Electric pulse generation systems using capacitive coupling

In accordance with the present disclosure, exposure of a sample to one or more electric pulses via capacitive coupling is described. In certain embodiments, the sample may be a biological sample to be treated or modified using the pulsed electric fields. In certain embodiments, the electric pulses may be delivered to a load using capacitive coupling. In other embodiments, the electric pulses may be bipolar pulses.

BACKGROUND

The subject matter disclosed herein relates to electric pulse generations systems for biomedical applications and, more specifically, to methods and systems that may employ capacitive coupling to alter shapes in the electric pulsing.

Pulsed power has numerous industrial applications, such as medical treatments, biotechnology, food processing, water treatment (e.g., water purification), exhaust gas treatment, ozone generation, and ion implantation. For example, transfection is a medical technique used to permeabilize cell membranes to facilitate DNA plasmid entry into the cell. This technique, also known as electroporation, typically involves applying electric pulses with sufficient strength and duration to permeabilize the cell membrane while maintaining viability of the cell. Once the cell membrane is rendered “leaky,” DNA (e.g., DNA, DNA plasmid, DNA single strands, DNA fragments, etc.) in a surrounding buffer solution passes into the cell. Certain in vivo and ex vivo platelet activation methods also utilize pulsed electrical stimulation.

Oftentimes in medical techniques employing pulsed power, the pulse generation system is directly coupled to the container (e.g., a cuvette) that holds the sample being stimulated. In a directly (i.e., conductively) coupled system, the current associated with the electric pulse flows directly through the sample. Typically, square wave pulses are utilized for electroporation, where one could adjust the pulse width, the pulse amplitude, number of pulses and the frequency. This may be facilitated by using special containers made of a conductive material (i.e., metal), which may be expensive or which may not be suitable for biological or biochemical specimens.

BRIEF DESCRIPTION

In a first embodiment, an electric pulse generation system includes memory, a display, and a user input device. The pulse generation system also includes a sample holder which includes a first and second electrode disposed on either side of a container containing a sample. The pulse generation system includes pulse generating circuitry configured to supply a pulse to the first and second electrodes, and a capacitive element disposed between the pulse generating circuitry and the second electrode. The pulse generating circuitry is capacitively coupled to the container. The pulse generation system also includes a processor configured to execute instructions stored on the memory to control the pulse generating circuitry.

In a second embodiment, an electric pulse generation system includes a memory, a display, and a user input device. The pulse generation system also includes a sample holder that includes a first and second electrode disposed on either side of a container containing a sample. The pulse generation system includes pulse generating circuitry configured to supply a pulse to the first and second electrodes, and a capacitive element disposed between the pulse generating circuitry and the second electrode. The capacitive element may be removable or may be bypassed during operation of the electric pulse generation system. The pulse generation system also includes a processor configured to execute instructions stored on the memory to control the pulse generating circuitry and whether the pulse generating circuitry is directly or capacitively coupled to the sample.

In a third embodiment, a method includes collecting blood from a patient. A configuration of a sequence of one or more electric pulses is specified based on a desired parameter associated with growth factor release. The blood sample or a platelet rich plasma sample derived from the blood sample is then exposed to the sequence of one or more pulsed electric fields via a capacitively coupled pulse generation system to trigger release of a growth factor in the blood sample or the platelet rich plasma.

In a fourth embodiment, an electric pulse generation system may include a memory, a display, and a user input device. The electric pulse generation system may also comprise a sample holder including a first electrode and a second electrode disposed on opposite sides of the sample holder, wherein the sample holder is configured to receive a sample container and pulse generating circuitry configured to supply a first pulse and a second pulse to the first and second electrodes. The first pulse has a pulse duration and a first electric field strength and the second pulse has the pulse duration and a second electric field strength. The first electric field strength and the second electric field strength are additive inverses. The electric pulse generation system may further include a processor configured to execute instruction stored on the memory to control the pulse generating circuitry.

