Patent Application
20110189650
1. Field of the Invention
This invention relates to biological cell sorting and purification systems. Certain embodiments are particularly adapted for use in microfluidic plumbing arrangements to selectively kill one or more entire population of undesired cells.
2. State of the Art
It is sometimes desirable to sort one or more selected population of biological particles from a sample containing a plurality of different populations of particles. For example, it may be desired to select for culture only a subset of particles that are present in a mixture of particles. If physical cell sorting is not done, selective cell killing may sometimes be done instead. However, commercially available killing devices and methodologies, such as lethal reagents that may be added to a fluid sample, are less flexible and precise than desired.
Conventional cell sorting devices tend to be complex, bulky, and expensive. An exemplary cell sorter based on a cytometric device with sheath flow is disclosed in U.S. Pat. No. 7,392,908 to Frazier. A particle analyzer including side-scatter detection and a cytometric device with capillary fluid flow is disclosed in U.S. Pat. No. 7,410,809 to Goix, et al. Causing magnetic beads to bind to selected cells is a known useful step in a technique to “hold back” and remove the bound cells from a population of cells, as disclosed in U.S. Pat. Nos. 7,417,418 and 7,579,823 to Ayliffe. The latter two utility patents also disclose microfluidic devices that are useful to interrogate biological particles as such particles flow through a thin film sensor.
It would be an improvement to provide a device, and a method of its use, for rapidly, inexpensively, and accurately manipulating a viable population of biological particles by discriminately changing a portion of the particles in a sample. One such change would desirably include purifying a viable population of biological particles by discriminately killing all of, or substantially all of, the undesired particles. An alternative desirable change would include providing structure effective to permit electroporating a selected portion of the sample.
This invention provides an apparatus that may be used for interrogating and modifying (including “purifying”) a sample of fluid that carries biological particles. The purification process may include killing all, or substantially all, biological particles that do not reside in a population of desired, or at least tolerable, particles. Preferred embodiments of the invention include alignment structure, detection structure, discrimination structure, manipulation structure, and a trigger operable to actuate the manipulation structure responsive to input received from one or both of the detection structure and the discrimination structure.
A workable alignment structure is configured and arranged to urge biological particles, which are carried in a fluid, toward substantially single-file travel through an interrogation zone. Workable alignment structure comprises a fluid sheath (such as provided in cytometry devices), a capillary device, or a fluid-carrying channel, such as may be formed in a thin film layer. An interrogation zone may broadly be defined as an area or volume in which information may be gathered about particles carried in a fluid diluent. Sometimes, an interrogation zone is carried on a disposable device that is adapted for one-time-use. A currently preferred such disposable device is embodied as a microfluidic cartridge. An exemplary such cartridge may be formed from a stack of thin film layers arranged to define a labyrinth channel through which fluid may be urged to flow.
Detection structure may include any structure operable to detect the presence of a first biological particle in the interrogation zone. Exemplary detection structure comprises a plurality of electrodes disposed in operable association with an orifice effective to permit detecting the presence of a particle in the interrogation zone by way of the Coulter principle. Certain detection structure may also characterize one or more particle characteristic, such as particle size. Alternative detection structure includes a radiation source disposed to impinge radiation comprising substantially a first frequency into the interrogation zone; and a radiation detector disposed to detect a Stokes' shift in the first frequency. Another alternative detection structure comprises a radiation source disposed to impinge radiation comprising substantially a first frequency into the interrogation zone; and a radiation detector disposed to detect side-scatter of the radiation.
Discrimination structure is operable to distinguish the first biological particle as either residing inside a defined population of particles, or not. Manipulation structure is configured and arranged substantially discriminately to manipulate a selected biological particle in a manipulation zone that is associated with the interrogation zone.
One workable trigger is adapted to operate the manipulation structure in the case when a detected biological particle of interest is both present in the killing zone; and resides inside the defined population of particles. In other cases, a workable trigger is adapted to operate the manipulation structure in the case when a detected biological particle is both: present in the killing zone; and resides outside the defined population of particles.
A particle manipulation zone may be disposed as a sub-portion of the interrogation zone, overlap a portion of the interrogation zone, or encompass the entire interrogation zone. Sometimes, a manipulation zone may extend, or be entirely disposed, downstream of the interrogation zone by a known time-of-flight for a biological particle to be manipulated. Sometimes, a manipulation zone may be disposed downstream of detection structure by a known time-of-flight for a biological particle to be manipulated.
