Technical Field
The present application relates to a centrifugal capture system for use in capture of particles such as beads or cells, and more particularly relates to a system comprising a rotatable capture chamber comprising a plurality of capture sites. The capture sites are geometrically dimensioned to receive one or more particles which are biased to the capture sites through a rotation of the capture chamber.
Description of the Related Art
Microfluidic systems for capturing and manipulating small numbers of cells or even single cells are a field of growing interest. Applications include single cell culture and treatment for drug screening and cell fusion. Existing techniques use pressure driven systems in which the geometrical capture structures themselves lead to an induced non-axial component of the flow field leading to a significant decrease in capturing efficiency. There is therefore a need to provide an improved system.
These and other problems are addressed in accordance with the present teaching by a centrifugal capture system that is operable under stagnant flow conditions. In a first configuration the system comprises a capture chamber comprising a plurality of capture sites defined therein. The system is configured such that a fluid comprising particles of interest may be introduced into the capture chamber. In a first configuration, the system is operable under stagnant flow conditions such that while particles are being biased towards individual capture sites. This means that there is no flux of the fluid within the capture chamber during a rotation of the capture chamber. In another configuration, a flow within the chamber may be present, albeit exerting less impact on the particle trajectories than the effect of the centrifugal force that is biasing the particles towards the individual capture sites such that a combination of sedimentation and slow flow is also possible. A rotation of the capture chamber induces a centrifugal force which induces motion onto the particles such that they are biased in straight lines in a radial direction away from the axis of rotation of the chamber and are sedimented into the capture sites. It will be appreciated that the particles experience a force related to the Stokes drag which also affects their capture within the individual capture sites. A plurality of capture sites may be provided within the chamber, the capture sites being provided at different distances away from the axis of rotation. In a first configuration the capture sites are provided in an array of individual rows, each row being a defined distance from the axis of rotation. Individual rows may be staggered relative to one another.
The present application will now be described with reference to the accompanying drawings in which:
The present teaching will now be described with reference to exemplary arrangements which will assist the person of skill in an understanding of the benefits and features of system incorporating the present teaching. By providing a system and methodology in accordance with the present teaching it is possible to provide a simple and highly efficient way to capture (typically micron-sized) particles, e.g. beads or biological cells or a combination of the two, on a centrifugal microfluidic platform in geometrical traps under stagnant or throttled flow conditions. In the exemplary arrangements which are described herein the dimensions of the capture sites are scale-matched with the dimensions of the particles being captured. The number of particles (between one and multiple) per capturing site can be set by the geometry and spatial alignment of the capturing elements. Scale matching between the capturing elements and the particles allows the capture down to one single bead or cell per site. The present teaching thus enables the investigation of single or small numbers of particles in an array format. High capture efficiencies as well as the capability to isolate defined numbers of particles down to a single-particle level are enabled by the interplay of stagnant or throttled flow conditions with micron-scale capture sites. In the same centrifugal setup, the captured particles may be exposed to a sequence of other liquids such as culture medium, wash buffers and drugs. The particles may also be exposed to other force fields which known from the state-of-the-art to have an effect on the particles, e.g. dielectrophoretic or magnetic or ultrasound. Also biochemical assays, particle counting and analysis/identification may be performed on the captured particles.
The capture chamber is defined within or provided on a rotatable substrate (121—see
As shown in
In a third step shown in
As shown in
Another variant to the cup-like capturing elements is a capture site defined by a small depression extending perpendicular to the centrifugal field and parallel to gravity.
Each capturing element or capture site is designed such that it can retain a defined number of particles, at least one. Certain configurations may be dimensioned to allow not more than one particle to occupy the capture site. Once a capturing element is filled to maximum capacity, subsequently arriving particles will not be captured but propagate to the next capturing element (
Initial experiments showed that >90% of all initially present particles can be captured with this system. Also capture elements designed to capture different numbers of particles can be aligned in the same array. It may also be possible to implement filtering on a polydisperse suspension of particles by size exclusion from small capture elements. The array itself can be of square, rectangular or any other shape including a spatially varying grid distance. In a fourth step, the captured particles can be examined or the environmental conditions can be influenced by changing the liquid in the camber or adding substances such as culture medium, wash, staining or elution buffers, and drugs, or combinations thereof.
