, the gradient chamber 100 includes a branch structure 101 and a monitoring chamber 102. The branch structure 101 has a plurality of interconnected branches (also called micro fluidics) for mixing the solutions provided via the fluid inlets 10, 11, into said concentration gradient. Via a concentration gradient outlet 103 the generated concentration gradient is provided to a concentration gradient inlet 104 of the monitoring chamber 102, into which cells are loaded via the cell inlet 12. An electrical sensor 2 is arranged within said monitoring chamber 102.

Fig. IB shows a schematic block diagram of another complex prior art device. In this example, the gradient chamber 110 includes a branch structure 112 and a number of monitoring chambers 114. The branch structure 112 has a plurality of interconnected branches for mixing the solutions provided via the fluid inlets 116, 118, into said concentration gradient. The generated concentration gradient is provided into the monitoring chambers 114, and then to the respective concentration gradient outlets 120.

These prior art microfluidic network approaches have significant drawbacks. The network needed to create the gradient occupies a considerable fraction of the device. The size of the network is essentially proportional to the resolution of the gradient. As a result, the potential of the device for miniaturization and high throughput experimentation is limited. Moreover, the narrow microfluidic channels forming the network increase the chance of leakage and are easily blocked by entrapped air bubbles, debris, etc. Fig. 2 shows a plan view of an embodiment of a microfluidic device 200 for producing a chemical gradient in accordance with embodiments of the present invention. The microfluidic device 200 includes a microfluidic channel 202 having two or more inlets 204, 206 for introducing fluid solutions, two or more channel segments 208, wherein at least one segment 208 includes a ridged element 210 including a plurality of ridges 212 in an interior channel wall 214. The ridged element 210 is adapted to create a localized secondary flow effect and a local mixing effect in that segment 208 of the channel 202 for the formation of said concentration gradient over the width W of the channel. Only a single segment 208 is shown for clarity. However, the channel 202 may include a number of segments 208, 224, 226, 228. Each of the two or more channel segments 208, 224, 226, 228 may include a dissimilar ridged element 210, 216, 218, 220, shown in Figs. 3-5, for creating localized secondary flow effects and local mixing effects in respective segments of the channel 202.

The ridged element 210, 216, 218, 220 may be included in a channel floor 214. Some of the ridged elements 216, 218, 220 may include a formation of at least two parallel ridges 212 separated by a groove 222 that is substantially even with a channel floor 214. With respect to a width W of the channel 202 perpendicular to fluid flow direction F, the ridged element 210 may be in a middle portion of a channel floor 214 only. This arrangement is illustrated in Fig. 2.

With respect to a fluid flow direction F perpendicular to the width W of the channel 202, the segments 208, 224, 226, 228 may include a plurality of parallel zones. Figs. 2-5 illustrate nine parallel zones each, Z1-Z9, but the number of zones are arbitrary and are intended to aid in understanding the function of the device 200.

As illustrated in Fig. 2, the segment 208 does not include a ridged element 210 in the three zones Z1-Z3, Z7-Z9 adjacent each side wall 230 of the channel 202. This segment 208 illustrates that the ridged element 210 may be in only the middle three zones Z4-Z6. In addition, the ridged element 210 may include two distinct arrangements 232, 234 that are staggered or offset with respect to the width W of the channel 202. The segment 208 may include the distinct portions 238, 240 of the ridged element 210 in only two of the three middle zones Z4-Z6 each. Thus the arrangement of Fig. 3 may provide fluid mixing with respect to a central portion of the fluid flow through the channel 202.

The grooved/ridged element 210 illustrated in Fig. 2 is arranged such that mixing occurs, with respect to the width W of the channel, only in the zones corresponding to the location of the ridged element 210. The ridged elements 210, 216, 218, 220 are oriented at a certain angle α with respect to the main flow direction F. The angle α illustrated is about 45°, but it could be between 30°and 60°. The width direction of the first arrangement of ridges 238 may be about 2/3 the total width of zones for the entire ridged element 210. The height of the ridges 236 or depth of the grooves may be between 20 and 40% of the total channel height, which may be about 25 μm. The dimensions of each ridge 236 in a streamwise direction may be comparable to the channel height, which may be about 50 μm. The pattern shown in Fig. 2 consists of two distinct arrangements 238, 240 of repeating grooves/ridges, oriented at 45° and -45° with respect to the main flow F, and displaced or shifted somewhat perpendicular to flow direction F.