DETAILED DESCRIPTION

Present embodiments relate to a pulse generation system for applications employing pulsed power. Specifically, the embodiments described herein relate to a pulse generation system wherein biological samples are placed in a cuvette or other suitable vessel or container. The pulse generation system may be coupled to the corresponding load by capacitive coupling, and in some embodiments, by both capacitive and direct coupling. If the load may be coupled to the pulse generation system by both capacitive and direct coupling, an operator may select which type of coupling to use. Although the embodiments described herein relate to a specific application, it should be appreciated that these are merely examples of possible uses of the subject matter. Accordingly, the disclosed techniques may be implemented, for example, in other medical treatment applications, biotechnology, food processing, water treatment (e.g., water purification), exhaust gas treatment, ozone generation, and ion implantation. In particular, the samples exposed to the electric pulses may be samples used in medical treatment, biotechnology, food processing, water treatment (e.g., water purification), exhaust gas treatment, ozone generation, and/or ion implantation techniques.

With the foregoing in mind,FIG. 1illustrates a pulse generation system10. The pulse generation system10may include pulse generating circuitry12and a load14. The load14may include electrode sets (or array of electrodes)16and18; the electrodes16and18may be designed to conduct high amounts of current, such as in the range of 0.01 kA-35 kA. In the depicted embodiment, the electrodes16and18are spaced apart on opposite sides of a cuvette20. That is, the cuvette20is disposed between and contacted by the electrodes16and18and the electrodes are coupled to the pulse generator via contacts22. In one embodiment, the cuvette20is configured to hold a biological or biochemical sample24, such as a blood sample. In certain embodiments, the cuvette20is disposable and/or is removable from a sample holder26. Accordingly, insertion of the cuvette20and contact of the electrodes16and18with the contacts22allows the pulse generator to produce an electric pulse, and the sample24within the cuvette20is exposed to the pulses. Although the illustrated embodiment depicts a cuvette20, it should be appreciated that a cuvette is but one example of a sample container, and that any suitable container configured to hold a sample may be disposed between the electrodes16and18. In certain embodiments, the cuvette20or the corresponding sample holder may conduct the electric pulses. The cuvette20separates the electrodes16and18from one another. Though the preceding description describes the cuvette holding a biological sample, it should be appreciated that the load14may include any suitable sample that benefits from exposure to electric pulses and the corresponding sample holder.

In certain embodiments, the system10may include suitable control and input circuitry and may be implemented in a dedicated housing or may be coupled to a computer or other processor-based system. The system10may include a processor28that controls the pulse generating circuitry12. Additional components of the system10may include a memory30storing instructions executed by the processor28. Such instructions may include protocols and/or parameters for the electric pulses generated by the pulse generating circuitry12. The processor28may include, for example, general-purpose single-or multi-chip microprocessors. In addition, the processor28may be any conventional special purpose processor, such as an application-specific processor or circuitry. The memory30may be a mass storage device, a FLASH memory device, removable memory, etc. In addition, a display32may provide indications to an operator related to the operation of the system10. The system10may include a user input device34(e.g., a keyboard, mouse, touchscreen, trackball, hand held device or controller or any combination thereof) for activating the pulse generating circuitry12and/or selecting appropriate parameters.

In the depicted embodiment, the system10is used for ex vivo platelet activation. For example, the sample may be a blood product that has been removed from the body and processed to enrich the platelet concentration (e.g., platelet rich plasma). In other embodiments, the system10may be used for in vivo techniques. Accordingly, the system10may be implemented as a wand or other handheld device with spaced electrodes that delivers an electric pulse in or on a load.

