Patent Application
20060020371
The present disclosure relates generally to methods and apparatus for manipulating, detecting, imaging, and/or identifying particles or objects via electromagnetic fields. In various examples, integrated microsystem methods and apparatus are disclosed, involving electric and/or magnetic field-generating devices fabricated using conventional semiconductor techniques (e.g., Si, SiGe, CMOS, GaAs, InP) and configured to direct, sense, image, and/or identify particles or objects of interest via electric and/or magnetic field interactions. In some examples, such field-generating devices are integrated together with a microfluidic system to further facilitate movement, sensing, imaging and/or identification of particles or objects of interest.
In biological and medical sciences, it is often useful to be able to manipulate (e.g., move or direct) a biological sample (e.g., one or more cells) along a prescribed path. Manipulation of biological systems based on magnetic fields is one conventionally used method to accomplish this task. In one conventional implementation involving magnetic fields, a small magnetic bead with a chemically modified surface can be coupled to a target biological system, such as a particular cell or microorganism. Depending on the type of coating of a given bead, and the relative sizes of the bead and the target cell or microorganism, the bead may be bound to the surface of the cell or organism (exterior coupling), or ingested by the cell or organism (interior coupling). Such a “bead-bound” sample then may be suspended in a host liquid to constitute a “microfluid,” and the suspended sample in the microfluid can then be manipulated using an external magnetic field. Devices based on this principle often are referred to as “magnetic tweezers” and have been conventionally used, for example, to trap small particles (e.g., DNA) suspended in a liquid for study.
Because magnetic fields and the magnetic beads themselves are typically biocompatible, this process is non-invasive and generally not damaging to the sample. However, conventional magnetic tweezers fail to provide individual control of multiple magnetic beads because these devices typically produce only a single field peak that may be moved; thus only a single bead or, simultaneously, a group of beads in close proximity, may be conventionally controlled within a microfluid.
Another area related to the movement and manipulation of biological samples, particles, or other objects suspended in liquid involves a phenomenon referred to as “dielectrophoresis.” Dielectrophoresis occurs when an inhomogeneous electric field induces a dipole on a particle suspended in liquid. The subsequent force on the dipole pulls the particle to either a minimum or a maximum of the electric field. Almost any particle, without any special preparation, can be trapped or moved using dielectrophoresis when it is exposed to the proper local electric field. This is an advantage of electric field-based operation over the magnetic field-based manipulation described above, as the latter mandates marking biosamples or other objects of interest with magnetic beads. However, a potential disadvantage of the dielectrophoresis is that a relatively strong electric field may damage the cell, particle or other object of interest in some circumstances.
Yet another area related to the movement and manipulation of biological samples that enables various applications in medical diagnostics and life sciences is referred to as “microfluidics.” Microfluidics is directed to the containment and/or flow of small biological samples by providing a micro-scale biocompatible environment that supports and maintains physiological homeostasis for cells and tissues. Microfluidic systems may be configured as relatively simple chambers or reservoirs (“bathtubs”) for holding liquids containing cells/biological samples of interest; alternatively, such systems may have more complex arrangements including multiple conduits or channels in which cells, particles, or other objects of interest may flow. By controlling the flow of fluids in micro-scale channels, a small quantity of samples can be guided in desired pathways within a microfluidic system. Integration of various microfluidic devices, such as valves, filters, mixers, and dispensers, with microfluidic channels in a more complex microfluidic system facilitates sophisticated biological analysis on a micro-scale. Fabrication of even some complex conventional microfluidic systems generally is considered to be cost-effective, owing to soft-lithography techniques that allow many replications for batch fabrication.
Once fabricated, however, conventional microfluidic systems (especially more complex systems) do not offer an appreciable degree of flexibility, and specifically suffer from insufficient programmability and controllability. In particular, conventional microfluidic systems that are used for analytic operations such as cell sorting are manufactured to have a specific number and arrangement of fixed channels and valves. Operation of the valves controls the flow of cells into the channels, thereby sorting them. Function of the system generally is based on a statistical approach of differentiating amongst relatively larger numbers of cells, and not sorting one cell at a time. Because the arrangement of channels and valves is determined during fabrication of the microfluidic system, each system is designed for a specific operation and typically cannot be used in a different process without modifying its basic structure.
Integrated circuit (IC) technology is one of the most significant enabling technologies of the last century. IC technology is based on the use of a variety of semiconductor materials (e.g., Silicon Si, Silicon Germanium SiGe, Gallium Arsenide GaAs, Indium Phosphide InP, etc.) to implement a wide variety of electronic components and circuits. Perhaps one of the most prevalent examples of IC technology is CMOS (Complimentary-Metal-Oxide-Semiconductor) technology, with which silicon integrated circuits are fabricated.
CMOS technology is what made possible advanced computation and communication applications that are now a routine part of everyday life, such as personal computers, cellular telephones, and wireless networks, to name a few. The growth of the computer and communication industry has significantly relied upon continuing advances in the electronic and related arts in connection with reduced size and increased speed of silicon integrated circuits, whose trend is often quantified by Moore's law. Currently, silicon CMOS chips can contain over 100 million transistors and operate at multi-gigahertz (GHz) speeds with structures as small as 90 nanometers. CMOS microfabrication technology has matured significantly over the last decades, making silicon integrated circuits very inexpensive. Despite several advantages, however, neither CMOS nor any other semiconductor-based IC technology has been widely used (i.e., beyond routine data processing functions) to implement structures for biological applications such as sample manipulation and characterization.
Applicants have recognized and appreciated that integrated circuit semiconductor-based technology (e.g., Si, SiGe, GaAs, InP, etc.), and especially CMOS technology, provides a viable foundation for the realization of systems and methods for manipulating and characterizing biological materials and other objects of interest. Furthermore, Applicants have recognized and appreciated that by combining CMOS or other semiconductor-based technology with microfluidics, a wide variety of useful and powerful methods and apparatus relating to biological and other materials may be realized.
In view of the foregoing, various embodiments of the present disclosure are directed to methods and apparatus for one or more of manipulation, detection, imaging, characterization, sorting and assembly of biological or other materials on a micro-scale, involving an integration of CMOS or other semiconductor-based technology and microfluidics.
For example, one embodiment is directed to an IC/microfluidic hybrid system that combines the power of an integrated circuit chip with the biocompatibility of a microfluidic system. In one aspect of this embodiment, various components relating to the generation of electric and/or magnetic fields of such a hybrid system are implemented on an IC chip that is fabricated using standard protocols (e.g., CMOS) in a chip foundry. In another aspect, the field generating components themselves may be formed using standard CMOS protocols and hence do not require any micromachining techniques (e.g., as in micro-electro-mechanical structures, or MEMS implementations). The electric and/or magnetic fields generated from such an IC chip may be used to manipulate and/or detect one or more dielectric and/or magnetic particles and distinguish different types of particles.
In particular, in one embodiment, an array of microelectromagnets, or “microcoils,” are implemented on an IC chip and configured to produce controllable spatially and/or temporally patterned magnetic fields. In one aspect, the IC chip also may include a programmable digital switching network and one or more current sources configured to independently control the current in each microcoil in the array so as to create the spatially and/or temporally patterned magnetic fields. In another aspect, the IC chip may further include a temperature regulation system to facilitate biocompatibility of the hybrid system.
In another embodiment, an array of microelectrodes, or “microposts,” are implemented on an IC chip and configured to produce controllable spatially and/or temporally patterned electric fields to manipulate particles of interest based on dielectrophoresis principles. In one aspect, the IC chip also may include a programmable digital switching network and one or more voltage sources configured to independently control the voltage across each micropost in the array so as to create the spatially and/or temporally patterned electric fields. As in the previous embodiment, in another aspect, the IC chip may further include a temperature regulation system to facilitate biocompatibility of the hybrid system.