The pattern of grooves/ridges comprising a ridged element 210, as depicted in the Fig. 2 may be called one "cycle" and may consist of a first arrangement of ridges 238, e.g., six "type 1" grooves/ridges, followed by a second arrangement of ridges 240, e.g., six "type 2" grooves/ridges. In practice, the number of repeated grooves/ridges of a certain type may be smaller or larger, and may be typically between 5 and 20. The "cycle" of Fig. 2 may be repeated several times, i.e., between 5 and 20 times, to obtain a good homogenization of the fluids within the desired zones.

Figs. 3-5 illustrate arrangements of segments 224, 226, 228 where each ridged element 216, 218, 220 includes two rows of generally parallel ridged elements 213 arranged side-by-side. In addition, the arrangement of ridges of each segment 224, 226, 228 is slightly different. Viewed sequentially, each of the segments 224, 226, 228 includes the two generally parallel rows 213 of ridged elements 216, 218, 220 where the parallel rows 213 of the arrangement shown in Fig. 4 are spaced farther apart than in the arrangement shown in Fig. 3, and the arrangement shown in Fig. 5 includes wider spacing than in Fig. 4. Thus, if segments 224, 226, 228 are placed sequentially in a row in a single channel 202, each subsequent segment includes wider spacing between the parallel rows 213 than the preceding segment. As explained previously, the purpose of the grooved/ridged elements 210, 216,

218, 220 indicated in Figs. 2-5 is to provide homogenization of the dissimilar fluids within the targeted zones, while the fluids in the other zones are not affected. Testing has shown this device to be effective in creating the desired results.

Fig. 6 illustrates a plan view of individual ridges 236. In one embodiment, the height of the ridged elements 210, 216, 218, 220 and each individual ridge 236 above a floor 214 of the channel 202 is approximately 20-40% of the total height of the channel 202. Each ridge 236 may include a profile that is squared to the channel floor 214 or each ridge 236 may have a rounded profile, depending on the desired fluid mixing effect desired. The individual ridges 236 and the ridged elements 210, 216, 218, 220 may be sized to provide an appropriate number of zones, and to accommodate the length and height limitations within the channel 202. In one embodiment, the width of each ridge 236 perpendicular to fluid flow F is approximately 74 μm and the length of each ridge 236 parallel to fluid flow F is approximately 50 μm, with a spacing of approximately 50 μm as well. However, the actual and relative sizes of the ridges 236 and ridged elements 210, 216, 218, 220 may be varied to suit the particular application. The ridges 236 of the ridged elements 210, 216, 218, 220 may be angled α with respect to the fluid flow direction F. The angle α may be within a range from 30° to 60°.

As illustrated in Fig. 2, the first arrangement of ridges 238 in each segment 208, 224, 226, 228 may be angled clockwise from the fluid flow direction F, and the second arrangement 240 may be angled counterclockwise from the fluid flow direction F. This arrangement provides a localized mixing effect in one direction for the first half of the segment and a local mixing effect in a second direction for the second half of the segment. This arrangement may be reversed as well. The gradient may be created with a wide variety of chemical fluid solutions, e.g., a concentrated growth factor solution and a pure buffer solution.

The method of creating a concentration gradient with a micro fluidic device 200 comprises constructing a microfluidic channel 202 that includes two or more fluid inlets 204, 206 and two or more channel segments 208, 224, 226, 228. At least one segment 208, 224, 226, 228 includes a ridged element 210, 216, 218, 220 that includes a plurality of ridges 212 in an interior channel wall 214. Fluid solutions may be introduced into the inlets 204, 206 and through the channel 202, to create a localized secondary flow effect and a local mixing effect in the fluid solutions via the ridged elements 210, 216, 218, 220 to form a concentration gradient. Fig. 7 provides exemplary concentration profiles of two fluids A, B at specific points along the channel 202, as a result of the localized mixing effects, according to the method presented. Fig. 7 illustrates the distribution and relative concentrations of two fluids across the width W of a channel 202. Fig. 7A illustrates the relative fluid arrangements immediately after the fluids enter the channel 202 at D, where there is no mixing between the two liquids at all. Figs. 7B-7E illustrate the relative fluid arrangements and concentrations after a number cycles, i.e., 5 -20 cycles across the grooved/ridged elements 210, 216, 218, 220.