One operable manipulation structure is embodied as killing structure that includes a radiation source having sufficient discharged energy density to permit exposing a biological particle, during the time that biological particle is passing through a killing zone, to at least that quantity of energy sufficient to kill the biological particle. One exemplary killing structure comprises a laser. Alternative killing structure within contemplation nonexclusively includes electric elements capable of causing voltage or current spikes, LEDs, and Arc lamps of various types.
Certain embodiments of the invention may be structured to form a microfluidic device including alignment structure configured and arranged to urge biological particles, which are carried in a fluid, toward substantially single-file travel through an interrogation zone. One such device also includes detection structure operable to detect the presence of a first biological particle in the interrogation zone using electrical impedance in accordance with the Colter principle. Further, that device includes discrimination structure operable to distinguish the first biological particle as either residing inside a defined population of particles, or not. The exemplary device may also include killing structure configured and arranged substantially discriminately to kill a selected biological particle in a killing zone that is associated with the interrogation zone. Alternatively, the device may include electroporation structure effective to electroporate one or more particle, as desired. Finally, an exemplary device also may include a trigger operable to discriminately actuate certain particle manipulation structure responsive to input received from both of, or either of, the detection structure and the discrimination structure.
A device structured according to certain principles of the instant invention may be used in a method to identify and manipulate selected biological particles. The method broadly includes providing a microfluidic device comprising: alignment structure, detection structure, discrimination structure, particle manipulation structure, and a trigger operable to actuate the manipulation structure responsive to input received from one or both of the detection structure and the discrimination structure. Broadly, the alignment structure should be configured and arranged to urge biological particles, which are carried in a fluid, toward substantially single-file travel through an interrogation zone. Workable detection structure includes any structure operable to detect the presence of a first biological particle in the interrogation zone. Exemplary discrimination structure is operable to distinguish the first biological particle as either residing inside a defined population of particles, or not. Operable manipulation structure is configured and arranged substantially discriminately to manipulate substantially a single selected biological particle in a manipulation zone that is associated with the interrogation zone. Preferred manipulation structure is effective to cause a change to essentially a single particle, within realistic constraints imposed by coincidence. The method continues by introducing a fluid sample, comprising biological particles carried by a dilutant fluid medium, for flow of the sample past the alignment structure. Then, the method includes operating the trigger to actuate the manipulation structure effective to manipulate a selected portion of biological particles responsive to input received from one or both of the detection structure and the discrimination structure as the sample flows through the device. Manipulation within contemplation nonexclusively includes: killing, lysing, and electroporating a particle. Sometimes, the selected portion is defined by a common characteristic that is directly detected by the discrimination structure. Other times, the selected portion is defined by a common characteristic that is not directly detected by the discrimination structure.
In the drawings, which illustrate what are currently considered to be the best modes for carrying out the invention:
Reference will now be made to the drawings in which the various elements of the illustrated embodiments will be given numerical designations and in which the invention will be discussed so as to enable one skilled in the art to make and use the invention. It is to be understood that the following description is only exemplary of the principles of the present invention, and should not be viewed as narrowing the claims which follow.
Currently preferred embodiments of the present invention provide low-cost, disposable, sensors operable to perform analyses of various sorts on particles that are carried in a fluid. Sensors structured according to certain principles of the instant invention may be used once, and discarded. However, it is within contemplation that such sensors may alternatively be reused a number of times.
Examples of analyses in which embodiments of the invention may be used to advantage include, without limitation, counting, characterizing, or detecting members of any cultured cells, and in particular blood cell analyses such as counting red blood cells (RBCs) and/or white blood cells (WBCs), complete blood counts (CBCs), CD4/CD8 white blood cell counting for HIV+ individuals; whole milk analysis; sperm count in semen samples; and generally those analyses involving numerical evaluation or particle size distribution for a particle-bearing fluid (including nonbiolgical). Embodiments of the invention may be used to provide rapid and point-of-care testing, including home market blood diagnostic tests. Certain embodiments may be used as an automated laboratory research cell counter to replace manual hemocytometry.
Broadly, preferred embodiments are adapted to perform one or more operation on one or more selected particle that is entrained in a fluid carrier. Exemplary such operations nonexclusively include: detecting, counting, characterizing, killing, and/or modifying cells, such as by way of an electroporating process. Certain preferred embodiments of the invention are adapted to provide a low-cost fluorescence activated cell sorter (FACS) that may be used to selectively kill biological particles and thereby “purify” a fluid sample. Other preferred embodiments may be used to transfect a population, or a subset of a population, of cells.