In yet another embodiment, an array of microcoils implemented on an IC chip may be configured to produce both controllable, spatially and/or temporally patterned, electric fields and/or magnetic fields. In one aspect, the IC chip also may include a programmable digital switching network, together with one or more current sources and one or more voltage sources, configured to independently control the current in and voltage across each microcoil in the array to create the spatially and/or temporally patterned magnetic fields and electric fields. In another aspect of this embodiment, the microcoils effectively act as microposts when a voltage is applied across them, thereby functioning to manipulate particles of interest based on dielectrophoresis principles as in the previous embodiment. Again, the IC chip according to this embodiment may further include a temperature regulation system to facilitate biocompatibility of the hybrid system.
In connection with any of the foregoing embodiments related to electric and/or magnetic field generation, according to yet another embodiment of the present disclosure, a microfluidic system may be fabricated either directly on top of the IC chip, or as a separate entity that is then appropriately bonded to the IC chip, to facilitate the introduction and removal of cells in a biocompatible environment, or other particles/objects of interest suspended in a fluid. In this manner, the patterned electric and/or magnetic fields generated by the IC chip can trap and move biological cells or other objects inside the microfluidic system.
Other embodiments of the present disclosure are directed to sensing/imaging methods and apparatus utilizing one of the IC-based magnetic and/or electric field generating arrays as introduced above, or other arrangements of magnetic and/or electric field-generating devices. For example, in various aspects of these sensing embodiments, a microcoil array, a micropost array, or other arrangement of field-generating devices (e.g., see the various structures described in PCT Application No. PCT/US02/36280, filed Nov. 5, 2002, entitled “System and Method for Capturing and Positioning Particles,” International Publication No. WO 03/039753 A1) may be controlled using signals of various frequencies so as to be capable of detecting one or more cells, particles or objects of interest, and even the type of particle or object of interest, by measuring resonance characteristics associated with interactions between samples and one or more of the field-generating devices.
In some embodiments, radio frequency (RF) signals are employed to facilitate detection, imaging and/or identification. One of the principles underlying these RF embodiments is that an RF field is capable of interacting with virtually any particle (biological or otherwise) that conducts electricity at the RF signal frequency, or is polarizable electrically or magnetically. Accordingly, in these RF sensing embodiments, the interaction between an RF field and an object in the vicinity of the RF field may be exploited to determine the position of one or more objects of interest so as to facilitate imaging of the object(s). In this manner, semiconductor-based/microfluidic hybrid systems and methods as disclosed herein may be configured to detect and image biological cells, particles and other objects of interest via purely electrical/magnetic means using RF signals, and without resorting to chemical agents or optical techniques. Based on such RF imaging techniques, various implementations of a hybrid system according to the present disclosure may incorporate feedback control mechanisms, whereby samples of interest may be manipulated based on acquired images of the samples.
In some aspects, the RF techniques disclosed herein may be used not only to detect and image particles, but also to identify different types of particles/objects of interest. This type of identification may be accomplished, for example, by measuring spectral responses of RF field/particle interactions over a broad range of frequencies and comparing these responses to known frequency dependent behavior of various materials in electromagnetic fields. In other aspects, RF techniques disclosed herein also may be used to conduct local measurements of magnetic resonance (including ferromagnetic resonance) in a uniform magnetic field applied to a sample or object of interest to thereby identify the material of the sample based on characteristic oscillating frequencies of spins (e.g., Electron Spin Resonance or “ESR”) or magnetic domains (e.g., Nuclear Magnetic Resonance or “NMR”). Accordingly, methods and apparatus according to various embodiments of the present disclosure may be employed to effectively implement a Magnetic Resonance Imaging (MRI) system on a chip.
In view of the manipulation, detection, imaging and identification techniques discussed above and in greater detail below, Applicants have recognized and appreciated that semiconductor-based/microfluidic hybrid systems and methods as disclosed herein facilitate a wide variety of new types of investigations in biomedicine and systems biology, as well as other applications.
For example, another embodiment of the present disclosure is directed to cell sorting methods and apparatus by employing IC/microfluidic hybrid methods and apparatus, as well as RF sensing/imaging methods and apparatus as introduced above. In one aspect, cell sorting methods and apparatus according to this embodiment facilitate molecularly-precise identification and rapid, highly-accurate sorting of cells. In particular, biological cells may be sorted individually with ultrahigh accuracy and with molecularly-precise identification. Such precision sorting facilitates the separation of specific (e.g., “rare”) cell types or pathogens (e.g., stem cells for bone marrow reconstitution procedures in cancer patients) for clinical applications. Such precision sorting also facilitates parsing a tissue's demographics and evaluating each cell type separately, rather than collecting gene expression data on tissue from an ensemble of different cell types.
Yet another embodiment of the present invention is directed to methods and apparatus for assembling micro-scale engineered tissues. In one aspect of this embodiment, a two-dimensional cell trap array based on an IC/microfluidic hybrid system is configured to be capable of micro-scale tissue assembly with precise control of cellular demographics and spatial distribution (e.g., artificial tissues from heterotypical distributions of cells may be assembled one cell at a time). Such a technique according to one embodiment of the present disclosure represents a new way to develop novel in vitro assays for studying communication networks amongst different cell types, drug efficacy, and for fundamental physiological study in a standardized, repeatable manner.
Semiconductor-based IC/microfluidic hybrid systems and methods according to various embodiments of the present disclosure have several important technological advantages. First, a semiconductor-based/microfluidic hybrid system may be fabricated in an appreciably cost-effective manner with high yield using a mature CMOS technology and inexpensive lithographic techniques for formation of the microfluidic system portion. Such CMOS implemented systems may be made significantly small in size and appropriately packaged to withstand various environmental hazards. Advanced low-power integrated circuit techniques also facilitate the fabrication of battery-powered devices. In view of the foregoing, such systems can be made as rugged single-use disposable devices, and may be employed in a variety of applications, including potentially adverse and/or emergency situations, that would otherwise be precluded using conventional methods and apparatus. For example, small, inexpensive, battery-powered, rugged hybrid systems according to various embodiments of the present disclosure may be easily and effectively employed in emergency medical situations to quickly screen an individual's health using saliva, breath, sweat, or blood samples. Such systems also may be employed to detect biologically harmful substances in a given environment.
Additionally, as compared to conventional magnetic manipulation methods using simple magnetic tweezers or external magnets, or conventional dielectrophoresis techniques, semiconductor-based/microfluidic hybrid systems and methods according to the present disclosure can manipulate single or multiple biological cells, particles or other objects of interest in a large quantity with easy, precise, and rapid control. Furthermore, semiconductor-based IC/microfluidic hybrid systems and methods according to various embodiments of the present disclosure offer significant flexibility over conventional microfluidic systems. In particular, somewhat more complex conventional microfluidic systems control biological samples in a fixed channel network using predetermined valve controls; hence, different operations require different specific microfluidic systems. In contrast, semiconductor-based/microfluidic hybrid systems and methods according to various embodiments of the present disclosure are capable of performing various and sophisticated cell/particle manipulation operations without necessarily requiring a complex microfluidic system structure.
For example, in one embodiment, a programmable hybrid system according to the present disclosure may be implemented using a relatively simple microfluidic system having only a single chamber (a “bathtub”) integrated with a semiconductor-based system that provides programmable and independently controllable electromagnetic fields. In this implementation, cells may be moved through the chamber along virtually any path under computer control of the electromagnetic fields. In this manner, the topology of a “virtual micro-scale plumbing system” for samples of interest may be flexibly changed for a wide variety of operations based on the programmability afforded by computer control. This provides an extremely powerful tool for precision cell/object manipulation in both relatively simple and more sophisticated operations.
In sum, one embodiment according to the present disclosure is directed to an apparatus, comprising a plurality of CMOS fabricated field-generating components, a microfluidic system configured to contain a fluid in proximity to the plurality of CMOS fabricated field-generating components, and at least one controller configured to control the plurality of CMOS fabricated field-generating components to generate at least one electric or magnetic field having a sufficient strength to interact with at least one sample suspended in the fluid.
Another embodiment according to the present disclosure is directed to a method, comprising an act of generating at least one electric of magnetic field from a plurality of CMOS fabricated field-generating components, the at least one electric or magnetic field having a sufficient strength to interact with at least one sample suspended in a fluid contained in a microfluidic system in proximity to the plurality of CMOS fabricated field-generating components.