As the fluids A, B first enter the channel 202 at point D (see Fig. 2), very little mixing, if any, occurs. This fluid concentration profile, measured across the width W of the channel 202 is presented in Fig. 7A. Fluids A, B are side-by-side in a laminar flow, but have not been mixed. Diffusion is not a significant factor. Fig. 7B illustrates a concentration profile that would be observed immediately after the fluids A, B passed through segment 208. Fig. 2 illustrates a ridged element 210 in the middle of the channel 202 to provide a local mixing effect in the center of the channel 202. Thus, after passing through the first segment 208, only the center part of the fluid flow will be mixed to provide a partial concentration gradient as shown in Fig. 7B.

Next, the fluid passes through a second segment 224 having a ridged element 216 similar to that illustrated in Fig. 3. The ridged element 216 does not affect the fluid at the center of the channel 202 as the first segment 208, but affects the two new "boundaries" between the center fluid and the relatively unmixed fluids near the sides of the channel from Fig. 7B. Restated, the second step is the local mixing of the fluids A, B at the edges of the region homogenized in the first step. This is achieved with a grooved/ridged element 216 depicted in Fig. 3. Thus after the fluids A, B pass through the second segment 224, the overall appearance is a more gradual transition as illustrated in Fig. 7C.

Next, the fluid passes through a third segment 226 having a ridged element 218 similar to that illustrated in Fig. 4. The ridged element 218 does not affect the fluid in the same place as the second segment 226, but affects the two new "boundaries" between the previously mixed fluids and the relatively unmixed fluids near the sides of the channel from Fig. 7C. Thus after the fluids A, B pass through the third segment 226, the overall appearance is a more gradual transition as illustrated in Fig. 7D.

Finally, the fluid passes through a fourth segment 228 having a ridged element 220 similar to that illustrated in Fig. 5. The ridged element 220 does not affect the fluid in the same place as the third segment 228, but affects the two new "boundaries" between the previously mixed fluids and the relatively unmixed fluids near the sides of the channel from Fig. 7D. The third and fourth steps, illustrated in Figs. 7C-7D, results in mixing of the fluids in zones moved successively outward from previously homogenized zones, towards the side walls of the channel 202.

Thus after the fluids A, B pass through the fourth segment 228, the overall appearance is a more gradual transition as illustrated in Fig. 7E.

It is clear that, as illustrated in Figs. 7B-7E, the two fluids are mixed locally in the part of the channel 202 in which the grooved/ridged elements 210, 216, 218, 220 are present, but not away from the zones including the outside the grooved/ridged elements 210, 216, 218, 220. A local "homogenization" occurs, but not a general system-wide homogenization.

The eventual result of the local mixing in the four steps just described is shown in Fig. 7. As illustrated, the fluids are "homogenized" progressively, from the center of the channel 202 towards the side walls of the channel 202, leading to a linear concentration profile built up in a series of finite steps corresponding to the width of the zones defined in Fig. 2. To achieve this, it is important that the zones in which mixing/homogenization takes place in consecutive steps are overlapping partially.

Any number of segments, and inlets, may be assembled to create custom chemical gradients to meet the needs to a particular application. However, the goal is to create a predictable concentration gradient in a simple, repeatable manner. The sharpness of the chemical concentration gradient may be further adjusted based upon the concentrations of each of the chemicals introduced into the system. The dimensions used in the provided examples are specific, but the principle will also work with other dimensions. For the grooved/ridged elements 210, 216, 218, 220 to be effective, there are some dimension guidelines. The depth of the grooves/height of the ridges 236 should be between 20% and 40% of the channel height. The width of the grooves/ridges parallel to W should be between 50 % and 80% of the width W of the fluid stream to be homogenized. The streamwise dimension, parallel to fluid flow F, of the grooves/ridges should be smaller than their width- wise dimension. Depending upon the specific results desired, the total length of the channel 202 is defined by the dimensions and number of repeats of the grooves/ridges. In the particular case of a total channel width of 500 μm, a ridge streamwise dimension of 50 μm, and four segments containing five cycles that consist of 12 ridges each, the total length is 4x6,000=24,000 μm, i.e., about 50 times the channel width. Other geometrical designs of the grooved/ridged elements would lead to different concentration profiles at each step and/or overall, e.g., a non-linear concentration profile.