For convenience in this disclosure, the invention will generally be described with reference to its use as a particle detector and killer Such description is not intended to limit the scope of the instant invention in any way. It is recognized that certain embodiments of the invention may be used simply to detect passage of particles, e.g. for counting. Other embodiments may be structured to determine particle characteristics, such as size, or type, thereby permitting discrimination analyses. Furthermore, for convenience, the term “fluid” may be used herein to encompass a fluid mix including a fluid base formed by one or more diluents and particles of one or more types suspended or otherwise distributed in that fluid base. Particles are assumed to have a characteristic “size”, which may sometimes be referred to as a diameter, for convenience. Currently preferred embodiments of the invention are adapted to interrogate particles found in whole blood samples, and this disclosure is structured accordingly. However, such is not intended to limit, in any way, the application of the invention to other fluids including fluids with particles having larger or smaller sizes, as compared to blood cells.
In this disclosure, “single-file travel” is defined different than literally according to a dictionary definition. For purpose of this disclosure, substantially single-file travel may be defined as an arrangement of particles sufficiently spread apart and sequentially organized as to permit reasonably accurate detection and discriminate killing of particles of interest. When two particles are in the interrogation zone at the same, it is called coincidence, and there are ways to mathematically correct for it. Calibration may be performed using solutions having a known particle density (e.g. solutions of latex beads having a characteristic size similar to particle(s) of interest). Also, dilution of the particles in a fluid carrier may contribute to organizing particle travel. As a non-limiting example, the desired particle density to urge single-file travel and reduce or avoid coincidence is approximately between about 3×103 to about 3×105 cells/ml, where the particle size is on the order of the size of a white blood cell.
The term “microfluidic” is used in this disclosure somewhat more broadly than might be its conventional definition. As used herein, the term “microfluidic” is intended to broadly encompass fluid flow arrangements that urge particles of interest, which are carried by a fluid stream, into substantially single-file travel through an interrogation zone. Exemplary devices to accomplish such behavior may contain a fluid flow constriction having a characteristic size on the order of between about a few microns to about millimeter scale, and sometimes, even larger.
As illustrated in
Detection structure 55 encompasses any device, or assembly of devices and elements, operable to detect the presence of a biological particle in an interrogation zone 68. Broadly, an interrogation zone 68 is an area in which information about a particle may be determined. Exemplary such information includes particle size, type, and presence. Desirably, alignment structure 50 cooperates with, and sometimes may encompass, an amount of sample dilution to reduce particle coincidence to an acceptable level and urge particles into single-file travel through the interrogation zone 68.
A particle manipulation zone that is “associated” with the interrogation zone 68 means the particle manipulation zone may be directly present in the interrogation zone, or may be located at a position that is determinable based upon operational characteristics of the device, e.g at a known distance from, and with a known (or determinable) particle time-of-flight downstream from, detection structure 55.
In
Discrimination structure 57 encompasses any device, or assembly of devices and elements, operable to distinguish biological particles as either residing inside a defined population of particles (e.g. particles of interest), or not. In
Particle manipulation structure 60 encompasses any device, or assembly of devices and elements, configured and arranged to cause a “particle manipulation”. “Particle manipulation” encompasses lysing, killing, and/or electroporation of particles, among other physical changes that may be imposed onto a particle. Sometimes, particles may even be sorted in the traditional sense (i.e., separating or removing specific cells from the population or dividing cells into separate groups). Desirably, such particle manipulation may be performed on a discriminating basis to less than the entire population of particles in a sample. Most preferably, such particle manipulation may be performed on substantially a particle-by-particle basis. That is, preferred embodiments are effective to manipulate substantially a selected particle vs. essentially millions of particles at a time.
One exemplary particle manipulation structure 61 is adapted to kill a selected biological particle in a killing zone that is associated with the interrogation zone. Operable killing structure 61 nonexclusively includes lasers and other energy-outputting devices. Although it is not required, typically a dedicated killing structure 61, such as a laser, is selected having a significantly different wavelength compared to the excitation radiation source 71. For example, a killing laser is typically selected to emit in the ultraviolet (UV) spectrum, or infrared (IR) spectrum. In contrast, an excitation radiation source 71 typically emits radiation in the visible spectrum. However, it is within contemplation that the intensity of the excitation source 71 could simply be increased sufficiently to effect a kill when desired.
Assemblies structured according to certain principles of the invention also include a trigger operable to actuate a particle manipulation structure 60 responsive to analysis of data received from one or both of a detection structure 55 and a discrimination structure 57. With reference still to
For example, and with further reference to
With reference now to
The thickness, T1, of an opaque member and characteristic size, D1, of an orifice 108 are typically sized in agreement with a size of a particle of interest to promote single-file travel of the particle through the opaque member, and to have substantially only one particle inside the orifice at a time. In the case where the apparatus is used to interrogate blood cells, the thickness of the opaque member may typically range between about 10 microns and about 300 microns, with a thickness of about 125 microns being currently preferred. The diameter, or other characteristic size of the orifice, may range between about 2 and 200 microns, with a diameter of about 50 microns being currently preferred for analysis and/or manipulation of blood cells.