It is envisioned that the pulse generation system10as provided herein may be implemented as a single-purpose device (e.g., solely for platelet activation) or as a multi-purpose device that may be used for other electric field exposure applications, such as electroporation, in addition to platelet activation, as discussed herein. Further, the system10may be configured to generate an electric pulse according to one or more protocols. The protocols may be based on user inputs of configurable values or parameters and/or may be stored in the memory30as pre-set protocols to be selected by the user. In one embodiment, the system10may operate without any user input to the activation protocol other than an input to start activation once the sample24is loaded. In such an embodiment, the pulse generating circuitry12may operate under control of the processor28to operate a single protocol with predetermined electric field strength, pulse length, and/or total exposure time. Such a protocol may be determined by empirical or theoretical studies. In other embodiments, the system10may be configured to receive a user input related to the electric field strength, pulse length, and/or total exposure time. Further, the system10may be configured to generate a particular pulse shape or to generate a series of pulses that may differ from one another according to a user input and/or a stored protocol setting.

The pulses generated by the system10may have a duration from about 1 nanosecond to about 100 microseconds, and an electric field strength from about 0.1 kV/cm to 350 kV/cm, depending on the application. The spacing between the electrodes16and18may influence the strength of the electric field, which is defined as the ratio of the applied voltage and the electrode gap distance. For example, if a cuvette provides a 1 cm gap between the electrodes, exposing the cuvette to 1 kV yields an electric field strength of 1 kV/cm. While the pulses generated by the system may be at least 10 kV/cm, 50 kV/cm, etc., they should not exceed the breakdown field of the sample24.

In conventional systems, a pulse generation system would be directly coupled to the corresponding load, such that current would flow directly from the pulse generating circuitry to and through the sample. As such, the cuvette, or, generically, the sample container, may be made from a conductive (i.e., metal) material, which may be expensive or otherwise undesirable, such as due to the nature of the sample. Further, the sample may become contaminated due to contact with metallic surfaces. The cuvette20may also need to have certain characteristics that reduce the chance of electrical breakdown (e.g., arcing).

To reduce or eliminate the complexity of the sample holder26, and in the depicted embodiment, the cuvette20, the pulse generation system10may be capacitively coupled to the load14. The system10may include a capacitive element36disposed between the pulse generating circuitry12and the sample24, as illustrated inFIG. 1. In some embodiments, the capacitive element36may be disposed between the pulse generating circuitry12, and the electrode16. In the capacitively coupled system10, the capacitive element36prevents direct current (DC) from flowing through the sample24, and forces bipolar impulsive currents through the sample.

The capacitive element36may be any suitable component or material that acts as a capacitor and is disposed in series with the sample24. For example, the capacitive element36may be a capacitor disposed at the end of the pulse generating circuitry12, as illustrated inFIG. 2. A capacitor36may also be disposed between the electrode16and the sample24, as illustrated inFIG. 3. For example, a capacitor36may be attached to a compartment located between the electrode16and the sample holder26in a cuvette.

In some embodiments, the capacitive element36may be a structure disposed in the cuvette20or a structure that is a part of the cuvette20itself. For illustrative purposes,FIG. 9provides an illustration of a traditional cuvette152without a capacitive element. The traditional cuvette152may have a cavity104used for sample placement (e.g., a sample cavity). Cavity104may be formed by a body106of the cuvette. The body106of the cuvette may be constructed using a nonconductive material (e.g., quartz, plastic). The body of the cuvette may have two opposite walls, wall124A and124B. When disposed in a sample holder26, the walls124A and124B may be adjacent to electrodes16and18of the sample holder26, respectively. To form an electrical circuit with the pulse generating system10, the sample wall124A may have an electrode156A wall124B may have a second electrode156B. In some embodiments, the sample holder26may have a spring-loaded mechanism that pushes electrodes16and18of the sample holder26against electrodes156A and156B of the cuvette152. In the traditional cuvette152, electrodes156A and156B provide a conductive (e.g., resistive, non-capacitive) path between the cavity104and the electrodes16and18of the sample holder through walls124A and124B of the body106. To that end, electrodes156A and156B may, each, have an internal surface exposed to the cavity.

