HK1171409A - A biological microfluidics chip and related methods - Google Patents

Abstract

A biological microfluidics chip (100) comprising a substrate (102), a microfluidic inlet port (104) defining an opening in a surface of the substrate (102), and a microfluidic outlet port (106) defining an opening in a surface of the substrate (102). The biological microfluidics chip (100) also comprises a plurality of wells (108) extending from a top surface (110) of the substrate, wherein each well (108) is bounded by one or more walls, and an inlet opening (112) and an outlet opening (114) are provided in a wall of each of the plurality of wells (108). An inlet microfluidic channel (116) is provided in the substrate (102) to connect the microfluidic inlet port (104) to each of the inlet openings (112) in the walls of the wells, and an outlet microfluidic channel (118) is provided in the substrate to connect each of the outlet openings (114) in the walls of the wells to the microfluidic outlet port (106).

Description

Biological microfluidic chips and related methods

Technical Field

The present invention relates to a biological microfluidic chip and a method of using the same. For convenience, a biological microfluidic chip may be referred to as a "biochip".

Background

In scientific research in biology and biomedicine, researchers often culture cells or embryos in culture vessels for the purpose of research. One very common form of culture vessel is the so-called microtiter plate (microtitre plate), which usually comprises straight-sided cylindrical wells (wells) formed in the plate. Microtiter plates with standard shape and size are used to lock the retentate (retention) in the analysis device or robotic processor.

In practice, microtiter plates typically comprise an array of microtiter wells in a grid-like pattern. One well-known arrangement comprises 96 micro-titration wells, defining an array of 8 rows of 12 micro-titration wells each. This 96-well plate design has become an industry standard format prescribed by the Society for Biomolecular Screening.

Microtiter plates are typically made from transparent polymers, such as acrylonitrile-butadiene-styrene ('ABS'). This transparency characteristic enables researchers to perform various optical tests on cells, embryos or larvae cultured in microtiter wells. Furthermore, micro-titer wells are suitable for conducting a large number of assays and studies that do not involve cellular material.

The microtiter wells are open at their upper ends when in use. An electronically controlled meter (dosignapapratus) can be used to inject culture solution and, for example, reagents, enzymes or other additives into each well of a microtiter plate to study the effect of these reagents, enzymes or other additives on cells in the microtiter wells.

For convenience, terms used in the present invention, such as "upper", "above", "lower", "vertical", "horizontal", "upwardly", and "downwardly", are explained with reference to a microtiter well or a biological microfluidic chip in an operating orientation. Thus, these terms are understood when the biochip is laid flat on a horizontal surface such as a test bed with the opening of the well facing upward. It will be recognized, however, that when a biochip or other cell culture container is used, its orientation may be changed, for example, by centrifuging or otherwise shaking, or tilting or inverting the biochip or other cell culture container as part of an experimental or observation procedure. However, the above terms and related terms should not be construed to limit the scope of the present invention to a particular orientation of the culture vessel or to any particular mode of use.

A Microfluidic device consisting of a flow layer and a control layer made of flexible polymers is disclosed in "Microfluidic system for on-chip-through-hole-in-animal monitoring and screening at sub-cellular resolution", published by Christopher B.Rohde et al, Vol.8, 28, 2007 in the journal of the national academy of sciences (PNAS) No. 104, No.35, 13891, 13895. The flow layer includes microchannels for manipulating nematodes (c. elegans), immobilizing them for imaging, and for delivering culture media and reagents. The flow layer also contains microchambers for culturing animals. The control layer is comprised of microchannels that, when pressurized, flex the membrane into the flow channel to impede or redirect the flow of fluid. Animals inside the flow tube (flow line) can be imaged using a high resolution microscope through a transparent glass substrate.

The listing or discussion of a prior-published document or any background material in this specification should not be taken as an admission that the document or background material is part of the state of the art or is common general knowledge. One or more aspects/embodiments of the present invention may or may not address one or more of the background issues.

Disclosure of Invention

According to a first aspect of the present invention, there is provided a biological microfluidic chip comprising:

a substrate;

a microfluidic inlet defining an opening on the substrate surface;

a microfluidic outlet defining an opening on the substrate surface; and

a plurality of apertures extending from the top surface of the substrate, wherein the boundary of each aperture is defined by one or more walls, an inflow opening and an outflow opening being provided on the walls of each aperture;

one or more microfluidic inflow channels within the substrate connecting the microfluidic inlet to the each inflow opening on the pore wall; and

one or more microfluidic outflow channels within the substrate, the microfluidic outflow channels connecting the outflow openings on the pore walls to the microfluidic outlets.

In conventional use, the inflow channel carries fluid into the bore and the outflow channel carries fluid out of the bore. However, the manner of flow may be reversed if desired.