An operable opaque member 102 may function, in part, to reduce the quantity of primary radiation 118 (or sometimes characterized as excitation radiation) that is emitted by source 104, which is received and detected by radiation detector 106. Primary radiation 118 is illustrated as a vector having a direction. Desirably, substantially all of the primary radiation 118 is prevented from being detected by the radiation detector 106. In any case, operable embodiments are structured to resist saturation of the detector 106 by primary radiation 118. In certain embodiments, primary radiation 118 may simply pass through orifice 108 for reception by the radiation detector 106. Therefore, as will be further detailed below, certain embodiments may employ one or more selective radiation filters as a measure to control radiation received by detector 106, or alternatively, direct primary radiation 118 at an angle with respect to the detector 106.
The opaque member 102 illustrated in
A workable core 122 for use in detecting small sized particles can be formed from a thin polymer film, such as PET having a thickness of about 0.005 inches. Such polymer material is substantially permeable to radiation, so one or more coatings, such as either or both of coating 124 and 126, can be applied to such core material, if desired. A workable coating includes a metal or alloy of metals that can be applied as a thin layer, such as by sputtering, vapor deposition, or other well-known technique. Ideally, such a layer should be at least about 2-times as thick as the wavelength of the primary radiation, e.g. about 1 μm in one operable embodiment. The resulting metallized film may be essentially impervious to transmission of radiation, except where interrupted by an orifice. Aluminum is one metal suitable for application on a core 122 as a coating 124 and/or 126.
The apparatus 80 illustrated in
It should be noted, for purpose of this disclosure, that the term “wavelength” is typically employed not necessarily with reference only to a single specific wavelength, but rather may encompass a spread of wavelengths grouped about a characteristic, or representative, wavelength. With reference to
With reference still to
Sometimes, and as illustrated in
A radiation source 104 may be formed from a broad spectrum radiation emitter, such as a white light source. In such case, it is typically preferred to include a pre-filter 188 adapted to pass, or transmit, radiation only in a relatively narrow band encompassing the characteristic value required to excite a particular fluorescing agent associated with a particle of interest. It is generally a good idea to limit the quantity of applied radiation 118 that is outside the excitation wavelength to reduce likelihood of undesired saturation of the radiation detector, and consequent inability to detect particles of interest.
In one embodiment adapted to interrogate blood cells, it is workable to use a red diode laser, and to include a short pass filter (after the diode laser), or excitation filter, that passes primary light radiation with wavelengths shorter than about 642 nm. A currently preferred embodiment adapted to interrogate blood cells uses a green diode laser, and includes a short pass filter, or excitation filter, that passes primary light radiation with wavelengths shorter than about 540 nm. It is also currently preferred to include a band pass filter (prior to the photodetector) with a peak that matches a particular selected fluorescence peak. Commercially available dyes may be obtained having characteristic fluorescent peaks at 600, 626, 660, 694, 725, and 775 nanometers. Long pass filters are also often used in place of band-pass filters prior to the photodetector. The pipette tip “cap layer” and “substrate” can also be designed to act as optical filters to aid or eliminate the need for the traditional excitation and emission filters. In this disclosure, “Post filter” may more conventionally be referred to as an “emission filter”.
With continued reference to
Certain particle manipulation structure 60, such as laser 194, is disposed to permit impinging lethal radiation 196 onto biological particles that are members of one or more undesired population. Detection of the presence of a particle can be determined by radiation detector 106, or with alternative detection structure. Information 73 from radiation detector 106 may be input to discrimination structure 57. When the particle is determined to be a member of a population that is desired to be removed to “purify” the sample, trigger 75 may enable discharge of the killing laser 194. Power for the killing laser 194 can be provided by way of wires generally indicated at 198.
It is within contemplation that one or more additional elements may be included in an embodiment such as illustrated in
It is within contemplation that two or more of the illustrated layers may be concatenated, or combined. Rather than carving a channel out of a layer, a channel may be formed in a single layer by machining or etching a channel into a single layer, or by embossing, or folding the layer to include a space due to a local 3-dimensional formation of the substantially planar layer. For example, illustrated layers 202 and 204 may be combined in such manner. Similarly, illustrated layers 208 and 210 may be replaced by a single, concatenated, layer.
