The present technology relates generally to systems for disrupting biological samples and associated devices and methods. Many embodiments of the present technology relate to systems for lysing cells and associated devices and methods.
Diagnosis is the first hurdle in disease management, enabling expedited appropriate treatment in developed settings where sophisticated equipment and trained personnel are available. For example, in the United States, in-vitro diagnostic procedures represent about 1.6% of Medicare spending, yet influence 60-70% of medical decisions. Nucleic acid amplification tests (NAATs) performed in the laboratory represent the pinnacle of sensitive and specific pathogen detection. Unfortunately, this state of the art is also expensive and complex, requiring infrastructure and instrumentation not available in all settings.
The lack of adequate diagnostics is especially troublesome in the case of tuberculosis (TB), which infects approximately one-third of the world's population according to the World Health Organization (WHO). Sixty percent of TB patients only have access to a peripheral level of the health system, where no suitable TB diagnostics exist. Conventional TB diagnostics in low-resource settings, mainly sputum smear microscopy and cell culture, lack the ideal specificity and timeliness. Also, the required equipment is rarely available.
Microfluidic devices have shown promise to enable the type of point-of-care device that could bring NAATs to the point of care in low-resource settings, but sample preparation, such as cell lysis, remains the weak link in microfluidics-based bioassays. Mechanical lysis methods, such as bead beating, are desirable in that one can avoid the need to purify the sample from a chemical lytic agent before the downstream bioassay, but these methods traditionally suffer from relatively complex, user- and power-intensive instruments and protocols.
Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating clearly the principles of the present disclosure.
The present technology is generally related systems for disrupting biological samples and associated devices and methods. A system for disrupting biological samples includes a vessel configured to receive the biological sample, a permanent magnet configured to be positioned within the vessel. An electromagnet is configured to be positioned proximate the vessel and a current source is operably coupled to the electromagnet and configured to transmit an alternating current. In some embodiments, when the biological sample is placed within the vessel and the alternating current is transmitted to the electromagnet, the electromagnet produces an alternating magnetic field that causes the permanent magnet to rotate within the vessel, thereby lysing at least one of the cells of the biological sample.
Specific details of several embodiments of the present technology are described below with reference to
As shown in
The vessel 120 can be a tube (e.g., a laboratory tube) having a generally cylindrical sidewall 122 and a conical bottom portion 126 that together define an interior portion of the vessel 120. The vessel 120 may, for example, be in the shape of a micro centrifuge tube (e.g., an Eppendorf tube), a centrifuge tube, a vial, etc. As shown in
It will be appreciated that although the vessel 120 shown in
The system 100 can optionally include a plurality of beads 112. The beads 112 may be pre-loaded into the vessel 120, or the user may add the beads 112 during the lysis procedure. The beads 112 can be any particle-like and/or bead-like structure that has a hardness greater than the hardness of the cells targeted for lysis. The beads 112 may be made of plastic, glass, ceramic, metal and/or any other suitable materials. In certain embodiments, the beads 112 may be made of non-magnetic materials. The beads 112 can be rotationally symmetric about at least one axis (e.g., spherical, rounded, oval, elliptic, egg-shaped, droplet-shaped particles, etc.). In other embodiments, however, the beads 112 can have a polyhedron shape. In some embodiments, the beads 112 can be irregularly-shaped particles and/or include protrusions. In certain embodiments, the mass of beads 112 added to the vessel 120 can be between about 1 mg to about 10,000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, and about 1 mg to about 10 mg, etc. In a particular embodiment, the bead mass can be between about 200 mg to about 400 mg. In certain embodiments, the individual beads 112 can have a diameter in the range of about 10 μm to about 1,000 μm, about 20 μm to about 400 μm, or about 50 μm to about 200 μm. The system 100 can include beads 112 having the same or different diameters.
Without being bound by theory, it is believed that the size and mass of beads 112 present in the vessel 120 during the lysis procedure affects the viscosity of the lysing medium, which affects the lysing efficiency. Lysing efficiency can be quantified, for example, by measuring the amount of DNA recovered from the lysed sample (e.g., via quantitative nucleic acid amplification). As demonstrated by the graphs shown in
Referring back to
In some embodiments, the inner surface of the vessel 120 and/or the outer surface of the permanent magnet 130 may include one or more protrusions (not shown) or may be otherwise texturized to increase the surface area of the respective surface and improve lysing efficiency. For example, one or more protrusions can be adhered to or formed on the outer surface of the permanent magnet 130 and/or inner surface of the vessel 120 (e.g., via adhesive, soldering, welding, electrodeposition, etc.). The protrusions can have any suitable shape, size and/or configuration (e.g,. spherical, cubical, cylindrical, half-spherical, polyhedron, non-polyhedron, etc.).