Biological microfluidic chips (biochips) can be used to culture and study cells, embryos, and larvae in wells; the microfluidic channel may be used to provide one or more microfluidics to the well and may also be used to remove one or more microfluidics from the well. In one example, drugs or other compounds and/or nutrients may be provided to the wells for use in the experiment. In some embodiments, biological waste, metabolites, and/or bacteria that can affect the performance of the assay in the well can be removed from the well via the microfluidic outflow channel.

The biochip according to the embodiment of the invention can improve scientific experiments performed in the wells of the biochip and can obtain more accurate and reliable results at lower cost and faster speed.

The walls of the plurality of wells may include lateral boundaries (which may be considered as side walls) and may also include a bottom wall (which may be considered as a bottom of the well). The walls may be flat/planar or curved.

It is to be understood that the term "microfluidics" relates to the movement patterns, precise control and manipulation of fluids, and that the fluids are geometrically constrained to small, typically sub-millimeter dimensions.

The length of the microfluidic channel between the microfluidic inlet and the inflow opening in the wall of the well is approximately equal for each well or a portion of the well. That is, the length of the channel between the inlet and the inflow opening is substantially the same for each hole, although some holes are farther from the inlet.

In this way, the pressure and flow rate of fluid provided by the microfluidic channel to each individual well is substantially the same. This reduces the chance of cross-contamination of the contents of different wells and ensures that the same environmental conditions are provided for each well. Thereby enabling one or more of the disclosed biological microfluidic chips to perform reliable and repeatable experiments simultaneously in a plurality of different wells under the same conditions.

Also, the length of the microfluidic channel between each outflow opening and the microfluidic outlet on the wall of the well is approximately equal for each well. In the same manner, as described above, it is ensured that the fluid is removed from each of the holes at the same flow rate.

In some examples, the length of the microfluidic channel may be varied such that the aperture furthest from the inlet and the aperture closest to the inlet receive the same flow rate of fluid at the same time. In each case, the same variable length may be used for the respective microfluidic channel and each inflow/outflow opening within the well.

The biological microfluidic chip may further comprise:

a temperature control inlet and a temperature control outlet; and

a temperature control channel for conveying, in use, a temperature control fluid from the temperature control inlet to the temperature control outlet along a path proximate to one or more of the plurality of bores such that heat may be exchanged between the temperature control liquid and the contents of the bores.

The provision of such a built-in temperature control system enables the movement of the biochip during the experiment, while the use of a separate temperature control means enables the biochip to be confined to a specific location.

Heat can be exchanged so that the biochip heats or cools the contents of the wells during use and a stable and constant temperature can be provided between all wells.

The temperature control liquid may be an aqueous medium, an oil, or any other fluid suitable for maintaining a desired temperature while flowing along the temperature control channel.

One or more of the temperature control inlet, temperature control outlet and temperature control passage may be provided for a portion of the plurality of orifices, such that the temperature of each or a portion of the orifices may be individually controlled.

In some examples, apertures associated with different temperature control inlets and/or temperature control channels may share one temperature control outlet. The temperature control passages from multiple inlets may meet before opening to the outlet to mix together the temperature control fluid of each passage that has completed the heating or cooling hole function. This is advantageous for reducing the number of ports required for the biochip and is also considered acceptable because the temperature control fluid from each temperature control inlet has already completed its role before mixing together to exit the biochip.

The plurality of wells may constitute an array of wells and a separate temperature control inlet and temperature control channel is provided for each row of wells of the array of wells. In this way, the temperature of the contents of the row of holes can be independently controlled from the temperature of the holes of the other rows.

The inflow opening on the bore wall may be located lower than the outflow opening. This enables microfluidics entering or exiting the well through the microfluidic channel to be used efficiently within the well. For example, a first opening (inlet) lower than a second opening (outlet) can maintain a desired fluid depth within the bore and can ensure that microfluidics effectively pass through the bore.

The biological microchip may further comprise a second microfluidic inlet, and a second inflow opening located on a wall of each of the wells. The second inflow opening may be in fluid communication with the second microfluidic inlet through a second microfluidic inflow channel. In this way, more complex experiments can be performed in the bore, for example by mixing different fluids from different inflow inlets in the bore. In one embodiment, different compounds may be provided to the same well, e.g., through different inflow openings located on different sides of the well. A gradient effect may also occur between different fluids/mixtures received in the well from the first microfluidic inlet and the second microfluidic inlet.

The second inflow opening on the bore wall may be located lower than the outflow opening on the bore wall. This ensures that the fluid received from the first inflow opening and the second inflow opening efficiently flows via the aperture to the outflow opening.

It is to be understood that a biological microfluidic chip according to embodiments of the present invention may have a well with a plurality of inflow openings and/or outflow openings, and a plurality of inlets and/or outlets. It is also to be understood that in some embodiments, the inlet and inflow openings may serve as the outlet and outflow openings, and vice versa.