With continued reference to
Certain sensor embodiments employ a stimulation signal based upon driving a desired current through an electrolytic fluid conductor. In such case, it can be advantageous to make certain fluid flow channel portions approximately as wide as possible, while still achieving complete wet-out of the stimulated electrodes. Such channel width is helpful because it allows for larger surface area of the stimulated electrodes, and lowers total circuit impedance and improves signal to noise ratios. Exemplary embodiments used to interrogate blood samples include channel portions that are about 0.10″ wide and about 0.003″ high in the vicinity of the stimulated electrodes.
One design consideration concerns wettability of the electrodes. At some aspect ratio of channel height to width, the electrodes may not fully wet in some areas, leading to unstable electrical signals and increased noise. To a certain point, higher channels help reduce impedance and improve wettability. Desirably, especially in the case of interrogation electrodes, side-to-side wetting essentially occurs by the time the fluid front reaches the second end of the electrode along the channel axis. Of course, wetting agents may also be added to a fluid sample, to achieve additional wetting capability.
Still with reference to
In general, disposing the electrodes 220 and 222 closer to the tunnel portion 214 is better (e.g., gives lower solution impedance contribution), but the system would also work with such electrodes being disposed fairly far away. Similarly, a stimulation signal (such as electrical current) could be delivered using alternatively structured electrodes, even such as a wire placed in the fluid channel at some distance from the interrogation zone. The current may be delivered from fairly far away, but the trade off is that at some distance, the electrically restrictive nature of the extended channel will begin to deteriorate the signal to noise ratios (as total cell sensing zone impedance increases).
With continued reference to
Certain embodiments may be used to perform an operation on certain particles. Sometimes, the cells that are operated on can be a subset of a population, and other times the entire population of cells in a sample may be effected. For example, a sensor, such as sensor 200, may be used to electroporate desired cells. The electrical signal applied by generator 228 may be changed as needed, or a different signal generator may be used. Still with reference to
As illustrated in
Cell detection using the Coulter principle is preferably done by making a differential voltage measurement between electrode 220 and electrode 222 using a known constant current applied between electrode 224 and electrode 226. With reference to
In one case illustrated in
Fortunately, the diluent in which cells can live falls within an acceptable range for transmission of electrical signals (e.g. for cell detection and/or electroporation. The shape of the preferred electroporation signal is a square wave, although other shapes (like sinusoidal) may work. Faster/sharper rise times are believed to be desirable. Signal amplitude may also be an important variable. While the optimum signal amplitude is not yet isolated, it has been determined that an electroporation signal amplitude of 100V is workable.
It is currently believed that at least about 3 pulses of about 100 volts are required to be imparted to a cell to accomplish suitable electroporation. In an exemplary device 200, a cell flows through the interrogation zone in about 200 μsec. Therefore, a 20 kHz electroporation stimulus is believed appropriate under such conditions.
Electrodes may be positioned at a plurality of useful locations along a fluid channel. One or more electrical property may be monitored between strategically positioned electrodes to obtain information about the sample, and/or particles carried in the fluid. For example, with reference to
As illustrated in
An exemplary sensor 200 may be formed, at least in part, from a plurality of stacked and bonded layers of thin film, such as a polymer film. In an exemplary sensor component 200 used in connection with interrogation of blood cells, it is currently preferred to form top and bottom layers 202 and 210 from Polyamide or Mylar film. A workable range in thickness for Polyamide layers for such application is believed to be between about 0.1 micron to about 500 microns. A currently preferred Polyamide layer 202, 210 is about 52 microns in thickness. It is further within contemplation that a pair of top and/or bottom layers can be formed from a single layer including fluid channel structure formed e.g. by molding, etching, or hot embossing. Sometimes, a sensor structured according to certain principles of the invention may be made reference to as a cartridge, or cassette.
It is currently preferred to make the spacer layer 206 from Polyamide also. However, alternative materials, such as Polyester film or Kapton, which is less expensive, are also workable. A film thickness of about 52 microns for spacer layer 206 has been found to be workable in a sensor used to interrogate blood cells. Desirably, the thickness of the spacer layer is approximately on the order of the particle size of the dominant particle to be interrogated. A workable range is currently believed to be within about 1 particle size, to about 15 times particle size, or so. A double-sided adhesive polymer film is currently preferred as a material of composition for combination bonding-channel layers 204 and 208. Layers 204 and 208 in a currently preferred sensor 200 are made from double-sided Polyamide (PET) tape having a thickness of about 0.0032 inches. Alternatively, a plain film layer may be laminated to an adjacent plain layer using heat and pressure, or adhesively bonded using an interposed adhesive, such as acrylic or silicone adhesive.