The following references are incorporated herein by reference:
U.S. Non-provisional application Ser. No. 10/894,674, filed Jul. 19, 2004, entitled “Methods and Apparatus Based on Coplanar Striplines;”
U.S. Non-provisional application Ser. No.10/894,717, filed Jul. 19, 2004, entitled “Methods and Apparatus Based on Coplanar Striplines;” and
PCT Application No. PCT/US02/36280, filed Nov. 5, 2002, entitled “System and Method for Capturing and Positioning Particles,” International Publication No. WO 03/039753 A1.
It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.
FIGS. 3(a)-(d) illustrate a microelectromagnet wire matrix which provides one example of magnetic field-generating components that may be included in the hybrid system shown in
FIGS. 5(a) and (b) illustrate a micropost array which provides one example of electric field-generating components that may be included in the hybrid system shown in
FIGS. 7(a) and 7(b) show perspective and exploded views, respectively, of a multiple-layer microcoil that may be employed in the arrays of
FIGS. 11 (a)-(e) show five exemplary scenarios for the neighboring microcoils of
FIGS. 39(a)-(d) illustrate exemplary implementations of cell detection via RF sensing techniques as discussed above in connection with
Following below are more detailed descriptions of various concepts related to, and embodiments of, methods and apparatus according to the present disclosure for one or more of manipulation, detection, imaging, characterization, sorting and assembly of biological or other materials. It should be appreciated that various aspects of the subject matter introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the subject matter is not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.
I. System Overview
One embodiment of the present disclosure is directed to a semiconductor-based/ microfluidic hybrid system that combines the power of microelectronics with the biocompatibility of a microfluidic system. In some examples below, the microelectronics portion of the hybrid system is implemented in CMOS technology for purposes of illustration. It should be appreciated, however, that the disclosure is not intended to be limiting in this respect, as other semiconductor-based technologies may be utilized to implement various aspects of the microelectronics portion of the systems discussed herein.
In one aspect of this embodiment, as shown in
In the hybrid system 100, according to one embodiment, the microfluidic system 300 may be configured as a relatively simple chamber or reservoir for holding liquids containing samples of interest. For example, as illustrated generically in
In other aspects of the embodiment shown in
For example, in one implementation, as illustrated in
It should be appreciated that the foregoing exemplary arrangements are provided primarily for purposes of illustration, and that a variety of arrangements of a microfluidic system and field-generating components (including linear or two-dimensional arrays of field-generating components, or other arrangements of discrete field generating components) are contemplated according to other embodiments to provide multi-dimensional manipulation of samples. In general, according to the various concepts discussed herein, samples of interest may be moved through the microfluidic system along virtually any path, trapped or held at a particular location, and in some cases rotated, under computer control of the electric and/or magnetic fields generated by the field-generating components 200. In this manner, the topology of a “virtual micro-scale plumbing system” for samples of interest may be flexibly changed for a wide variety of operations based on the programmability and computer control afforded, for example, by the processor(s) 600. This provides an extremely powerful tool for precision cell/object manipulation in both relatively simple and more sophisticated operations.
In various embodiments of the hybrid system 100 shown in
Examples of magnetic field-generating components 200 that may be included in the hybrid system 100 shown in
In one embodiment based on the wire matrix shown in FIGS. 3(a)-(d), each conductor in the wire matrix (or alternatively predetermined groups of conductors) may be connected to a controllable current source (discussed further below) so that all conductors (or groups of conductors) can have independent current flows. By independently modulating the magnitude of the currents in the conductors, various dynamic magnetic field patterns can be produced in proximity to (e.g., above) the wire matrix. For example, the currents can be controlled such that the wire matrix can create a single magnetic peak that is moving continuously, multiple peaks with each peak controlled independently, or varying magnetic fields to rotate or twist a target sample.
Yet other examples of devices that may serve as magnetic field-generating components in the hybrid system shown in
It should be appreciated that for virtually any hybrid system 100 according to the present disclosure based on a microelectronics portion configured to generate controllable spatially and/or temporally variable magnetic fields, a parallel implementation may be realized using configurations for generating controllable spatially and/or temporally variable electric fields, or a combination of variable magnetic fields and variable electric fields.
For example, in one embodiment, the field-generating components 200 of the hybrid system shown in
As discussed above, dielectrophoresis occurs when an inhomogeneous electric field induces a dipole on a particle suspended in liquid. The subsequent force on the dipole pulls the particle to either a minimum or a maximum of the electric field. Almost any particle, without any special preparation, can be trapped or moved using dielectrophoresis when it is exposed to the proper local electric field. In this manner, according to one embodiment, one or more samples of interest suspended in liquid in the microfluidic system 300 may be manipulated via operation of the micropost array 210 to generate electric fields appropriate for this task.
More specifically, in one embodiment based on the micropost array 210 shown in FIGS. 5(a) and (b), each micropost in the array (or alternatively predetermined groups of microposts) may be connected to a controllable voltage source (discussed further below) so that all microposts (or groups of microposts) can have independent voltage potentials across them. By independently modulating the magnitude of the voltages across the respective microposts, various electric field patterns can be produced in proximity to (e.g., above) the micropost array 210 to facilitate manipulation of one or more samples of interest contained in the microfluidic system. To provide a ground for the respective micropost potentials, one exemplary geometry includes fabricating a ground plane adjacent to and above the micropost array (e.g., on a bottom surface of a microfluidic chamber), such that substantially all generated electric field lines point in the same direction. Alternatively, electric field maxima may be generated by applying different voltage potentials (e.g., plus and minus connections) to different (e.g., neighboring) microposts within the array, thereby obviating a ground plane.
In yet another embodiment, an array of microcoils may be configured to produce both controllable, spatially and/or temporally patterned, electric fields and/or magnetic fields. More specifically, in one implementation discussed further below in Section II, respective independently controllable voltages may be applied across the microcoils of a microcoil array, such that the individual microcoil structures behave essentially like the microposts of the micropost array 210 shown in FIGS. 5(a) and (b), namely, by generating electric fields that are capable of interacting with samples contained in the microfluidic system. According to one aspect of this embodiment, respective independently controllable currents also may be applied to the microcoils of the microcoil array, to additionally generate magnetic fields that are capable of interacting with magnetic samples contained in the microfluidic system. These and other types of electric field-based or electric/magnetic field-based implementations may be employed for a variety of applications relating to manipulation, sensing and imaging systems that integrate microelectronics and microfluidics.
As mentioned above and also shown in
In general, whether the field control components 400 include one or more current sources 420, one or more voltage sources 440, or both, according to one embodiment the field control components also include various switching or multiplexing components 460 to facilitate the appropriate application of currents and/or voltages to individual field-generating components or groups of field-generating components. In various implementations discussed in greater detail below, the switching or multiplexing components 460 may be configured as a programmable digital switching network (e.g., under control of the one or more processors 600) such that the output(s) of one or more current and/or voltage sources are applied in a prescribed independently controllable manner to the field-generating components, so as to create the spatially and/or temporally patterned electric and/or magnetic fields that facilitate sample manipulation.
As also shown in
In various aspects, the RF/detection components 480 provide for sample detection, imaging and characterization techniques that are purely based on electromagnetic fields, without requiring chemical elements that may possibly be harmful to samples of interest, or bulky optical microscopes. Nevertheless, it should be appreciated that, according to some techniques involving various concepts disclosed herein, sample detection and imaging may be assisted by chemically treating/targeting specific types of samples.
In general, as is well known based on Maxwell's Equations, an RF field is capable of interacting with virtually any particle (biological or otherwise) that conducts electricity at the RF signal frequency, or is polarizable electrically or magnetically. Accordingly, in various embodiments of the present disclosure, the interaction between RF electric and/or magnetic fields and samples of interest may be exploited not only to move samples but also to determine the position of the sample (e.g., to facilitate imaging). Moreover, spectral responses arising from the RF field/sample interaction may be used in some cases to identify or characterize different types or classes of samples.