An advantage of having more than one inflow opening and more than one outflow opening is that if one opening is blocked, fluid can still flow into the hole through the other openings.

The bio-microfluidic chip may further comprise a cap for sealing the hole. The lid may be slidable, self-sealing, removable, and/or heated. The cover may be constructed of plastic, rubber, silicone or other such polymer film and bonded to the glass by heat or adhesive. The lid may also be bonded to the chip via vacuum applied by dedicated microchannels within the chip. The cover is also used for controlling the pressure in the hole, protecting the upper opening of the hole from contacting foreign matters and avoiding fluid loss caused by evaporation; and/or the cover may open the upper opening of the well when it is desired to add certain objects (such as products, cells or cell clusters, other microfluidics) to the well. The heated cover may reduce the likelihood of condensate forming on the cover, thereby enabling more accurate imaging operations through the cover.

The biological microfluidic chip may be located within a scaffold.

When the lid is in place, each aperture is sealed relative to the other apertures. Thus, there is no risk of cross-contamination (e.g., bacteria or other pathogens, drugs, and other compounds) from well to well through the upper opening of the well. By means of the microfluidic channel, the risk of cross-contamination between two or more wells is reduced/prevented, since the channel is long and contains flowing fluid. Thus, the transfer of contaminants from one orifice to another would be against the direction of fluid flow.

It is to be understood that according to embodiments of the invention, a lid is not an essential feature of the biological microfluidics, and those examples described herein can operate in either an open mode (no lid in place) or a closed mode (i.e., a lid covering the upper opening of a well).

The biological microfluidic chip is reusable, e.g., for use in a large number of identical or different assays. In some examples, after the biochip is used, the biochip can be cleaned by flowing a wash solution through the microfluidic channels and wells. Cleaning the biochip may be an automated or semi-automated procedure to clean the biochip to a reproducible standard. The chip may also be sterilized with fluid, radiation, or ultraviolet light. Thus, the frequency of replacing the biochip according to the embodiment of the present invention is lower than that of the related art, and thus a more economical biochip can be provided.

The substrate may be made at least in part of D263 glass. This type of glass has been found to reduce autofluorescence compared to known polystyrene products, and can be used in at least a portion of a biochip through which imaging operations are performed.

Said plurality of holes having, in vertical cross-section, a shape comprising two truncated cones joined end to end; wherein the narrower end portions of the frusto-conical shape are configured together. The holes may have an "hourglass" shape with a circular or square horizontal cross-section. The frusto-conical shape may be frusto-conical or pyramidal, such as a square base pyramidal.

The substrate of the biochip may include a top layer and a bottom layer, and the two truncated cones are configured together at a boundary between the top layer and the bottom layer. Also, the first microfluidic channel and/or the second microfluidic channel may be disposed between a top layer and a bottom layer of the substrate. These features facilitate the fabrication of biochips.

One or more of the microfluidic channels may comprise a microfluidic valve. These microfluidic valves are used to control the flow of fluids into or out of the wells, depending on the requirements of the experiment to be performed in the wells. For example, a desired amount of fluid may be delivered to the wells at a desired time, depending on the particular experiment. The microfluidic valve may also be used to reduce the chance of cross-contamination between the contents of different wells.

According to another aspect of the present invention, there is provided a method of using the biological microfluidic chip, the biological microfluidic chip comprising:

a plurality of holes;

a microfluidic inlet; and

a microfluidic outlet;

the method comprises the following steps:

providing cells to one or more of a plurality of wells, wherein the cells are used in an experiment;

providing a fluid to the microfluidic inlet such that the fluid enters the one or more apertures;

removing the microfluidics from within the one or more wells through the microfluidics outlet; and

imaging the contents of the one or more wells to obtain experimental results within the one or more wells.

Examples of experiments may include:

experiments on zebrafish embryos/larvae, such as development of embryos/larvae over time; other embryos that can be used include embryos of other animals and plants;

in other embodiments, experiments may be performed on monolayers of cells, such as cardiac stem cells;

tissue and cells may be grown on a matrix or membrane within the pores to enable two-dimensional or three-dimensional growth of the cells and/or tissue.

The method may further comprise providing a temperature control fluid to a temperature control inlet of the bio-microfluidic chip to control the temperature of the contents of the one or more wells.

Providing cells to one or more of the plurality of wells may comprise injecting cells into an upper opening of the one or more wells, or introducing cells through the microfluidic channel and port.

Drawings

Preferred embodiments of the invention are described below, by way of non-limiting example, with reference to the accompanying drawings, in which:

FIG. 1 shows a biological microfluidics chip according to an embodiment of the invention;

FIG. 2 shows a vertical cross-sectional view of one well of a biological microfluidic chip according to an embodiment of the present invention;

FIG. 3 shows a biological microfluidic chip according to another embodiment of the present invention;

FIG. 4 shows further details of the biological microfluidics chip of FIG. 3;

FIG. 5 shows further details of the biological microfluidics chip of FIG. 3; and

FIG. 6 shows a biological microfluidic chip according to an embodiment of the invention in use;

FIG. 7 shows a biological microfluidic chip within a scaffold according to the invention.