The channel portion 214 is typically laser drilled through layer 206, although alternative hole-forming techniques are workable. A diameter of 35 microns for channel 214 is currently preferred to urge blood cells into single-file travel through the interrogation zone. Other cross-section shapes, other than circular, can also be formed during construction of channel 214. Naturally, the characteristic size of the orifice formed by drilling channel 214 will be dependent upon the characteristic size of the particles to be characterized or interrogated. Counter-boring can be performed on thicker layers to reduce the “effective thickness” of the sensing zone, if desired.
One multi-layered channel embodiment, generally indicated at 240 and illustrated in
Plumbing arrangement 240 includes five layers configured and arranged to form a channel system effective to direct flow of particle bearing fluid from a supply chamber 242, through orifice 108 in an opaque member 102, and toward a waste chamber 244. Desirably, a depth of fluid guiding channels 246 and 248 is sized in general agreement with a size of a particle 250, to resist “stacking” particles near the orifice 108. Fluid can be moved about on the device 240 by imposing a difference in pressure between chambers 242 and 244, or across orifice 108 disposed in opaque member 102. For example, a positive pressure may be applied to the supply chamber 242. Alternatively, a negative pressure (vacuum) may be applied to the waste chamber 244. Both positive and negative pressures may be applied, in certain cases. Alternative fluid motive elements, such as one or more pumps, may be employed to control particle travel through opaque member 102.
Although both of supply chamber 242 and waste chamber 244 are illustrated as being open, it is within contemplation for one or both to be arranged to substantially contain the fluid sample within a plumbing device that includes a multilayer embodiment 240. Also of note, although a top-down fluid flow is illustrated in
The multilayer plumbing arrangement 240 illustrated in
During assembly of a device, bonding may be effected by way of an adhesive applied between one or more layer, or one or more layer may be self-adhesive. It is currently preferred for channel layers 256 and 258 to be manufactured from double-sided tape. One workable tape is made by Adhesive's Research (part no. AR90445). Heat and pressure may also be used, as well as other known bonding techniques. Desirably, the thickness of at least the channel layers 256, 258 is on the order of the characteristic size of particles of interest to promote single-file travel of particles through an interrogation zone. A workable thickness of such layers in currently preferred devices used to interrogate blood cells typically ranges between about 10 microns and about 300 microns.
In certain cases, at least a portion of bottom layer 260 is adapted to form a bottom window 262, through which radiation 118 may be transmitted into an excitation zone. Similarly, top layer 254 includes a portion forming a window 264, through which fluorescence may be transmitted. Therefore, the assembly 240 is arranged to form a window permitting radiation to pass through its thickness. Such window includes window portions 262, 264, certain portions of channels 246 and 248 disposed in the vicinity of orifice 108, and the orifice 108 itself. Radiation can therefore be directed through the thickness of the assembly 240 in the vicinity of the orifice 108.
Emitted fluorescence may be detected by radiation detector 106 of detection structure 55. Presence of a cell may be detected by monitoring a radiological property such as side-scatter, reduction in transmitted radiation due to blockage of aperture 108, or fluorescence. In the event that a cell is detected in the interrogation zone, discrimination structure 57 is operable to distinguish in which population the cell resides. Desirable cells are permitted to pass through the interrogation zone without incident. However, cells in undesired population(s) are killed by particle manipulating structure 60, which is discriminately controlled by trigger 75. The resulting collected sample is therefore “purified”, in that the remaining viable cells are all members of a desired population of cells. The “purified” sample may then be manipulated or further interrogated as desired.
An embodiment structured according to certain principles of the instant invention and permitting either radiological and/or electrically based interrogation of a fluid sample is indicated generally at 274 in
In currently preferred embodiments, device 274 is made from, or includes, layers of thin film. Workable films include polymers such as Kapton, Mylar, and the like. Sometimes, one or more layer may be formed from a material, such as injection molded plastic, having an increased thickness to provide enhanced bending stiffness to facilitate handling of the device 274, provide one or more larger known-volume chamber, or for other reasons.
In one exemplary use of device 274, the device is inserted into engagement in an interrogation platform configured to provide the appropriate and desired interrogation capabilities. An interrogation platform typically includes a vacuum source, and one or both of electrical and radiological instrumentation. A fluid sample is placed into sample well 292, where it flows into a chamber defined by chamber-forming voids 294, 294′, and 294″. The fluid is then drawn from channel 294″ through aperture 296 in layer 280, and into channel 298 in layer 278. As illustrated, fluid in channel 298 flows in succession over interrogation electrodes 300 and 302.