For example, conducting samples have circulating currents induced by an RF field that in turn produce their own magnetic field, and interact strongly with an applied field. This is the basis of operation of conventional electric motors (e.g., a “squirrel cage” rotor with no electrical contacts). This interaction can be used to move samples and also detect their presence. In one mechanism discussed in greater detail below, magnetic polarization of a sample changes the inductance of a coil (e.g., a microcoil of an array) in proximity to the sample; accordingly, damping of oscillations of the magnetic polarization causes detectable losses in a circuit including the microcoil. In yet another example, electrical polarization of a sample gives rise to the forces responsible for dielectrophoresis (DEP). This polarization can be detected via a change in capacitance between the sample and the electrodes of an electric-field generating device (e.g., a micropost or microcoil with an applied voltage) with no dissipation, or by a change in damping due to the oscillating electric polarization in the sample. The foregoing examples provide various mechanisms by which the location of a sample can be detected, and thus imaged.
Based on such RF imaging techniques, various implementations of a hybrid system according to the present disclosure may incorporate feedback control mechanisms, whereby samples of interest may be manipulated based on acquired images of the samples. For example, in one embodiment, the hybrid system may be programmably configured (e.g., via the one or more processors 600) to first obtain an image of a distribution of samples contained in the microfluidic system. Thereafter, based on the imaged distribution, one or more particular samples may be manipulated based on a prescribed algorithm.
Various concepts disclosed herein relating to RF fields likewise may be employed for identification and characterization of samples of interest. For example, frequency dependent changes in either the electric or magnetic polarization of samples can be used to identify the type of sample, using knowledge of the behavior of various materials in electromagnetic fields from conventional solid state physics. These changes may be characterized over a broad range of frequencies. Accordingly, in one embodiment, by sweeping the RF frequency of signals applied to field-generating components (or using more sophisticated signal processing techniques), the frequency response (e.g., absorption spectrum) of the sample can be measured at a particular location, and the sample may be identified or characterized based on the measured response.
In yet other embodiments relating to the application of RF fields and sensing of field/sample interactions under the control of the RF/detection components 480, an RF field can be used to conduct local measurements of magnetic resonance in a uniform magnetic field applied to a sample. In particular, the spins or magnetic domains of a given sample oscillate with characteristic frequencies, which can be used to identify the type of spin or the sample itself. Magnetic resonance types include ferromagnetic resonance (FMR) (small YIG spheres can be used as magnetic beads, wherein a YIG sphere has a single magnetic domain that rotates freely at GHz frequencies because the bead is spherical). Additionally, Electron Spin Resonance (ESR) techniques may be employed to identify the g-factor of the spins involved to characterize their origin (i.e., the sample), as well as Nuclear Magnetic Resonance (NMR) to identify the g-factors of the nuclear spins. Thus, according to the principles discussed herein, a Magnetic Resonance Imaging (MRI) system may be implemented on a chip.
While not explicitly shown in
Finally,
Having provided a general overview of a hybrid system according to the present disclosure for manipulation, detection, imaging and characterization of samples using electromagnetic fields, more detailed descriptions of various concepts related to different portions of the hybrid system, as well as some exemplary applications for such a system, are set forth below.
II. Microcoil Array
Like the microelectromagnet wire matrix 200A discussed above in connection with FIGS. 3(a)-(d), a microcoil array 200B similar to that shown in
As compared to the microelectromagnet wire matrix 200A, the microcoil array 200B generally is more efficient for at least some of the following exemplary reasons. First, the fields generated in the microcoil array are more highly localized than in the microelectromagnet wire matrix, thereby providing a relatively higher spatial resolution for trapping and transporting samples. Second, the microcoil array has a finer degree of magnetic field control than does the microelectromagnet wire matrix and can thus handle a larger number of samples simultaneously; specifically, a N×N microcoil array can effectively provide N2 independent simultaneous local magnetic fields (based on N2 independent currents), whereas an N×N wire matrix can provide only 2N independent simultaneous fields (based on 2N independent currents). Third, as discussed in greater detail below, a microcoil provides a better platform for RF detection owing to its well-defined inductance. Fourth, parasitic magnetic fields due to electrical leads generally are less significant in the microcoil array than in the microelectromagnet wire matrix.
One issue in the design of a two-dimensional microcoil array 200B according to one embodiment of the present disclosure relates to the magnetic force that can be generated in a plane immediately above and parallel to the array. This plane is indicated generally in both
With the foregoing issues in mind, one embodiment of the present disclosure is directed to a microcoil array fabricated on a semiconductor (e.g., Si) substrate using conventional CMOS process technology. In one aspect of this embodiment, various field control components, including control electronics for the microcoil array, are integrated together with the microcoil array and fabricated as a CMOS IC chip, so as to provide for the generation of spatially and/or temporally variable magnetic fields for sample manipulation, as well as RF fields to facilitate sample detection, imaging and characterization. In particular, in exemplary implementations, the microcoils themselves are formed using standard CMOS protocols and hence do not require any micromachining techniques (e.g., as in micro-electro-mechanical structures, or MEMS implementations).
More specifically, to address the design issues noted above, according to one embodiment multiple metal layers available in a CMOS fabrication process are employed in the microcoil configuration to allow generation of adequate magnetic field strengths sufficient to effectively trap and transport samples. FIGS. 7(a) and 7(b) show perspective and exploded views, respectively, of an exemplary three-layer microcoil 212 according to this embodiment, and
As illustrated in
As discussed generally above, the principle of operation of the microcoil array 200B for magnetic sample manipulation is to create and move one or more magnetic field peaks by modulating currents in the respective microcoils 212 of the array. For example, consider first “turning on” (i.e., passing current through) only one microcoil 212 of the array (e.g., the microcoil shown in
F=Vχ/μo∇B2, (1)
where V is the volume of the magnetic bead 112, χ is the effective magnetic susceptibility of the bead, μo is the magnetic permeability of a vacuum, and B is the generated magnetic field magnitude. If this microcoil is then “turned off” while an adjacent microcoil of the array is turned on, the magnetic field peak is moved to the center of the adjacent microcoil, thereby transporting the magnetic bead to the new peak location.
The magnetic field B required to generate a particular trapping force F is proportional to the current flowing through the microcoil and the inductance of the microcoil; the inductance of the microcoil is in turn proportional to the number of turns of the microcoil and the size (diameter) of the microcoil. Accordingly, a microcoil design that provides a relatively high inductance generally is desirable to provide for a magnetic field of sufficient strength to trap samples. At the same time, to maintain a fine spatial resolution amongst the microcoils of the array and facilitate sample transport between adjacent microcoils, it is generally desirable to have a relatively small inter-coil spacing or pitch 216 and relatively small diameter 214 of the upper portion 212A of a microcoil, as indicated in
Accordingly, in various aspects of this embodiment, the overall number of turns of the microcoil and the diameter of each coiled portion is appropriately selected to provide an appropriate array pitch, as well as an appropriate microcoil inductance to generate sufficient magnetic fields, to facilitate sample trapping and transport between microcoils. To this end, the multiple layer microcoil structure shown in
More specifically, in the exemplary microcoil shown in
As also illustrated in FIGS. 7(a) and (b), the middle portion 212B and the lower portion 212C of the microcoil may have larger diameters than the upper portion. In one aspect, the larger diameters of the middle and lower portions is possible because the spacing between adjacent middle and lower portions of adjacent microcoils in the array may be smaller than the spacing between adjacent upper portions without compromising the resolution of the generated magnetic fields (i.e., the resolution of the generated magnetic fields is largely determined by the top metal layer). Thus, the middle and lower portions generally may include a greater number of turns and/or a larger diameter than the upper portion, thereby providing for a relatively higher microcoil inductance. Additionally, as shown in FIGS. 7(a) and (b), the lower portion 212C may include tabs 228 to facilitate connection of the microcoil 212 to a current (or voltage) source, as discussed further below. In one exemplary implementation, each of the middle and lower portions may include three conductor turns, wherein a diameter 220 of the middle portion 212B may be on the order of approximately 20-25 μm, and a diameter 218 of the lower portion 212C may be on the order of approximately 15-20 μm (the relatively smaller diameter of the lower portion permits the inclusion of the tabs 228). In other implementations, different numbers of conductor turns and/or different dimensions may be used for respective coil portions, and may be determined empirically or based on numeric simulations of desired magnetic fields for different applications.