Detailed Description

One or more embodiments described herein relate to a biological microfluidic chip having a plurality of wells/grooves, wherein each well is in fluid communication with a microfluidic inflow channel and a microfluidic outflow channel. In this way, fluid can be passed through the holes to remove any bacterial or biological waste that has accumulated over time. Likewise, drugs or nutrients may be delivered into the wells via the microbial channels for culturing and studying embryos, larvae and adults of multicellular organisms, single/multi-layered tissues/organs, cells or cell lines. Alternatively or additionally, a drug or other compound may be introduced into each well through the upper opening of the well.

In some embodiments, the microfluidic inflow channel between the microfluidic inlet and the opening into the well is substantially the same length for each well or a portion of the wells. This allows the pressure between the orifices to be equalized for each orifice or a portion of the orifices, and maintains the flow rate between the orifices. In some instances, this may advantageously reduce cross-contamination between the contents of different wells, and may provide a clean fluid for each well.

In other embodiments, the length of the microfluidic channel may be different so that the pressure between the inlet and the one or more orifices is equal and the flow rate therebetween is maintained. It is to be understood that the physical characteristics of the microfluidic inflow channel and the microfluidic inlet may be designed in any way to be able to provide a plurality of wells with fluids at the same pressure and at the same flow rate. Examples of microfluidic inflow channels and/or microfluidic inlet physical features include length, diameter, cross-sectional shape, and surface features that can affect fluid flow.

In some embodiments, microfluidic outflow channels and microfluidic outlets may be provided in a similar manner to microfluidic inflow channels and microfluidic inlets to remove fluid from one or more wells.

Fig. 1 shows a biological microfluidic chip (biochip) 100 according to an embodiment of the present invention.

The biochip 100 includes a substrate 102 having a microfluidic inlet 104 and a microfluidic outlet 106, with the inlet 104 and outlet 106 defining openings on a top surface 110 of the substrate 102. Microfluidic inlet 104 and microfluidic outlet 106 may serve as inlets for microfluidic channels to the external environment. A plurality of grooves/holes 108 extend downwardly from a top surface 110 of the substrate 102.

In the embodiment shown in FIG. 1, only two apertures 108a and 108b are shown in order to clearly illustrate the features of aperture 108. It is understood that in actual practice, the biochip 100 may comprise 32 wells, 96 wells, 869 wells, or any desired number of wells. An advantage of embodiments of the biochip 100 is that a large number of wells and their associated microfluidic channels can be provided in a small area. For example, a biochip according to an embodiment of the present invention may accommodate 869 wells, whereas a conventional microtiter plate may accommodate only 96 wells in the same area.

Each aperture 108 has a first opening 112 into a sidewall of the aperture 108 and a second opening 114 into an opposing sidewall. The first opening 112 is an example of an inflow opening (let opening), and the second opening 114 is an example of an outflow opening (outlet opening). In this example, the aperture 108 has a square horizontal cross-section. In other embodiments, apertures 108 having various cross-sectional shapes may be used, and the sidewalls may take the form of any configuration of side, such as flat/planar or curved.

A microfluidic inflow channel/conduit 116 connects the microfluidic inlet 104 to each inflow opening 112 within the bore 108. Likewise, a microfluidic outflow channel 118 connects the microfluidic outlet 106 to each outflow opening 114 on the sidewall of the hole 108.

In this example, the outflow opening 114 in the sidewall of the hole 108 is higher than the first opening 112, i.e., the outflow opening 114 is closer to the upper surface from which the hole 108 extends downward. This configuration of inflow openings 112 and outflow openings 114 may advantageously force liquid from inflow openings 112 into apertures 108 and then out of apertures 108 through outflow openings 114 without stagnating within apertures 108. The configuration of openings 112 and 114 may result in efficient and economical fluid throughput through apertures 108.

FIG. 2 shows a cross-sectional side view of a well 202 of a biochip 200 according to an embodiment of the invention.

The biochip 200 comprises a first/top layer 204 of substrate, a second/middle layer 206 of substrate and a third/bottom layer 218 of substrate. As will be appreciated from the description below, fabricating the substrate as three layers may facilitate the microfluidic channels to be located within the body of the substrate, and between layers.

The hole 202 in this example has a circular horizontal cross section when the hole 202 is viewed from above. The structure shown in fig. 2 is equally applicable to holes 202 having different cross-sectional shapes. The hole 202 shown in fig. 2 can be considered to be "hour glass" shaped in vertical cross-section.