Valves may be used to swap between the two inlets enables to reverse the concentration profile, and may be done in an oscillatory way if needed. The induced concentration gradient is independent of the speed of fluid flow through the channel. This results because the secondary flow caused by the grooved/ridged elements 210, 216, 218, 220 scales with the streamwise flow speed. For very slow flows, diffusion may play a part in the general homogenization of the fluid over the channel width. This effect will be negligible, as long as the so-called Peclet-number Pe is large. Pe defined as Pe = ULI D , in which U is the characteristic flow velocity, L is the characteristic length scale, in this case the width of the channel, and D is the typical diffusion coefficient. Pe indicates the relative importance of convection to diffusion, and its value can be interpreted as the number of channel widths the fluid needs to travel in the channel direction to get complete mixing by diffusion. The diffusion coefficient of the buffer solution/growth factor solution can be estimated as D~\ x 10 ^~6 Cm ² S ¹ . For L the channel width W=500 μm may be taken as a typical value, and U is the mean axial velocity. The condition Pe>50 (i.e. only after a travelling distance of 50 channel widths diffusion will play a significant role, recall that the total length for the specific case given above is 50 times the channel width) gives the condition U>10 μm/s. For flows with a lower mean speed, diffusion may come into play. This is an order-of-magnitude estimate that may take a different value for different channel dimensions.

Thus the microfluidic device 200 may be applied in situations in which a chemical concentration gradient of a species needs to be established, e.g., a growth factor gradient that can be used to measure chemotactic properties of cells. Another potential application is free flow iso-electrical focusing where a salt or conductivity gradient is required. Another potential application is DEP-focusing (Dielectrophoretic) where a conductivity gradient is required. Another application is the measurement of the properties of all kind of cells, including bacteria, in a chemical concentration gradient of e.g. antibiotics. The measurement of the motility of monocytes in a chemical concentration gradient is important to determine the potential risk for Coronary Artery Disease (CAD). As described above, a concentration gradient can be established due to the mixing of two fluids by ridged elements 210, 216, 218, 220 on a substrate, e.g., a microfluidic channel 202. These ridged elements 210, 216, 218, 220 may cause a secondary flow perpendicular to the main flow resulting in a controlled mixing mechanism.

It was found that when inserting cells L, e.g., white blood cells, via the cells inlet 240 as depicted in Fig. 8, the cells L may be picked up by the fluid stream F and flow to the end 242 of the microfluidic device without stopping. This can be a problem in chemotaxis studies. If the inserted cells L do not stop in the channel 202, they will not have the opportunity to adhere and migrate in the direction of the concentration (growth factor) gradient due to the main flow F. Therefore a solution for capturing cells is needed so that their motility can be studied.

It was found that the integration of a perpendicular grooved/ridged element 244 into a cell capture area 250 after the cells inlet 240 enables the cells L to be captured in the perpendicular grooves 248 between the perpendicular ridges 246, instead of simply flowing through the microfluidic device 200 to the exit 242 and leaving the microfluidic system 200. The perpendicular grooves 248 and ridges 246 should be perpendicular to the main flow direction F, see Fig. 9, so as to avoid disturbing the established gradient.

Before setting up the concentration gradient and thus a flow F, the cells L may be inserted via the cells inlet 240. The cells L will flow through the last part of the microfluidic system 200, before the perpendicular grooved/ridged element 244, and there the cells L will be captured by the perpendicular grooves/ridges 246, 248. When the flow F with the cells L is minimum, e.g., 1-2 μl/min, the cells L will sink to the bottom of the channel 202 and the perpendicular grooved/ridged element 244 due to gravitation. The cells L can adhere to the surface between the perpendicular ridges 246, as shown in Fig. 9. Laminar flow above the perpendicular grooves/ridges 246, 248 carries the fluid with the concentration gradient, e.g., a growth factor, over the perpendicular ridges 246 and diffusion from this flow into the perpendicular grooves 248 provides a concentration gradient to the cells L. Chemotactic motion of the cells L can now take place and may be observed. This embodiment with the perpendicular grooves/ridges 246, 248 mimics the

3D vascular system where cells can adhere onto the substrate on multiple sides and where the main flow mimics the blood stream.

Thus, this invention presents a micro-fluidic device in which a concentration gradient of a (chemical) species is induced by geometrical patterns placed on the channel wall(s); a microfluidic system in which the concentration gradient can be reversed

(continuously) in time by the integration of additional valves in the system; a microfluidic system in which the chemical gradient is a growth factor gradient for the measurement of chemotactic mobility of cells; and a microfluidic system in which the chemotactic mobility of monocytes can be measured, for the diagnosis and risk stratification of Coronary Artery Disease (CAD).

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.

In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single element or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage.

Any reference signs in the claims should not be construed as limiting the scope.