Therefore it will be appreciated that in accordance with the present teaching that it is possible to easily split an initial sample consisting of a multitude of particles into spatially separated groups of particles, each consisting of a defined number of particles. A special application of this invention is the study of single cell behavior. In this case instead of particles, biological cells are used. Cells can be investigated by common means, such as optical instrumentation, e.g. microscopy, or by external or integrated sensors, e.g. based on impedance measurements. The grid or array can also be used to study inter-cell communication. Also a sequence or cell suspensions might be introduced to the array, e.g. to study the interaction of different cell types or between treated and untreated cells in a single capture element.
It will be appreciated that the present teaching employs a unique combination of centrifugal sedimentation, stagnant flow conditions and precisely fabricated microstructures. Microfabrication enables scale-matching such that defined numbers of (monodisperse) particles can be captured in each element. Centrifugal action allows propelling the particles under stagnant flow. The stagnant flow itself avoids that, as prescribed by the continuity of flow lines, non-radial velocity components arise in the vicinity of the capture elements which tend to carry the particles away. Nevertheless, the structure may also be operated in flow mode, e.g. during capture or exposure to fluids once captured. If desired, release of the cells may be enabled by gravitational or centrifugal sedimentation in the opposite direction, e.g. by orienting the chip correspondingly.
In modifications to that described heretofore, the present teaching advantageously provides for a varying of lateral spacing between capture sites within the same line, a varying of the number of capturing elements in different lines and/or vary the spacing between capturing lines.
In some setups it might be advantageous to vary the shape of the capturing elements. While it is not intended to limit the present teaching to any one specific geometrical configuration some possible shapes are shown in FIG. 6, which shows a V-shaped capture site (601), a cup-shaped capture site (602) and a collar or torc shaped capture site (603). It will be appreciated that each of these capture sites defines a capture area (604) that is open towards the proximal end of the chamber. One chamber can contain either only one type of capturing element or a multitude of different capturing elements.
In some setups it might be advantageous to structure the capturing elements such that liquid can flow through the capturing elements by introducing sieving elements such as pores, slits, holes or the like within capturing structures and/or by creating a slit above or below the capturing element.
Another arrangement shown in
It may be desirable to concentrate the passage of the particles, e.g. through a mid-region of the chamber. This may be provided by providing baffles or guides—generically termed biasing means—to preferentially direct particles away from the side walls and towards that mid region. This may be provided to effect a lateral distribution of beads in a homogenous fashion across the chamber. In addition are as an alternative to the physical baffles, agitation in the inlet and the free sedimentation path prior to the capture region may be used to induce to a more homogeneous or more focused distribution of incoming particles prior to their exposure to individual capture sites.
The invention described above can easily be integrated in a more complex setup, where the inlet(s) of the capturing chamber is connected to one or more upstream structures, that perform for example tasks such as sample preparation. The outlet(s) of the chamber can for example be connected to a structure that performs a detection of certain biological markers, e.g. secreted from (stimulated) biological cells or eluted off the captured beads. Another variant to change between stagnant flow mode during capture and flow mode to expose the captured particle to a sequence of reagents is to close the chamber with a valving element, e.g. sacrificial valves opening upon exposure to radiation or heat. The valving might also be implemented by frequency-controlled valves such as a siphon primed by capillary action or overflow. Of course, other up and downstream process steps well documented in the literature of lab-on-a-chip or centrifugal “lab-on-a-disk” technologies can be imagined easily.