The upper portion of the hole 202 (i.e., the portion of the hole 202 that passes through the upper layer 204) extends downward from the upper surface 214 of the first layer 204 of the substrate. In this example, the upper portion of the hole 202 is frustoconical, and wherein the taper associated with the upper portion of the hole points downward, i.e., away from the upper surface 214 of the first layer 204 of the substrate.

The hole 202 has a bottom that extends into the second layer 206 of the substrate. The horizontal cross-section of the lower portion of the bore 202 is also circular. In this example, the bottom of the hole 202 is also frustoconical in shape, but this time the cone associated with the frustoconical shape of the bottom of the hole 202 points upwards, i.e. in a direction towards the top surface 214 of the first layer 204 of the substrate.

The horizontal cross-section of the hole 202 at the boundary between the first layer 204 and the second layer 206 of the substrate is substantially the same for both layers 204 and 206, resulting in a continuous hole 202. It will be appreciated that the two pointed-to-pointed frustoconical portions of the bore 202 are "hourglass" shaped in vertical cross-section when the bore 202 is taken as a whole.

A hole 202 having an hourglass shape may facilitate imaging the contents of the hole 202. For example, a well 202 having this shape enables observation/analysis/measurement of the contents of the well 202 from the top or bottom of the biochip 200 without looking through unnecessary areas 204 and 206 in the substrate. Imaging is typically performed using a microscope and the microfluidic chip may be made of a glass layer for microscope grade. The boundaries between first layer 204, second layer 206, and third layer 218 do not affect imaging of the pore contents. In other embodiments, the aperture walls may be flat/planar such that the apertures have a constant cross-sectional shape and size along their length.

The bore 202 has a first opening 208 in fluid communication with the microfluidic channel 207. In this example, the microfluidic channel 207 is an inflow channel of fluid. The opening 208 is adjacent a lower surface 216 of the bore 202.

Further, a second opening 210 is provided on the sidewall of the hole 202 between the first layer 204 and the second layer 206 of the substrate. The second opening 210 is in fluid communication with a second microfluidic channel 212. In this example, the second microfluidic channel 212 is a microfluidic outflow channel. In use, embryos or larvae (such as zebrafish embryos or larvae) may be located within the bore 202; nutrients, drugs, or other compounds may be pumped into the well 202 from the microfluidic inflow channel 207 and the first opening 208, or these substances may be introduced from the upper opening of the well by a pipette.

One or more fluids may be removed from within the well 202 through a second opening 210 in the sidewall of the well 202, wherein the second opening 210 opens into a microfluidic outflow channel 212. It is to be understood that the fluid extracted from the well 202 may include any waste that may form within the well 202 over time, as well as products produced by embryos/larvae and any bacteria or other pathogens, or biological waste, or drugs or other compounds, or sloughed tissue or matrix.

FIG. 3 shows still another embodiment of a biochip according to an embodiment of the present invention.

The biochip 300 of fig. 3 has an array of wells/grooves 302. In this example, the array is a 4 x 8 array of 32 wells. Further, at one end of the biochip 300, 8 inlet/outlet ports 304 are arranged in a 4 × 2 array. It is to be understood that the ports and apertures may be in other configurations. The microfluidic channels extending from inlet/outlet 304 are not shown in fig. 3 to clarify the figure. While the fluid passages extending from the inlet/outlet 304 and their relationship to the bore 302 are shown in greater detail in figures 4 and 5. It is to be understood that the microfluidic channels shown in fig. 4 and 5, respectively, are actually present simultaneously within the biochip 300 shown in fig. 3, but are shown separately for clarity. In this example, the cross-section of the hole is square when viewed from above, but other shapes are possible.

Fig. 4 shows the biochip 300 of fig. 3, an associated microfluidic inflow channel extending from a microfluidic inlet 410 to the well 302, and a microfluidic outflow channel extending from a microfluidic outlet 412 to the well 302.

It is to be understood that microfluidic inlet 410 is adapted to be connected to any source of microfluidic fluid, such as a pharmaceutical or nutrient medium to be provided to well 302. As shown in fig. 4, microfluidic channels 414 extend in parallel from microfluidic inlet 410 to each row of holes 302. In this example, the wells are arranged in a 4 x 8 array, so four microfluidic channel branches 414 (one for each row) extend from the inlet 410.

Each microfluidic channel 414 for a given row of wells 302 is further branched to be in fluid communication with a first opening 418 within each well 302. Also, through this branching, it can be seen that microfluidic channels 414 provide fluid to each well 302 in parallel.