With particular reference to
After passing interrogation electrodes 300 and 302, fluid flows downward, through tunnel 228, to channel 308 in layer 282. Additional interrogation electrodes are typically disposed for contact with fluid in channel 308. Such interrogation electrodes may be used, for examples, to detect or interrogate particles moving through tunnel 228 using electrical impedance and the Coulter principle, and/or as one or more event indicator. For example, an event indicator may be used as a start/stop trigger for interrogating a predetermined volume of fluid. Arrival of a fluid wave-front causes a strong change in measured electrical impedance, and indicates the arrival of the wave-front at a first electrode location, which signal may be used to start a test. A subsequent electrode disposed downstream by a known volume may be employed to terminate the test.
As particles move past the tunnel 228, they may also, or alternatively, be interrogated radiologically (e.g. in accordance with Stokes' shift phenomena) at an interrogation zone generally associated with tunnel 228, which is structured to urge particles of interest into substantially single-file transit. As illustrated in
Impinging radiation in the illustrated transverse direction conveniently reduces the background noise applied to the detector 106, and also reduces need for filters. In alternative construction, an optical fiber may provided as a waveguide structure. It is also operable in certain alternatively structured embodiments to include radiation transmittable windows effective to permit simply impinging excitation radiation in a direction through the thickness of the interrogation cartridge, and to permit collection of Stokes' shift emitted radiation and/or side scatter radiation on the opposite side. One or more band-pass radiation filters would typically be employed in the latter configuration to reduce background noise received at detector 106.
With particular reference to
Making reference again to
As illustrated in
An operable plumbing arrangement structured according to certain principles of the instant invention may be manufactured using the following procedure to form an interrogation cartridge: 1. Lay optical fiber (a light pipe) down sandwiched into one of the layers of tape (i.e. laminate). It has been found convenient to use self-adhesive thin film tape, which can be die-cut. The various tape layers will include channels and apertures arranged on assembly to form a fluid conduit extending through the assembly and configured to form an interrogation zone through which particles of interest are urged to move in substantially single-file order. The layer the optical fiber is integrated into will typically have a receiving channel that is cut and sized to receive the fiber. 2. Additional laminate layers, or adhesive, may be added to keep the fiber in position. 3. The sub-assembly may then be sent to a laser drilling house to drill the cell sensing zone (CSZ) hole, or aperture, through the opaque layer. The hole will desirably be drilled relative to the location of the fiber (i.e., just off the end of the tip of the fiber). 4. The assembly is then typically finished when the final laminate cap layers (typically clear Mylar layers) are added. Sometimes, a stiffening substrate may be included to facilitate handling of the interrogation cartridge.
Certain components that are operable to construct an apparatus according to certain principles of the instant invention are commercially available. For example, one operable source of radiation 104 includes a red diode laser available under part number VPSL-0639-035-x-5-B, from Blue Sky Research, having a place of business located at 1537 Centre Point Drive, Milpitas, Calif. 95035. A preferred source of radiation 104 includes a green diode laser available under part number GDL7050L from Photop Technologies, Inc., having a place of business located at 21949 Plumber St., Chatsworth, Calif. 91311. Filter elements 188, 190 are available from Omega Optical, having a place of business located at 21 Omega Dr., Delta Campus, Brattleboro, Vt. 05301. Preferred filters include part numbers, 655LP or 660NB5 (Bandpass filter), and 640ASP (shortpass filter). An operable radiation detector 106 includes a photomultiplier tube available from the Hamamatsu Corporation, having a place of business located at 360 Foothill Rd., Bridgewater, N.J. 08807, under part number H5784-01. A workable killing laser 194 is available under part Number IQ 1C16 from Power Technology. Molecular Probes (a division of Invitrogen Corporation, www.probes.invitrogen.com) supplies a plurality dyes that are suitable for use in tagging certain particles of interest for interrogation using embodiments structured according to the instant invention. In particular, AlexaFluor 647, AlexaFluor 700, and APC-AlexaFluor 750 find application to interrogation of blood cells. In general, propidium iodide, PE, and CY3 find application to interrogation of cells. These dyes are also commonly used in flow cytometric applications and have specific excitation and emission characteristics. Each dye can be easily conjugated to antibodies for labeling, or tagging, different cell types. An operable fiber optic cable for forming a waveguide is available under part No. BK-0100-07 from Thor Labs, having a web site address of http://www.thorlabs.com. One useful fiber diameter is about 0.010″.