The electromagnet 140 includes a coiled magnet wire 142 embedded within or surrounded by a tubular support 144. In other embodiments, the magnet wire is in a spiral or helical configuration. In some embodiments, the electromagnet 140 only includes the magnet wire 142 (and not the support 144). The electromagnet 140 is configured to be electrically coupled to the current source 180 (e.g., via a cable 170). When activated, the current source 180 delivers an alternating current (e.g., an electrical audio signal) to the magnet wire 142 of the electromagnet 140.
The current source 180 can be a battery-powered portable electronic device (e.g., a mobile electronic device) capable of generating an electrical audio signal. For example, the current source 180 can be configured to generate an alternating current that alternates between about 10 Hz and about 90 Hz. In some embodiments, the current source 180 can generate an alternating current that alternates between about 20 Hz and about 60 Hz (e.g., about or equal to 30 Hz, about or equal to 40 Hz, about or equal to 60 Hz, etc.). The current source 180 can include a cell phone, a portable audio device (e.g., a portable mp3 player, a portable radio, a portable cd player, a tape player, etc.), a tablet, a laptop, or other suitable devices. In some embodiments, the current source 180 can include a display screen 182, an electrical output 188 (e.g., an audio jack), and one or more controls. In some embodiments, the display screen 182 is a touch screen. The display screen 182 can indicate to the user various signal parameters, such as the time elapsed, the frequency at which the current is alternating, and the waveform. The current source 180 can further include a power button 184 and optional control buttons 186 to adjust one or more of the parameters. In some embodiments, the control buttons 186 may be incorporated into a touch-screen display.
The current source 180 can further include a processor 190 and memory 192. The memory 192 can include one or more programs. Each of the programs can include one or more pre-set signal parameters. For example, a first program can output a 30 Hz signal with a sinusoidal waveform, and a second program can output a 40 Hz signal with a square wave waveform. The programs need not have different values for each parameter. In some embodiments, each of the programs can be tailored to a different lysis procedure. For example, lysis of stronger cells, such as mycobacterium tuberculosis (MTB), may require a higher frequency and/or a longer duration of agitation. As such, the current source 180 may contain a program specifically designed for lysis of MTB cells that includes a relatively higher frequency. In some embodiments, one or more programs can be downloaded to the current source 180 via a hard connection or wirelessly. For example, a frequency and waveform generator application, such as FreqGen (William Ames), can be downloaded to the current source 180 and supply a variety of waveforms at a wide range of frequencies. In some embodiments, the system 100 can further include an amplifier (not shown) to increase the power delivered by the current source 180.
A method for using the system 100 disclosed herein will now be described with reference to
Before or after delivering the lysing mixture to the vessel 120, the vessel 120 can be positioned within an operating distance of the electromagnet 140. As used herein, “operating distance” refers to a distance between the electromagnet 140 and the permanent magnet 130 whereby an alternating current traveling through the electromagnet 140 causes the permanent magnet 130 to move. As shown in
Upon positioning the vessel 120 and electromagnet 140, the user can then activate the current source 180 to deliver an alternating current to the wire 142 of the electromagnet 140. The vessel 120 and/or electromagnet 140 can be moved relative to one another at any point during the activation of the electromagnet 140. As the alternating current passes through the magnet wire 142, the direction of the magnetic field continuously alternates. In response to the alternating magnetic field caused by the electromagnet 140, the permanent magnet 130 is alternatingly attracted to and repelled from the electromagnet 140, thereby causing the permanent magnet 130 to rotate. In some embodiments, the permanent magnet 130 can rotate about its central axis such that the center of mass of the permanent magnet 130 remains substantially stationary. In other embodiments, the permanent magnet's 130 center of mass moves while it rotates (e.g., the permanent magnet 130 “bounces around” while it rotates). In such embodiments, the permanent magnet 130 may collide with the vessel wall.
Rotation of the permanent magnet 130 within the vessel 120 creates a region of high shear stress between the permanent magnet 130 and the interior surface of the vessel 120. The permanent magnet's 130 rotation also causes the lysing mixture to travel around at least a portion of the permanent magnet 130 and through the high shear regions. When traveling through these high shear regions, the cells encounter one or more destructive forces, such as shear stress and forces associated with collisions with the permanent magnet 130, beads 112 and/or the vessel 120. As such, over time one or more cells of the biological sample lyse.