With the foregoing in mind,FIGS. 10A, 10B, 10C, and 11illustrate non-limiting example of cuvettes that may include the capacitive element36. For example, the cuvette102inFIG. 10Amay include a capacitive element by employing conductive electrodes that are separated from the cavity104by a dielectric gaps114A and114B. Cuvette102may have a cavity104for placement of the sample. Cuvette102may further include a body106constructed using a nonconductive material. To form an electric circuity with electrodes16and18, cuvette102includes conductive contacts108A and108B. In cuvette102, the conductive contacts108A and108B are separated from the cavity104by dielectric gaps114A and114B, which form the capacitive element of cuvette102. The dielectric gaps114A and114B may provide a capacitive coupling between a sample in cavity104and electrodes16and18of the sample holder. The capacitance of the capacitive coupling is determined by the dielectric material along the dielectric gaps114A and114B, which may be the same as the material used in the body106(e.g., plastic, quartz). The capacitance of the capacitive coupling is also determined by the length of the dielectric gaps114A and114B and the height of the conductive contacts108A and108B. In fact, the distances of the dielectric gaps114A and114B may be adjusted to tune the capacitance, and may be chosen based on the suitability of the capacitance for a specific application, for example, platelet activation. The dielectric gaps114A and114B may, for example, be in a range between about 0.1 mm and about 5 mm.

The capacitance may be provided without the use of conductive contacts. Cuvette122inFIG. 10Bwithout conductive contacts is illustrated. In cuvette122, the walls124A and124B of the capacitor may form the dielectrics for the capacitive element36when connected to the electrodes16and18of the pulse generating system10. In such system, the thickness126A and126B of walls124A and124B, respectively, may be determine the capacitance of the capacitive coupling, as discussed above with respect to capacitor102. Moreover, the dielectric properties of the material used in the construction of the body106may further determine the capacitance. In fact, the thickness126A and the thickness126B may be adjusted to achieve a capacitance value that is suitable for specific applications such as, for example, platelet activation. Thicknesses126A and/or126B may, for example, in a range between about 1 mm and about 5 mm.

Cuvettes102and122, each, employ two separate dielectric regions (e.g., dielectric gaps114A and114B, thicknesses126A and126B) to form the capacitive coupling. Thus, in such systems, the nominal capacitance may depend on the accuracy of the manufacturing process. The cuvette142, inFIG. 10C, may have its capacitive coupling formed by a single dielectric region. Cuvette142may have a capacitive element formed by the thickness145along the single wall144. Thickness145may, for example, in a range between about 1 mm and about 5 mm. On the opposite wall, the cuvette142may have an electrode146that couples the interior of cavity104to the exterior of the cuvette using a conductive (e.g., resistive, non-capacitive) path. As such, when placed in the sample holder, cuvette142may form a capacitive coupling between a sample in cavity104along the wall145, and a resistive coupling between a sample in cavity104through electrode146. Note that, as discussed above, the capacitance in the capacitive coupling in cuvette142may be determined by the dielectric properties of the material used to form the body106, as well as the thickness145of wall144.

In some embodiments, the dielectric material that provides the capacitive coupling may be a material that is different from the nonconductive material used to form the body106. Cuvette162inFIG. 11illustrates a capacitive coupling that may be formed by a dielectric164that provides the capacitance. The thickness166of the dielectric164may determine the capacitance of the dielectric. Thickness145may, for example, in a range between about 1 mm and about 5 mm. The dielectric164may, for example, be a ceramic dielectric, a plastic dielectric, a crystal dielectric, or any other non-conductive material. Cuvette162may also include an electrode170that couples the interior of cavity104to the exterior of the cuvette through a conductive (e.g., resistive, non-capacitive) path. Note further that, in some embodiments, a sample collection device may be used as the capacitive element. For example, if the system10is used for platelet activation, a sample collection device, for example, the syringe used to collect the sample24(i.e., blood), may have electrodes, dielectric structures, or walls with well-defined thickness, which may be used as both a sample holder and a capacitive element. The overall capacitance provided by embodiments such as the cuvettes ofFIGS. 10A, 10B, 10C, and 11, may be in a range between 1 nF and 1 mF.