In this example, the microfluidic channel 414 does not extend directly from the inlet 410 to each first opening 418 within the well 302, but rather the channel 414 is configured such that the channel length between the microfluidic inlet 410 and each first opening 418 is substantially the same for each well in order to provide fluids for each well that maintain similar fluid flow rates. This may be accomplished by providing different channel lengths between the main channel of the microfluidic channel 414 and the first opening 418 in different wells 302. For example, the microfluidic channel may extend along a path that doubles back to the origin several times to provide the desired overall channel length between the inlet 410 and the first opening 418, as indicated by reference numeral 416 in fig. 4. It will be appreciated that for wells 302 that are farther from the inlet 410, the path length between the main channel of the microfluidic channel 414 and the first opening 418 should be short, such that the entire channel length provided between the inlet 410 and the first opening 418 provides a substantially uniform fluid flow rate to each well. This means that the resistance to the fluid is approximately the same for each orifice regardless of the distance from the port at which the orifice is supplied, and therefore the fluid can be supplied evenly to each orifice.

The microfluidic channels 414 and 416 so disposed enable the physical characteristics experienced by the fluid in its path to the wells 302 to be the same for each well 302. This provides each well 302 with fluid at the same pressure, thereby providing fluid at the same flow rate, and thus reducing the likelihood that the contents of the wells 302 will be forced back into the microfluidic inflow channels 414 and 416. This, in turn, can reduce the likelihood of cross-contamination between the contents of the individual wells 302.

The same structure is applied to microfluidic outflow channels 424 and 422, where microfluidic outflow channels 424 and 422 connect microfluidic outlet 412 to second opening 420 within bore 302.

Fig. 5 shows ports and channels for controlling the temperature within the wells 302 of the biochip 300. In this example, the cross-section of the hole 302 is square, as viewed from above, but may be other shapes.

In this example, there are four temperature control inlets 510a, 510b, 510c, 510 d: each row of the array of holes 302 has one temperature control inlet. In this way, the temperature of each row of holes 302 may be controlled individually. The biochip 300 comprises a single temperature controlled outlet 512. Other possible configurations include a single temperature control inlet and a single temperature control outlet, in which embodiment the entire biochip can be maintained at a uniform temperature.

A temperature control channel 506 extends from each temperature control inlet 510 to convey temperature control liquid from the inlet 510 to an outlet 512 along a path adjacent to the row of holes 302 associated with the temperature inlet 510. It is to be understood that "adjacent" means that the temperature control channel 506 is sufficiently close to the aperture 302 such that heat can be exchanged between the contents of the aperture 304 and the temperature control fluid within the temperature control channel 506. Heat may be exchanged into or out of the temperature control fluid to cool or heat the contents of the bore 302.

In this example, the temperature control channel 506 extends along a path adjacent three of the four sides of the horizontal square cross-section of the hole 302 to uniformly heat or cool the contents of the hole. It is to be understood that temperature control channel 506 may take any path associated with well 302 so long as heat may be exchanged between the temperature control fluid within temperature control channel 506 and the contents of well 302. In examples where the bore 302 has a circular horizontal cross-section, the temperature control passage may extend along part or almost all of the circumference of the circular bore wall.

The temperature control channels 506 of each row of holes meet together after passing through the holes to form a common temperature control return channel 508, wherein the temperature control return channel 508 is in fluid communication with a temperature control outlet 512.

It is known that prior art products use an external heating module to maintain the wells in the biochip at a stable experimental temperature. Thus, in prior art systems, the microtiter plate must always be fixed to the heating module, so that the microtiter plate cannot be moved during use. Thus, if a uniform temperature is required for each well, prior art microtiter plates are not readily available for high throughput screening in automated robotic processing systems. In contrast, the embodiments of biochips described in the present invention have built-in heating/cooling channels and therefore do not need to be fixed on a thermostatic module, and can be freely moved in a robotic system. This provides a biochip that is more flexible in use than prior art microtiter plates.

The embodiments described herein can be used to test zebrafish embryos/larvae in wells and to monitor the development of the embryos/larvae over time, such as untreated or treated with drugs or other compounds. In other embodiments, the assay may be performed on a monolayer of cells, membranes, matrices (matrices) or other substrates (substrates) within the wells.

In this example, the biochip uses glass and/or (fused) quartz as raw material. This starting material can significantly reduce autofluorescence compared to known polystyrene for 96-well microtiter plates. Glass is considered a relatively inexpensive material, but a mixture of glass and polystyrene is relatively expensive because of the coating techniques required. Further, glass is better than plastic in terms of abrasion resistance and resistance to repeated cleaning cycles.

In this example, the surface of the biochip is glass, which enables refocusing of the sample to be imaged in 0.1 seconds. In contrast, known biochips having a polystyrene surface can suffer from drift (drift) due to the rough surface of the biochip and can cause variations in the infrared wavelength as part of the imaging operation. This means that refocusing the prior art biochip requires more than 1 second per well, which greatly increases the time required to perform large batch embryo screening.

FIG. 6 shows a vertical cross-sectional view of a well 602 of a biochip 300 according to an embodiment of the invention in use; in which zebrafish embryos 604 are being tested.