With reference now to the IC vertical layer structure illustrated in
In one embodiment, the CMOS processing techniques employed to fabricate the vertical layer structure shown in
With respect to the middle and lower layers/portions 212B and 212C, the CMOS processing techniques may yield a thickness 224 for both the lower and middle layers/portions of approximately 0.5 to 1 μm. These layers may be patterned such that the line width in the x-y plane is also approximately 0.5 to 1 μm, yielding a metal conductor cross section for the lower and middle portions of approximately 0.5×0.5 μm2 to approximately 1×1 μm2. A distance 226 between the metal layers may be on the order of approximately 1 μm (it should be appreciated that, based on the TSMC 0.18 μm design rule, the distance between the metal layers may be as small as 0.46 μm).
Based on the foregoing general dimensions, a microcoil inductance on the order of approximately 1 nano Henry (1 nH) or higher may be achieved. By generally decreasing various dimensions relating to the metal conductors, the number of coil turns may be increased, resulting in inductances as high as 60 to 100 nano Henries (60-100 nH). It should be appreciated, however, that as the width of metal conductors becomes smaller, the parasitic resistance of the coil generally increases and the maximum allowable current through the coil generally decreases, which ultimately limits the strength of the magnetic field that may be generated; hence, there may be practical trade-offs between coil size and field strength.
More generally, it should be appreciated that the vertical layer structure shown in
As shown in the vertical layer structure of
Based on the general structure of a CMOS microcoil as outlined above, significant local magnetic fields may be generated above each microcoil of the array 200B to manipulate samples. To provide an illustrative range of values for magnetic field strength and sample trapping force, a two-layer microcoil structure having an overall diameter of approximately 20 μm and 4 coil turns per layer is considered. The exemplary microcoil includes an aluminum conductor having an average conductor cross-section of 1×1 μm2, wherein the line width is 1 μm, the gap between adjacent conductor turns of a given layer is 1 μm, and the distance between the two layers is 1 μm. The maximum current density for an aluminum conductor is approximately 200 mA/μm2; hence, the exemplary microcoil under consideration is capable of supporting approximately 200 mA of maximum current flowing through it.
If a sample of interest includes a cell coupled to a conventionally available magnetic bead (e.g., Dynabead) having a diameter of approximately 4-5 μm and a magnetic susceptibility χ of approximately 0.25, the force F exerted on the sample by the peak magnetic field of approximately 300 Gauss shown in
While the foregoing example is based on an exemplary maximum current through the microcoil, it should be appreciated that significantly lower currents (e.g., on the order of approximately 20 mA) nonetheless provide sufficient peak magnetic fields and resulting forces (e.g., on the order of approximately 10 pico Newtons) for the effective manipulation of a variety of magnetic samples. Generally, the magnitude of magnetic force generated by the microcoil increases with current through the microcoil. In some instances, as current is increased toward a maximum current, a high current density in a microcoil over a prolonged period may result in electromigration, a phenomenon in which a large current in a narrow conductor gradually results in metal void failures. Electromigration generally is more pronounced at higher temperatures, though. Hence, in the hybrid systems described herein (in which operating temperatures typically would be below 50 degrees C., and in some cases regulated for biocompatibility at 37 degrees C.), current densities that generate magnetic forces sufficient for effective sample manipulation generally would not cause significant electromigration.
Moreover, while the foregoing example demonstrates that microcoils similar to those shown in
According to yet another embodiment, “vertical” microcoils may be fabricated and used in manipulation and imaging of magnetized samples, similarly to the multi-layer microcoils described above. Presently available CMOS technologies support primarily planar metal layers, and hence the microcoils discussed above are essentially “planar” in that they are disposed along a plane parallel to the x-y axes indicated in the various figures, and generate magnetic fields perpendicular to the surface of the IC chip 102 (i.e., essentially along the z axis). However, in another embodiment, by employing micromachining and/or other three-dimensional assembly processes as post-fabrication steps, it is possible to tilt the planar microcoil away from the substrate surface (after removal of oxide), yielding a vertical microcoil. Such a vertical microcoil produces a magnetic field parallel to the surface of the IC chip 102 (i.e., essentially in a plane parallel to the x-y axes). By employing both vertical and planar microcoils in one implementation according to the present disclosure, three-dimensional sample manipulation is possible, including rotation in addition to linear transport. In the context of RF detection and imaging discussed in greater detail below, the vertical microcoil may allow large-signal RF perturbations for imaging, while the planar microcoil provides a DC field to manipulate the samples, thereby enhancing the capability of a hybrid system incorporating both vertical and planar microcoils.
Having discussed various aspects of the structure and fabrication of an exemplary microcoil according to the present disclosure based on conventional semiconductor fabrication processes, the interaction between neighboring microcoils in an array with respect to the generation of magnetic fields for sample manipulation is now considered in greater detail. As discussed above, the principle of operation of the microcoil array 200B shown in FIGS. 6(a) and (b) is to create and move one or more magnetic field peaks by modulating currents in the respective microcoils 212 of the array so as to move and/or trap magnetic samples. The magnitude of the magnetic field generated by a given microcoil of the array is based on the magnitude of the current flowing through the microcoil, and each microcoil in the array is capable of generating a local magnetic field peak above the microcoil. In this sense, the array 200B may be thought of generally in terms of “magnetic pixels,” wherein an N×N array of microcoils is capable of producing at least N×N magnetic peaks, or “pixels,” each capable of attracting and trapping a sample.
In one embodiment, not only may the magnitude of the current flowing through each microcoil be modulated to facilitate sample manipulation, but also the direction of the current flowing through a given coil may be altered, so as to facilitate a smoother transition of a sample from pixel to pixel, or effectively increase the spatial resolution for sample manipulation (i.e., effectively decrease the pitch 216 of the array). FIGS. 11(a)-(e) show five exemplary scenarios for the neighboring microcoils 212-1 and 212-2 of
In particular, in
In
As discussed above in connection with
As shown in
Finally,
In one aspect of the embodiment of
In one aspect of this embodiment, each of the base current sources 426-1A through 426-1H may be implemented in a conventional manner using MOS transistors, wherein the current provided by each base source is determined by the control voltage 469 (VCTRL). For example, in one exemplary implementation, the control voltage VCTRL may be applied to all of the base current sources such that a particular control voltage provides a corresponding current from each base source (e.g., a control voltage of 0.7 to 3.3 Volts generates a corresponding current in each base source of from 0 to 1.3 milliamperes). It should be appreciated that, in different implementations, the control signal VCTRL may be varied to provide for variable base currents or alternatively may be held constant (e.g., connected to Vdd).
Furthermore, it should be appreciated that although the variable current source 420-1 shown in
In the embodiments discussed above in connection with
In
To provide some exemplary illustrations of appropriate scanning time scales for sample manipulation, a commercially available magnetic bead (e.g., Dynabead) having a diameter of approximately 4-5 μm is considered in a liquid water environment as a representative magnetic sample. In general, samples suspended in a liquid experience a viscous drag as they move through the liquid; this viscous drag generally affects the speed with which a sample reacts to an external magnetic field (and hence the “response time” of the sample). For a magnetic sample suspended in a liquid, the response time τcutoff is given as
τcutoff≈O(μμo/χB2), (2)
where μ is the dynamic viscosity of the liquid. Accordingly, if the sample is exposed to a pulsed magnetic field having a frequency that is significantly higher than the sample's “cutoff frequency” (i.e., the reciprocal of the sample's response time), the pulsed magnetic field appears to exert an essentially continuous average magnetic force on the sample. In this manner, one current source may be multiplexed amongst multiple microcoils of an array (i.e., sequentially applied in time) at an appropriate rate to generate seemingly continuous magnetic forces from the perspective of the samples in question. The magnetic force resulting from a magnetic field was discussed generally in connection with Eq. (1) above. For a Dynabead in water having a diameter of approximately 5 μm under a magnetic field on the order of 30 Gauss, the response time Tcutoff is on the order of 10−2 seconds. Using a pulsed magnetic field having a frequency greater than the reciprocal of the sample's response time (e.g., >approximately 100 Hz), the resulting force is equal to the product of the duty cycle and the force given by Eq. (1).