The lysis systems disclosed herein can include additional features to improve lysing efficiency. For example, in some embodiments, the lysis systems disclosed herein can include a temperature control device. Additionally, in some embodiments, the lysis systems can include one or more feedback mechanisms. For example, in some embodiments the system 100 can include a current source having an electrical input jack (e.g., a microphone jack), an additional cable, and a microphone (e.g., a magnetic coil coupled to a diaphragm). During the lysis procedure, the cable can be coupled to the current source (e.g., via the input jack) and the microphone, and the microphone can be positioned adjacent the vessel 120 and/or electromagnet 140. Rotation of the permanent magnet 130 creates an electromagnetic field that can be monitored by the microphone and processed by the current source. For example, the additional voltage can be superimposed on the input signal which can be monitored by the current source to determine the voltage, frequency, and/or waveform of the superimposed signal. Abnormal changes in voltage can be detected by comparing the input voltage to a calibration curve (e.g., developed by manually spinning a magnet inside the coil of wire and measuring the voltage waveform). In some embodiments, the microphone can additionally or alternatively monitor the acoustic signature (frequencies, waveform) of the permanent magnet hitting the tube as it rotates.
Lysis systems configured in accordance with embodiments of the present technology provide several advantages over conventional mechanical lysis devices. First, the lysis system of the present technology achieves cell lysis with relatively inexpensive materials and at a significantly lower cost to the user. Second, the lysis system of the present technology is self-powered, and thus does not require an electrical outlet. Moreover, the lysis system disclosed herein consumes very little power, and thus (1) can operate for extended periods of time without needing to re-charge, and (2) can operate at lower temperatures (as compared to conventional devices), which can be beneficial for avoiding damage to any RNA and/or proteins that may be present in the biological sample.
The various embodiments described above can be combined to provide further embodiments. The embodiments, features, systems, devices, materials, methods, and techniques described herein may, in some embodiments, be similar to any one or more of the embodiments, features, systems, devices, materials, methods, and techniques described in U.S. Provisional Patent Application No. 61/289,156, filed Dec. 22, 2009, PCT Application No. PCT/US2010/061675, filed Dec. 21, 2010, U.S. Provisional Patent Application No. 61/501,055, filed Jun. 24, 2011, PCT Application No. PCT/US2012/044060, filed Jun. 25, 2012, U.S. patent application Ser. No. 13/518,365, filed Jun. 21, 2012, U.S. patent application Ser. No. 14/129,078, filed Mar. 24, 2014, U.S. Provisional Application No. 61/832,356, filed Jun. 7, 2013, U.S. Provisional Patent Application No. 61/861,055, filed Aug. 1, 2013, PCT Application No. PCT/US2014/012618, filed Jan. 22, 2014, U.S. Provisional Patent Application No. 61/929,769, filed Jan. 21, 2014, U.S. Provisional Patent Application No. 61/808,106, filed Apr. 3, 2013, U.S. Provisional Patent Application No. 61/832,536, filed Jun. 7, 2013, U.S. Provisional Patent Application No. 61/868,006, filed Aug. 20, 2013, and U.S. Provisional Patent Application No. 61/867,950, filed Aug. 20, 2013, all of which are incorporated by reference in their entireties. Aspects of the disclosed embodiments can be modified, if necessary, to employ concepts of the various patents, applications, and publications to provide yet further embodiments. For example, in some embodiments, the system may include a vessel having a first end, a second end opposite the first end, and a permanent magnet positioned therebetween. The first end of the vessel can be configured to receive one or more biological samples, and the second end of the vessel can be configured to be positioned in fluid communication with one or more of the microfluidic devices and systems detailed in one or more of the patent applications listed above. For example, the second end may be open such that the biological sample passes through the portion of the vessel housing the permanent magnet and exits into engagement with the microfluidic device or system. In some embodiments, the second end can include a filter, valve, or other device spanning at least a portion of the inner diameter of the vessel at the second end. When the current source of the system is activated, one or more cells of the biological sample may be lysed while passing through the portion of the vessel housing the permanent magnet.
From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of at least some embodiments of the invention. The systems described herein can perform a wide range of processes for preparing biological specimens for analyzing. Where the context permits, singular or plural terms may also include the plural or singular term, respectively. Unless the word “or” is associated with an express clause indicating that the word should be limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list shall be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a specimen” refers to one or more specimens, such as two or more specimens, three or more specimens, or four or more specimens.
This application claims the benefit of U.S. Provisional Application No. 61/929,769, filed Jan. 21, 2014, titled “Electromechanical Cell Lysis Using a Mobile Electronic Device,” which is incorporated herein by reference in its entirety.
This invention was made with U.S. government support under HR0011-11-2-0007, awarded by the Defense Advanced Research Projects Agency. The U.S. Government has certain rights in the invention.
Number | Date | Country | |
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61929769 | Jan 2014 | US |