In this example, the embryo 604 is surrounded by its chorion 606 and embedded within a low-melting agarose 608 (injected into the well by the robotic processor) to prevent the embryo from moving within the well. Agarose 608 solidifies into a gel, but does not damage the sample or prevent gas/nutrient exchange, and agarose 608 facilitates injection of the test drug through the extraembryonic membrane (chorion 606). Gel 608 may also limit the spread of potential infection. Each well 602 in the biochip 600 is supplied with a stable or constant defined buffer solution through the parallel extending microfluidic inflow channel 610, thereby reducing the risk of microbial cross-contamination and drug leakage to adjacent wells. In addition, the drug can be administered by a robotic pipettor through a plastic or glass sliding cover, wherein the sliding cover can be retracted to expose the opening of the well for injection of the drug. Alternatively, the cap may be a self-sealing cap, such as a rubber or polymer plug, a laminated film or tape. It is to be understood that agarose 608 is not essential, and in other embodiments, embryos may be freely within the fluid or any other substance in the well.

Some embodiments of the biochips described herein can include a cover for covering the opening of the well on the top surface of the biochip substrate. The lid may be a sliding lid integrated as part of the biochip. In this way, the lid can be slid to one side to an open position to expose an opening of the aperture for introduction of embryos prior to the start of the experiment, and/or for introduction of drugs during the experiment. During the experiment, the lid can be slid back to the covering position.

In some examples, the lid can seal the aperture such that the aperture is air and/or fluid tight, thereby enabling efficient microfluidic flow within the aperture. The removable lid enables recovery of the embryo after the experiment for further detailed analysis, such as Polymerase Chain Reaction (PCR), extraction of messenger ribonucleic acid (mRNA), etc., after the experiment within the biochip is completed. The removal of the cover facilitates the washing of the wells after the experiment, thereby making the biochip reusable.

In some embodiments, the lid may be heated to reduce the likelihood of condensate forming on the lid. The reduction in condensation allows for more accurate imaging operations through the cover in use. Alternatively, the lid may be composed of a plastic film or a porous film; wherein the plastic film is applied under heat and the perforations in the porous membrane are aligned with the upper opening of the well and with a glass cover placed over the porous membrane. When the cap is a film, the cap may be self-sealing after the needle has been passed through the cap access hole. In some cases, the lid is optically clear/transparent, enabling microscopic analysis of the contents of the well; in other cases, the cover may have a mirror-like upper or lower surface.

FIG. 7 shows a vertical cross-sectional view of a well 202 when a biochip 200 (see FIG. 2) according to an embodiment of the invention is placed in a holder according to the invention. The support comprises several layers of material, which when connected together by screws, can hold the biochip in a specific position. The top cover 1 is made of metal/plastic or other material. The plate 2 is a glass plate or a polymer seal and may be mounted on the top sealing layer 3. The top sealing layer 3 is made of, for example, silicone. Layer 4 is a bottom (silicone) seal. The layer 5 constitutes a bottom cover and may be made of metal and/or plastic or other material. The angle between the dashed lines 6 represents the bottom field of view for imaging. The dashed line 7 indicates the position of the hole in the layer 3 for imaging. The angle between the dashed lines 8 represents the top field of view for imaging. The stent of the present invention is not limited to this particular configuration.

Now, a detailed description will be made of a specific implementation process of the bio-microfluidic chip according to an embodiment of the present invention. Embodiments of the bio-microfluidic chip can be made of D263 glass, since it was found that D263 glass reduces autofluorescence compared to known polystyrene products.

The cross section of the inner hole of the biochip is 2mm²-4mm²This is in contrast to known 96-well microtiter plates, since the cross-sectional area of the wells of a 96-well microtiter plate is significantly larger, i.e. 33.18mm²(surface area (. pi.r)²) r is 3.25 mm; h 10 mm). Thus, embodiments of the biochip can reduce automatic "discovery" when used within an automated system&Time of the mark ".

In this example of the biochip, the capacity of a single well was 8mm³This is in contrast to known 96-well plates, of which the capacity of a single well is approximately 250mm (2 mm. times.2 mm)³-331mm³(33.18mm²(7.5mm or 10 mm)). Thus, embodiments of the biochip can reduce the cost of using compounds by an estimated 31% -41% (250/8-331/8).

In this example, the biochip is capable of accommodating 869 wells, whereas a conventional microtiter plate only accommodates 96 wells per plate in the same area. This is because the surface area of the 96-well plate (total area: 7823 mm)²(ii) a Width × depth: 72.3mm x 108.2mm) is approximately 0.012 holes/mm². In contrast, biochips according to embodiments of the invention can accommodate ≧ 0.11 wells/mm²Wherein the surface area of each well plus its associated microfluidic channel is 3mm x 3 mm.

The embodiments described herein can be used to perform experiments within the wells of a biological microfluidics chip. An experimental study object, such as an embryo, may be added into the upper opening of the substrate defining the aperture. Alternatively, the upper opening of the substrate may be covered with the above-described cover.