Once a sample is attracted to a local magnetic field, a sufficient magnetic potential energy must be maintained to trap the sample in the field. In particular, a sample suspended in a liquid moves chaotically due to random collisions of the sample with the surrounding liquid molecules, a phenomenon known as Brownian motion. Such Brownian motion can lead to diffusion of the sample; with random velocity, the sample can move in a random path (e.g., in a tangled zig-zag manner) away from its location at any given time due to Brownian motion. As discussed above, the kinetic energy associated with this motion is proportional to temperature (i.e., 3/2 kT). Accordingly, to maintain a trap, the average magnetic potential energy of the generated field must be sufficiently greater than the sample's thermal energy.
In view of the foregoing, once a sample is initially trapped based on a pulsed magnetic field, the sample may remain trapped in the pulsed magnetic field as long as the magnetic field is not off for a period of time that allows significant diffusion of the sample away from the “trapping area” above a given microcoil. An upper limit for the field off-time τoff is given approximately by τoff<d2/D, where d is the diameter of the microcoil and D is the diffusion constant of the sample (from the definition of D, for a given time t, a particle travels an average distance d=(Dt)1/2). The diffusion constant D of a sample (in meters2 per second) is given generally by
D=kT/3πηa (3)
where η is the viscosity of the liquid (in kg/m·s) and a is the diameter of the sample. In the exemplary scenario under consideration, the viscosity η of water is approximately 10−3 kg/m·s and the diameter of the Dynabead sample is 5 μm; accordingly, assuming a temperature T of approximately 300 K (i.e., room temperature), the diffusion constant D for the Dynabead sample in water is approximately 8.5×10−14 m2/s. If a microcoil diameter of 20 μm is assumed, τoff should be less than approximately 5000 seconds. From a practical standpoint, the foregoing example illustrates that multiplexing current to the microcoils at a rate of 10,000 Hz or higher (i.e., τoff<10−4 seconds) permits practically no appreciable diffusion of the sample due to Brownian motion; with an off-time τoff<10−4 seconds, the 5 μm Dynabead diffuses approximately only 3 nanometers.
In general, it should be appreciated that the configuration of current sources and microcoils illustrated in
It should also be appreciated that while the exemplary concepts discussed above in connection with
For example, first consider the microcoil array 200B of
In another example based on electric field generation, the microcoil array 200B of
III. Sample Detection, Imaging and Characterization
As discussed above in Section I, with reference again to
In general, as is well known based on Maxwell's Equations, an RF field is capable of interacting with virtually any particle (biological or otherwise) that conducts electricity at the RF signal frequency, or is polarizable electrically or magnetically. Accordingly, in various embodiments of the present disclosure, the interaction between RF electric and/or magnetic fields and samples of interest may be exploited not only to move samples as discussed above in Section II, but also to determine the position of the sample (e.g., to facilitate imaging).
For example, conducting samples have circulating currents induced by an RF field that in turn produce their own magnetic field, and interact strongly with an applied field. This interaction can be used to move samples and also detect their presence. In one mechanism discussed in greater detail below, magnetic polarization of a sample changes the inductance of a coil (e.g., a microcoil of an array) in proximity to the sample and, in turn, this inductance change can be detected using high frequency signals. In yet another example, electrical polarization of a sample gives rise to the forces responsible for dielectrophoresis (DEP). This polarization can be detected via a change in capacitance between the sample and the electrodes of an electric-field generating device (e.g., a micropost or microcoil with an applied voltage) with no dissipation, or by a change in damping due to the oscillating electric polarization in the sample.
The foregoing examples provide various mechanisms by which the location of a sample can be detected. Based on the capability to detect the position of a sample relative to a given field generating component, in one embodiment each of the field generating components 200 is analogous to an imaging pixel (e.g., consider a two-dimensional CCD array) that provides valuable information toward constructing a comprehensive image of a sample distribution suspended in a microfluidic system. In another embodiment, images of sample distributions in turn may be used as feedback to manipulate one or more samples according to a prescribed algorithm.
In one embodiment based on magnetic bead-bound samples, the effect of the bead's magnetism on the inductance of a microcoil is exploited to facilitate sample detection. For example, the inductance L of a given microcoil is proportional to an effective magnetic permeability μeff. Without any magnetic particles in the vicinity of the microcoil, μeff is equal to the magnetic permeability of a vacuum μo, but in the presence of a magnetic bead (e.g., a paramagnetic particle, or PMP) having some magnetic permeability μbead, the effective permeability associated with the microcoil is μeff=(1−a) μo+aμbead (where a<<1) thereby altering the inductance of the microcoil by some amount ΔL. Accordingly, by monitoring the inductance L of a microcoil via high frequency signals applied to the microcoil, such changes ΔL in the microcoil's inductance may be detected, thereby indicating the presence of a bead-bound sample in the vicinity of the microcoil.
Depending on the size and hence inductance of the microcoil and the magnetic permeability of the bead, changes in inductance ΔL may range from approximately 0.1% of L to 1% of L (e.g., a Dynabead having a diameter of approximately 4.5 to 5 micrometers and a magnetic permeability μbead of approximately 1.25μo can cause a change in inductance ΔL on the order of 0.1% of L). Also, the frequency response of the bead's magnetic permeability also should be taken into consideration; in particular, for the Dynabead example, μbead has a real value for frequencies below approximately 100 MHz. Hence, in one exemplary implementation, RF signals below or approximately 100 MHz are employed in the detection scheme.
For ease of illustration and to facilitate the following discussion, the remaining components in
As mentioned above, in the embodiment of
To explain the operation of the circuit shown in
As discussed in further detail below, the frequency locked loop is configured such that the control voltage Vc stabilizes at some DC value when θ1=θ2. Accordingly, from the above equations (4) and (5), a “lock frequency” ωlock for the frequency locked loop may be expressed as
From the foregoing, it may be appreciated that the lock frequency ωlock is essentially a function of changes in the microcoil inductance L, as CRF, RL, RC, R1, and R2, are known fixed values. In one exemplary implementation, a nominal microcoil inductance L on the order of 1 nH is considered, with a nominal coil resistance RL of approximately 50Ω. To ensure that ωlock is below or approximately 100 MHz, CRF is chosen at 1 pF, with a typical RC on the order of approximately 1 k Ω, R1 is chosen at approximately 50Ω and R2 is chosen at approximately 10 kΩ.
To measure changes in inductance ΔL due to the presence of a magnetic sample in proximity to a microcoil, an instantaneous lock frequency ωlock is measured and compared to a nominal lock frequency representing the absence of a magnetic sample. In exemplary implementations in which ωlock is nominally approximately 100 MHz in the absence of a magnetic sample, changes in the lock frequency Δωlock due to the presence of a magnetic sample may be on the order of approximately 50 to 100 kHz. In
In the circuit arrangement illustrated in
where z is a zero and p is a pole of the transfer function. From the foregoing, it can be seen that the transfer function Z(s) includes a pole at s=0 in the denominator, due to the presence of the capacitor 484A. An expression for the control voltage VC in the Laplace domain then may be given as
VC(s)=IZ(s)=Kθ(θ2−θ1)Z(s). (8)
From the foregoing, it may be observed that in steady state (s=0), Z(s) tends to infinity; hence, to ensure a stable control voltage VC, the quantity (θ2−θ1) must tend to zero in steady state. Accordingly, the capacitor 484A in the low pass filter 484 essentially ensures that the frequency locked loop stabilizes when θ2=θ1, thereby providing the expression for ωlock given above.