In some embodiments, the pulse generation system10using capacitive coupling may be configured to generate bipolar pulses. The processor28may control the pulse generating circuitry12such that two electric pulses, one after the other, may be generated. These two electric pulses may have the same pulse duration. However, the amplitude of the electric pulses may be additive inverses. For example, the first electric pulse may have an electric field strength of 50 kV/cm, while the second electric pulse may have an electric field strength of −50 kV/cm. As will be appreciated, the first pulse may have a positive polarity and the second pulse a negative polarity or vice versa, so long as the polarity of the first pulse is opposite that of the second pulse.

Pulse generation systems using capacitive coupling may have benefits related to the results of electrically stimulating the samples. For example, in platelet activation techniques using electrical stimulation, the rate of growth factor release may vary based on the types of electric pulses emitted by a capacitively coupled pulse generation system. For instance, an electric pulse a may cause a growth factor I to be immediately released, and a growth factor J to be subsequently released. On the other hand, an electric pulse b may cause a steady rate of release for growth factor I, while halfway through the process growth factor J is released. The characteristics for the pulses associated with varying growth factor release may be determined by empirical studies. These pulse configurations may be incorporated into the protocols stored on the memory30, or may be specified by user input.

A method40for triggering growth factor release, as illustrated inFIG. 4, may be used in conjunction with the system10. It should be understood that certain steps of the method40may be performed by an operator while other steps of the method may be performed by the system10. At step42, personnel (e.g., a doctor or nurse) draw blood from a patient, which is centrifuged to generate a platelet rich plasma (PRP) sample in step44. In the depicted implementation, personnel determine the pulse sequence and configuration of one or more pulses to apply to the PRP sample in the cuvette to trigger a specific or desired amount of released growth factors in step46. In some systems, the capacitance may be changed. This may take place, for example, in an embodiment having a switch or a removable capacitor. Change in capacitance may also be achieved by changing cuvettes that carry the capacitive element. In such system, step46may include an optional process in which the user may enter the configured capacitance. For example, a user may enter the state of the switch, the capacitance of the capacitor, or the capacitance associated with the cuvette. In some systems, the cuvette or the removable capacitor may have a tag (e.g., a label, a code, a bar code, a QR code, or a combination thereof) which may be provided to the pulse generating system. The tag may be associated with characteristics of the cuvette, such as the type of cuvette, capacitance associated with the cuvette, a material of the cuvette, or the prescribed use of the cuvette. The processor may adjust the configuration of the pulses based on the capacitance arranged in the system. In other embodiments, personnel may determine the correct sequence of pulses based on the desired type of released growth factors and/or desired rate of the release of growth factors. During step48, the PRP sample is exposed to the one or more pulses, which triggers growth factor release in step50. Finally, in step52, the growth factors are collected from the PRP sample.

While certain applications may benefit from capacitive coupling, others may benefit from direct coupling. As such, it may be desirable for the pulse generation system10to be able to couple capacitively or directly to the load14based on the intended biological application (e.g., platelet activation). For example, as mentioned above, the capacitive element36may be a capacitor disposed between the electrode16and the sample holder26. The capacitor36may be removable, such that the system10normally uses direct coupling, and when capacitive coupling is desired, the capacitor36is attached, in some embodiments, by an operator. Similarly, an operator may use a conductive sample holder26when direct coupling is desired and a nonconductive sample holder26when capacitive coupling is desired.