The described embodiments of the invention may secure an embryo or other subject within a well, which subject is then processed with one or more fluids received from a microfluidic channel; wherein the microfluidic channel has an opening to the wall of the well. As one example, the fluid may provide nutrients to the subject. And the subject is not moved into or out of the well via the microfluidic channel.

The wells in one or more of the biological microfluidic chips described herein may be considered as holding chambers for the growth of the subject and may also be considered as being associated with long-term culture experiments/systems. Wherein "long-term" may be several days, such as five days.

Biological microfluidics chips are considered to be a different technical field than microfluidic worm sorters; among them, the micro-fluid worm sorter traps worms in a sealed chamber by suction. Such microfluidic sorters may require different technical requirements than the biological microfluidic chip with wells described in the present invention.

Claims (20)

1. A biological microfluidic chip, comprising:

a substrate;

a microfluidic inlet defining an opening on the substrate surface;

a microfluidic outlet defining an opening on the substrate surface; and

a plurality of apertures extending from the top surface of the substrate, wherein a boundary of each aperture is defined by one or more walls, an inflow opening and an outflow opening being disposed on the walls of each aperture;

one or more microfluidic inflow channels within the substrate connecting the microfluidic inlet to each inflow opening on a wall of the well; and

one or more microfluidic outflow channels within the substrate, the microfluidic outflow channels connecting each outflow opening on the pore wall to the microfluidic outlet.

2. The biological microfluidics chip of claim 1, wherein, for each well, the length of the microfluidic channel between the microfluidic inlet and the inflow opening in the wall of the well is substantially equal.

3. The biological microfluidics chip of claim 1 or claim 2, further comprising:

a temperature control inlet and a temperature control outlet; and

a temperature control channel for transporting a temperature control fluid from the temperature control inlet to the temperature control outlet along a path proximate to one or more of the plurality of wells when the biological microfluidic chip is in use, such that heat can be exchanged between the temperature control fluid and the contents of the wells.

4. The biological microfluidics chip of claim 3, wherein one or more of the temperature control inlet, temperature control outlet, and temperature control channel are provided for a portion of the plurality of wells.

5. The biological microfluidics chip of claim 3 or claim 4, wherein the plurality of wells comprises an array of wells, and a separate temperature control inlet and temperature control channel are provided for each row of wells in the array of wells.

6. The biological microfluidics chip of any preceding claim, wherein the inflow opening on each pore wall is located lower than the outflow opening.

7. The biological microfluidics chip of any one of the preceding claims, further comprising:

a second microfluidic inlet; and

a second inflow opening located on the bore wall;

wherein the second inflow opening is in fluid communication with the second microfluidic inlet via a second microfluidic inflow channel.

8. The biological microfluidics chip of claim 7, wherein the second inflow opening in the pore wall is located lower than the outflow opening in the pore wall.

9. The biological microfluidics chip of any one of the preceding claims, further comprising a lid; wherein the lid is sliding, self-sealing, removable and/or heated.

10. The biological microfluidics chip of any preceding claim, wherein the substrate is at least partially D263 glass.

11. The biological microfluidics chip of any one of the preceding claims, wherein the plurality of wells comprise, in vertical cross-section, a shape comprising two truncated cones joined end-to-end; wherein the narrower end portions of the frusto-conical shape are configured together.

12. The biological microfluidics chip of claim 11, wherein the substrate comprises a top layer and a bottom layer, and the two frustoconical shapes are configured together at a boundary between the top layer and the bottom layer.

13. The biological microfluidic chip of claim 12, wherein a first microfluidic channel and/or said second microfluidic channel is disposed between a top layer and a bottom layer of said substrate.

14. The biological microfluidics chip of any preceding claim, wherein one or more of the microfluidic channels comprise a microfluidic valve.

15. A method of using a biological microfluidics chip according to one or more of claims 1-14, wherein the method comprises:

providing cells, embryos or larvae to one or more of the plurality of wells, wherein the cells, embryos or larvae are used in an experiment;

providing a fluid to the microfluidic inlet such that the fluid enters the one or more apertures;

removing fluid from within the one or more wells through the microfluidic outlet; and

imaging the contents of the one or more wells to obtain experimental results within the one or more wells.

16. The method of claim 15, further comprising the steps of:

culturing the cell, embryo or larva in one or more of the plurality of wells;

the cells, embryos or larvae are selectively treated with drugs or other compounds.

17. The method of claims 15-16, wherein the cells are stem cells.

18. The method of claims 15-17, wherein the assay is used for human, animal, microbial or plant research or screening.

19. The method according to claims 15-17, characterized in that a photosynthetic body, such as a plant cell, a chloroplast, is cultivated in said plurality of wells.

20. The method of any one of claims 15-17, further comprising providing a temperature control fluid to a temperature control inlet of the biological microfluidic chip to control the temperature of the contents of the one or more wells.