Based on the configuration of the bridge circuit 485 shown in
In one aspect of the embodiment of
In one aspect of the embodiment illustrated in
Having discussed the detection of a magnetic sample, various concepts disclosed herein relating to RF fields likewise may be employed for identification and characterization of samples of interest. For example, frequency dependent changes in either the electric or magnetic polarization of samples can be used to identify the type of sample, using knowledge of the behavior of various materials in electromagnetic fields from conventional solid state physics. These changes may be characterized over a broad range of frequencies. Accordingly, in one embodiment, by sweeping the RF frequency of signals applied to field-generating components (or using more sophisticated signal processing techniques), the frequency response (e.g., absorption spectrum) of the sample can be measured at a particular location, and the sample may be identified or characterized based on the measured response.
In yet other embodiments relating to the application of RF fields and sensing of field/sample interactions under the control of the RF/detection components 480, an RF field can be used to conduct local measurements of magnetic resonance in a uniform magnetic field applied to a sample. In particular, the spins or magnetic domains of a given sample oscillate with characteristic frequencies, which can be used to identify the type of spin or the sample itself. Magnetic resonance types include ferromagnetic resonance (FMR) (small YIG spheres may be used as magnetic beads, as each sphere has a single magnetic domain that rotates freely at GHz frequencies because the bead is spherical). Additionally, Electron Spin Resonance (ESR) techniques may be employed to identify the g-factor of the spins involved to characterize their origin (i.e., the sample), as well as Nuclear Magnetic Resonance (NMR) to identify the g-factors of the nuclear spins. Thus, according to the principles discussed herein, a Magnetic Resonance Imaging (MRI) system may be implemented on a chip.
IV. Temperature Regulation
As mentioned above in connection with
More specifically, according to one embodiment as illustrated in
In various implementations, the processor 600 may be configured to receive multiple temperature signals from respective different on-chip sensors, and process the multiple signals according to one or more predetermined algorithms (e.g., averaging, weighted averaging based on chip location, etc.) to provide some aggregate sensed temperature value, which then may be compared to the reference temperature. Based on a comparison of one or more sensed temperatures and the reference temperature, a control signal is provided to the off-chip temperature controller 500B, which heats up or cools down the package substrate 110 accordingly (e.g., a thermoelectric or “TE” cooler may be used as the off-chip controller 500B in one exemplary implementation). In another aspect of this embodiment, the thermal conductivity across all the layers and within each layer of the IC chip 102 is such that the whole system can be assumed to be at the same temperature. Thus, the regulation loop is sufficient to keep the temperature of the overall system at a constant value.
In the embodiment of
where the logarithm is base e, IS is the leakage current of the transistor, k is Boltzmann's constant, q is the electron charge, and T is the absolute temperature. The above equation indicates that the emitter voltage can be used as a direct measure of the chip temperature (Tchip). In one embodiment, the processor 600 compares this emitter voltage to a calibrated voltage representing the reference temperature (e.g., Tref=37° C.) using a 1-bit comparator. If Tchip>Tref, a control signal provided by the processor operates the temperature controller 500B to cool the chip, and vice versa.
In various implementations, the accuracy and long-term stability of the temperature regulator may be affected by mismatching of integrated components, drift of component parameters, I/f(flicker) noise, and mechanical stress. To improve the accuracy of the temperature regulator loop, in some embodiments various conventional analog integrated circuit design techniques may be utilized, such as auto-zeroing, adaptive calibration and dynamics element matching, and signal-chopping and averaging.
V. Microfluidic System
With reference again to
In other aspects of the embodiment shown in
Generally, the top layer of an CMOS chip includes a silicon nitride or polyimide passivation layer, whose purpose is to prevent chemical elements such as sodium from penetrating into the chip. According to one embodiment, a microfluidic system 300 may be further fabricated on the top of the CMOS chip passivation layer, wherein the microfluidic system includes micropatterned polyimide sidewalls in desired shapes so as to form channels, or “mini canals,” to guide samples.
In particular,
Once diced, the respective substrate portions 104 each including a single chip 102 may be spin-coated with polyimide and then patterned using conventional lithography techniques. Since the CMOS chip surface layer generally includes a polyimide passivation layer, micropatterned polyimide sidewalls can be fabricated with good adhesion to the similar-material passivation layer.
After the fabrication of the fluidic channel 316 in the polyimide layer 310, according to one embodiment the surface of the fluidic channel may be optionally coated (e.g., spin-coated) with a thin layer of polydimethylsiloxane, or PDMS. PDMS is a biocompatible material whose surface can be functionalized to either encourage or prevent cell adhesion. For example, in one aspect of this embodiment, treating the oxidized surface of polymerized PDMS with Fibronectin (FN) makes it amenable to micro-patterning of extracellular matrix proteins to facilitate cell adhesion and spreading. In another aspect, treating the surface of PDMS with Pluronic F127 can block protein absorption, thus preventing the adhesion of cells. These respective characteristics may facilitate different aspects of guiding biological samples down the microfluidic channels of a cell sorter according to one embodiment of the present disclosure (discussed further below in Section VI), and for directing the cells to specific locations during two-dimensional micro-scale tissue assembly according to another embodiment of the present disclosure (also discussed further below in Section VII). In various implementations, PDMS may be spin-coated to micron-thickness layers onto the surface of the fluidic channel, without compromising sample manipulation or imaging.
As illustrated in
Finally, as illustrated in
As illustrated in
Next, as illustrated in
According to another embodiment, the hybrid system 100 shown in
As shown in
In sum, according to various embodiments discussed above, an overall fabrication process for a CMOS/microfluidic hybrid system may include the following steps, in an appropriate order depending on the particular technique used: 1) silicon foundry fabrication of CMOS chip including microcoil array, digital switching network, imaging (e.g. RF) electronics and related circuitry, and temperature regulation electronics; 2) optional Permalloy deposition in appropriate microcoils to increase magnetic field strength; 3) fabrication of the microfluidic system (e.g., either on the chip directly via polyimide-based or ultraviolet epoxy-based techniques or separately with soft lithography PDMS mold); 4) PDMS coating of the CMOS chip surface with various agents for biocompatibility; 5) application of cover slip to from fluidic channel(s)/chamber; and 6) assembly of the CMOS/microfluidic hybrid system with an electrical board (e.g., package substrate) and a temperature controller (e.g., thermoelectric cooler).
VI. Sample Counting and Sorting
According to another embodiment, a hybrid system 100 including sample detection and imaging components as discussed above in Section III, and various configurations of a microfluidic system as discussed above in Section V, may be employed in a number of cell counting, sorting and identification applications.
For example, FIGS. 39(a)-(d) illustrate various exemplary implementations of cell detection via RF sensing techniques as discussed above in connection with
In another example, as shown in
Another embodiment according to the present disclosure is directed to precision cell sorting methods and apparatus based on a CMOS/microfluidic hybrid system including RF/detection components, pursuant to various embodiments discussed above. Isolating a homogeneous cell population with high accuracy from a dissoluted organ or tissue or from batches of pooled blood is important for conducting gene expression analysis, for cell and tissue engineering assays requiring a pure cell line, or for clinical applications (e.g., stem cell separation for bone marrow reconstitution procedures in cancer patients.). Many cells can be recognized due to the expression of unique cell surface receptors. In conventional approaches, magnetic beads coated with the ligand for these receptors have been used to engage the cells with magnetic tweezers and magnetic twisting cytometry. This technique has been used for cell sorting/separation as well, but the conventional magnetic separation technique employs a simple stationary magnet that statistically sorts a large group of bead-bound cells all at once, lacking controllability and precision. In contrast to conventional approaches, one embodiment of the present disclosure combines the high controllability of CMOS electronics with micro-scale manipulation and detection capabilities of the microcoil array to realize ultra-precise, high-throughput, and automated cell sorting methods and apparatus for individual biological cells attached to magnetic beads within heterogeneous suspensions.
In one exemplary implementation of this embodiment, as illustrated in
In
The cell sorting methods and apparatus exemplified in the arrangement of
Additionally, the cell sorting methods and apparatus discussed above facilitate parallel fluid processing with multiplexed microfluidic channels and CMOS circuits. CMOS electronics also makes possible automation in cell sorting. In comparison with fluorescence-activated cell sorters (FACS), a system according to the concepts discussed herein may be implemented in a much smaller and less expensive manner. Moreover, a cell sorting system according to the present disclosure requires minimal preparation of the cells for sorting (e.g., no transfection of fluorescent proteins). Additionally, in another aspect, it is arguably easier to maintain physiological homeostasis with a microfluidic system than any large volume device.