As was discussed above a system (100) in accordance with the present teaching may be integrated in a disk shaped substrate (121), an example being shown in
Such a disk may be considered as being similar to a compact disk having an aperture 801 for receiving the disk 121 onto the spindle 810 of a rotatable drive 820 which comprises a motor. The aperture 801 defines the axis of rotation of the disk. In the exemplary arrangement of
While the disk of
It will be appreciated that a system in accordance with the present teaching may comprises a disk substrate with a plurality of capture sites arranged radially within a capture chamber. In contrast to pressure driven systems, the cells are sedimented under stagnant, i.e. stopped flow conditions into the retention structures. During their entire approach the cells thus follow straight (radial) paths, implying a 100% theoretical capture efficiency in an interlaced capture array, such as that shown in
Experiments with 10-μm silica beads at a rotation frequency of 20 Hz have been performed with a characteristic size of the V-cups of 35 μm. Using a combination of SU-8 lithography and subsequent casting into PDMS it was possible to define individual capture sites within a capture chamber. By suitably dimensioning the individual capture sites, each capture site can only hold a certain, predefined maximum, number of beads and excess will beads propagate to the next capturing line within the array. The time dependent bead propagation through the capture lines is shown in the data in
Using the present teaching it is possible to provide a high level of control of the mean particle occupancy in arrays of scale-matched capture sites using centrifugal sedimentation. The induced centrifugal force may be combined ultrasound of other agitation of the particles. If the ultrasound is applied at the beginning of the capture regime through a sequence of intermittent bursts it is possible to reduce the mean particle distribution to a single occupancy and also to narrow the distribution width. By applying an ultrasound signal post capture, it is possible to allow for loosening of particle aggregates for a release of the trapped particles from the array. Once captured individual particles may be treated, stained and/or otherwise analyzed in situ while resting within the capture sites.
The sequence of images provided in
Once captured, the individual particles may be treated or otherwise analyzed. This ability to distribute, treat and eventually retrieve an ensemble of particles is of particular interest to systems biology. To demonstrate this,
It will be appreciated that heretofore has been described exemplary arrangement of particle capture system that can be used in the context of a lab-on-a-chip platform for particle- and cell-based assays. By varying the ratio between the sizes of particles to be captured and the dimensions of the capture sites selected for that capture, it is possible to impact the mean value as well as the width of the occupancy distribution. In this way by choosing appropriate operational parameters (e.g. size, geometry and spacing of capture sites, centrifugal frequency, overall number of particles applied, concentration of particles applied, (interspersed) agitation), the capture efficiency may be modified. For example it is possible to shape the captured particle distribution and shift its peak, even so far that it goes to 0, i.e. that all initially centrifugally captured beads may be retrieved.
By alternating periods of sedimentation and agitation through for example exposure to ultrasound, a strong bias towards single occupancy could be induced, the distribution width could be narrowed and the particles could eventually be retrieved from the array. In addition, cells could be captured, treated and stained, allowing study of individual or defined numbers of cells aligned in an array in systems biology. While exemplary geometrical configurations and methodologies have been described it will be appreciated that modifications can be made to that hereinbefore described without departing from the teaching of the present disclosure.
The capture chamber described herein is desirably a microfabricated or microengineered chamber with the capture areas having dimensions that are scale matched with the particles being captured. Within the present specification, the term microengineered or microengineering or micro-fabricated or microfabrication is intended to define the fabrication of three dimensional structures and devices with dimensions in the order of millimeters or a sub-millimeter scale.
The various embodiments described above can be combined to provide further embodiments. All of the commonly assigned US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including but not limited to U.S. patent application Ser. No. 12/855,579, filed Aug. 12, 2010 are incorporated herein by reference, in their entirety.
The words comprises/comprising when used in this specification are to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
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Number | Date | Country | |
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20150018192 A1 | Jan 2015 | US |
Number | Date | Country | |
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Parent | 12855579 | Aug 2010 | US |
Child | 14335683 | US |