Typically, it is recommended that a user dilute the sample to the point where statistically only one particle is in a detection zone, or “manipulation zone”, at any one time. The percentage of time that more than one particle is in a zone at any one time is referred to as “coincidence”. Coincidence is a statistical event based on the density of particles in solution and the physical size of, for example, the detection zone. The detection and manipulation zones provided by preferred embodiments are smaller than other known Coulter Counter type detection zones, so coincidence is reduced (smaller is better because the detection zone will contain less volume of sample at any one time). It is currently preferred that the user run samples that are diluted to a sufficiently low cell density to keep the coincidence down to under about a 10% correction level (i.e., one in ten detected “events” happens when more than one cell is in the detection zone, and for 9 in 10 events, only a single cell is present). Coincidence is a consequence of this type of measurement. All Coulter style systems have coincidence, to a certain degree.
While it is desirable to permit manipulation of particles of interest on a particle-by-particle basis, it is recognized that there might be 2, or 3, or perhaps even 5 particles of interest in a coincidence/manipulation zone of certain preferred embodiments, but not 1,000,000, 10,000, or 1,000. Preferred embodiments are structured and arranged to resist presence of 100 particles of interest, or even 10 particles of interest (at the same time), in a manipulation zone. Therefore, currently preferred embodiments include particle manipulation structure configured and arranged in harmony with alignment structure effective to impose a change on less than about five selected biological particles of interest, at one time, in a particle manipulation zone that is associated with an interrogation zone. More preferred embodiments include particle manipulation structure configured and arranged in harmony with alignment structure effective to impose a change on less than about three selected biological particles of interest, at one time, in a particle manipulation zone. Even more highly preferred embodiments include particle manipulation structure configured and arranged in harmony with alignment structure effective to impose a change on less than about two selected biological particles of interest, at one time, in a particle manipulation zone.
Of course, it should be recognized that certain smaller particles (compared to the size of particles of interest, e.g. molecules, cell fragments, or platelets compared to white blood cells that may constitute the particles of interest) may be present and carried in a fluid diluent along with particles of interest. Such smaller particles are not considered as being particles of interest, and are not considered as being present in a proper construction of the above manipulation thresholds.
In one method in accordance with certain principles of the invention, particles (e.g. blood cells) of interest are mixed with a commercially available or custom manufactured antibody-bound fluorescently labeled molecules (i.e., obtained from Invitrogen Corporation, Carlsbad, Calif.). The mixture is then incubated for a brief period of time (approximately 5 to 15 minutes) at a temperature typically between about room temperature and abut 39 degrees Celsius. For preparation of white blood cells for interrogation, a small amount of fluorescent dye (e.g. 10 microliters) is added to about 10 microliters of whole blood, vortexed and then incubated for about 15 minutes at room temperature in the dark. A lysing agent is then added to lyse the red blood cells. Once added, the mixture is again vortexed and then allowed to incubate for another 15 minutes (in the dark).
Fluorescent markers bind to target cells (or other biological particles of interest) in the sample during the incubation period. The particles suspended in solution are then passed through the orifice detection zone from one (supply) reservoir to another (holding) reservoir, typically by applying either an external vacuum source to pull the sample through or an external positive gas source to push the sample through. Fluorescently labeled particles are excited with primary radiation (light) as they traverse the opaque member (e.g. through the interrogation orifice of a device such as 274 in
In another method in accordance with certain principles of the invention, a user may run a “gating cassette” (e.g. a test cassette structured similarly to the embodiments of
In the context of this disclosure, a “gate” is intended to encompass a characteristic, such as cell size, type, or the like. It is within contemplation to have the interrogation system set the gates automatically. In one such scenario, the system may be programmed to look for two or more discrete populations within a sample and electroporate one of those sub populations using a priori information (e.g., electroporate the larger cells, or the fluorescent cells, or the non-fluorescent cells). It is further within contemplation to run just a larger volume cassette for a short time to analyze just some first fraction of the sample fluid (i.e., analyze some cells and then stop the flow). The user, or automated system, would then set the gates and run the remainder of the volume within the same cassette. If the fractional volume used to set the gates is small enough, it may be acceptable to ignore that un-electroporated (or un-manipulated) portion of the sample.
While the invention has been described in particular with reference to certain illustrated embodiments, such is not intended to limit the scope of the invention. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered as generally illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
This application is a continuation-in-part of U.S. utility application Ser. No. 12/699,745, filed Feb. 3, 2010, and titled “Microfluidic cell sorter and method”, the priority of which is hereby claimed.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12699745 | Feb 2010 | US |
| Child | 12872749 | US |