According to various aspects of the embodiment illustrated in
For example, by targeting endothelial cells in one exemplary implementation, PECAM is an ideal choice of cell surface molecules because of its unique expression in endothelial cells and because of its role in cell mobility and cellular adhesion; as a result, the likelihood of detachment of the bound magnetic bead during transit is reduced. In another implementation, endothelial cells may be sorted from a cell suspension also containing NIH 3T3 fibroblasts which do not express PECAM. The throughput rates and density of the cell suspensions may be calibrated for optimal sorting performance.
Also, in other implementations, an iterative process may be employed, wherein experimental parameters optimized in a first sorting process serve as the initial conditions for one or more subsequent sorting processes, such that cells may be sorted from suspensions containing multiple cell types. For example, in one process involving the neonatal heart, endothelial cells may be separated from cardiac myocytes, fibroblasts, immune cells that have extravasated prior to harvest, and neural tissue. The ‘noisy’ environment created by this mixed cell population in some cases determines the boundaries of cell sorting performance. In one aspect, diluting the cell suspension may increase the time required for sorting, but may increase sort accuracy. In another aspect, to assure sufficient dissolution, a suspension may be passed through a filter that selectively filters large cellular ensembles that have evaded dissolution by trypsin and collagenase.
VII. Tissue Assembly
In yet another embodiment according to the present disclosure, micro-scale assembly of engineered tissues may be realized using the various methods and apparatus discussed herein. For example, in one implementation, assembly of micro-scale, engineered cardiac tissues from heterotypic cell populations is accomplished utilizing a CMOS/microfluidic hybrid system 100 as discussed herein.
A complex signaling dialogue between multiple cell types in a tightly constrained space that is reorganizing with each developmental step mediates tissue morphogenesis. In the mature tissue, the spatial and demographic control of these cell populations is strenuously maintained but its loss marks the onset of the disease process in a recognizable fashion. What is unknown is how the subtle interactions of seemly controlled cell populations can potentiate pathogenic events. An excellent example of this is the cell-cell interactions between capillaries and cardiac muscle fibers in the heart, which alters action potential propagation, contributing to arrhythmogenesis. This is an important problem because there is currently no clinically reliable means of treating cardiac arrhythmias medicinally. Furthermore, antiarrhythmic drug pipelines at pharmaceutical and biotechnology companies are barren, in part due to a lack of experimental assays that support the identification of new drug targets. Thus, the ability to engineer micro-scale cardiac tissues of heterogeneous cell populations offers reliable, effective assays of cardiac arrhythmia for the discovery of new drug targets and the elucidation of answers to fundamental questions in cardiac electrophysiology.
More generally, heterotypic signaling between different cell populations defines the tissue micro-environmental changes in tumors, the heart, and liver. Therefore, micro-scale tissue assembly is important to study communication networks amongst different cell types, drug efficacy, and for fundamental physiological study in a standardized, repeatable manner. However, precise engineering of model tissues on micro-scale has proven difficult.
Several techniques for heterotypic cell culture with population control exist. Transwell plates have traditionally been used to study paracine signaling between two distinct cell populations. New techniques for mimicking the tissue microenvironment in vitro have relied on photolithographic techniques. One known strategy is based on using patterned photoresists or masks to allow cell attachment to select regions of a surface. Subsequent removal of the resist or mask reveals areas amenable to a second cell type's adhesion. A second strategy exploits dielectrophoresis to pattern and separate cervical carcinoma cells from red and white blood cells on a microelectrode array. Other strategies include microfluidic channels to direct cell suspensions to different locations on a surface, an electroactive mask that allows seeding of a second cell type to regions of a surface that were electrically activated to permit attachment, and gravity-enforced tissue assembly. These techniques have proven to be labor intensive, lacking precise population control, and slow. The technique based on dielectrophoresis is interesting, because it represents a strategy for cell sorting and micro-scale tissue reconstruction; however, it lacks the accurate cell population control required to do quantitative studies, the spatial control afforded by micropatterning technologies, and is reliant upon the cells having distinct polarizabilities for effective trapping and patterning. This prevents the guarantee of homogeneous cell populations, which can be assured only through molecular specificity.
In view of the foregoing, one embodiment according to the present disclosure is directed to the assembly of a two-dimensional tissue, as illustrated in
Subsequently, this endothelial tissue is assembled as an “embedded tissue” within a preexisting cardiac muscle tissue. In one embodiment, two-dimensional cardiac tissues may be built by culturing neonatal rat ventricular myocytes on micropatterned Fibronectin. Dissociated cardiac myocytes are cultured in micropatterned FN lines, as shown in
Using the microcoil array, capillary endothelial cells may be embedded in precise formations relative to the fiber orientation of the engineered cardiac tissue, as shown in
The small, focal adhesion-sized FN islands may not be amenable to myocyte adhesion and spreading because the spontaneous contraction of these myocytes tears them from a single, small FN island before they can sufficiently adhere. However, capillary endothelial cells bind these islands and extend lamellipodia to spread to occupy several simultaneously. Thus, regions that are micropatterned with small FN islands are capable of selectively hosting endothelial cells but not cardiac myocytes (See
According to the foregoing methodology, uniformity and geometric precision of the endothelial cell embed, as well as preventing the invasion of endothelial cells amongst the cardiac muscle fibers, may be accomplished. Applicants have recognized and appreciated that prepositioning of the cardiac myocytes on the micropatterned surface prior to the assembly of the endothelial embed is an important step in the process. In particular, cardiac myocytes require more time to attach and conform to extracellular matrix cues than other cell types. Additionally, capillary endothelial cells are quite migratory, whereas the cardiac myocytes are not. Thus, by prepositioning the cardiac myocytes, the cells of the endothelial embed may be effectively contained to their designated regions after assembly.
Conclusion
Various embodiments of a hybrid system as discussed herein incorporate elements of electromagnetics, microfluidics, semiconductor physics, lithographic techniques, high frequency (e.g., RF) electronics, analog/digital integrated circuits, feedback control and biology in a complementary system. In various exemplary implementations, such a hybrid system may be configured as a “biochip,” providing a versatile programmable device that can perform a wide range of biological experiments on a submicron scale, and thereby significantly benefit “lab-on-a-chip” development of industrial, scientific and military interests.
Having thus described several illustrative embodiments, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of this disclosure. While some examples presented herein involve specific combinations of functions or structural elements, it should be understood that those functions and elements may be combined in other ways according to the present invention to accomplish the same or different objectives. In particular, acts, elements, and features discussed in connection with one embodiment are not intended to be excluded from similar or other roles in other embodiments. Accordingly, the foregoing description and attached drawings are by way of example only, and are not intended to be limiting.
The present application claims the benefit, under 35 U.S.C. §119(e), the following U.S. provisional applications: Ser. No. 60/561,704, filed Apr. 13, 2004, entitled “Programmable Integrated Biochip;” Ser. No. 60/611,370, filed Sep. 20, 2004, entitled “An I/C Microfluidic Hybrid Microsystem for 2D Magnetic Manipulation of Individual Biological Cells;” and Ser. No. 60/627,940, filed Nov. 15, 2004, entitled “Methods and Apparatus for Manipulation and/or Detection of Biological Samples and Other Objects.” Each of the foregoing applications is hereby incorporated herein by reference.
Some of the research relating to the subject matter disclosed herein was sponsored by the following government grants, and the government has certain rights to some disclosed subject matter: NSF-PHY-0117795, NSF-DMR-98-09363, NSF-PHY-9871810, NSF-DMR-98-02242, DARPA-DAAD 19-01-1-0659, ONR-N0014-95-1-0104, and ONR-N00014-99-1-0347.
| Number | Date | Country | |
|---|---|---|---|
| 60561704 | Apr 2004 | US | |
| 60611370 | Sep 2004 | US | |
| 60627940 | Nov 2004 | US |