Alternatively, the pulse generating circuitry12may include circuitry that allows current to flow directly to the load14(i.e., direct coupling) or routes current through a capacitive element36(i.e., capacitive coupling) prior to the load14, as illustrated inFIG. 5. For example, the pulse generating circuitry12may include, in parallel, a direct coupling to the load14and the capacitive element36(e.g., a capacitor) in series with the load14(i.e., capacitive coupling). The processor28may control two switches54A and54B that allow current to flow to the load14via either direct coupling or capacitive coupling approach. The switches54A and54B may be any device capable of being selectively changed between an electrically conductive state and a nonconductive state, such as silicon-controlled rectifiers, power transistors, relay switches, or any other like devices. Alternatively, the processor28may control other devices, such as analog or digital multiplexors, that are capable of selecting the circuitry associated with the desired coupling approach or scheme. The processor28may receive a user input specifying which coupling scheme the system10should use. The protocols stored on the memory30specifying the characteristics of the pulses generated may also specify whether to use direct or capacitive coupling. Some applications may also benefit from a series of electric pulses delivered to the load14that alternate between direct and capacitive coupling. This may take place by having an active controller adjusting switches54A and54B, for example. Such configurations may be incorporated into the protocols stored on the memory30, or may be specified by user input.

EXAMPLES

Controlling the Amount of Growth Factor Release during Platelet Activation

FIG. 6depicts the amount of growth factor release in various types of blood samples exposed to electrical stimulation, along with a capacitive coupling approach. Results are shown for samples that include a platelet rich plasma (PRP) sample that has not been activated, a whole blood sample that has not been activated, and a PRP sample that has been activated via electrical stimulation in a capacitively coupled pulse generation system. The PRP samples were exposed to bipolar pulses with a voltage of 700 V (electric field strength of 3.5 kV/cm) and a current of 30 A for a predetermined duration (which may be between 1 ns and 1 s. As illustrated, the amount of platelet-derived growth factor (PDGF) present in the capacitively coupled PRP sample is about twice that of the non-activated PRP sample and the whole blood sample.

FIG. 7illustrates the amount of growth factor release in similar types of samples as inFIG. 6—but a higher capacitive coupling voltage triggers more growth factor release compared to the baseline, non-activated PRP and whole blood. Here, the PRP sample subjected to capacitive coupling was exposed to bipolar pulses with a voltage of 1200 V (electric field strength of 6 kV/cm) and a current of 60 A. The amount of PDGF released in the capacitively coupled PRP sample was six times more than that of the non-activated PRP sample and about thirteen times more than that of the whole blood sample. As shown, the voltage and current characteristics of the electrical stimulation affect the amount of growth factor released compared to the baseline when the pulse generation system is capacitively coupled to the sample. To further illustrate the effectiveness of the capacitively coupled pulse generation system,FIG. 8compares the amount of PDGF released in a non-activated PRP sample, a whole blood sample not exposed to electrical stimulation, a blood sample activated with bovine thrombin, and a capacitively coupled PRP sample. Note that, as illustrated inFIG. 8, the capacitive coupling may increase the amount of PRP sample that is activated.

One or more of the disclosed embodiments, alone or in combination, may provide one or more technical effects useful for providing pulsed power in various applications. Certain embodiments may allow operators to use nonconductive materials for sample holders in pulse generation systems. For example, the present capacitively coupled pulse generation system may use a syringe or other plastic container, instead of a cuvette, as a sample holder. These nonconductive samples holders may be less expensive, easier to sterilize, and more readily available than sample holders used in conventional pulse generation systems. Additionally, samples that are electrically stimulated using the present capacitively coupled pulse generation system may differ based on the types of pulses used. For instance, varying the pulse parameters for the present capacitively coupled pulse generation system for treating the sample, for example, in the platelet activation application, may modify the amount of growth factors released from the sample. Other embodiments may also allow operators to use direct or capacitive coupling in pulse generation systems. For example, the present pulse generation system may contain suitable control and pulse generating circuitry that allows current to flow directly to the sample (i.e., direct coupling) or reroutes the current through a capacitive element (i.e., capacitive coupling). The technical effects and technical problems in the specification are exemplary and not limiting. It should be noted that the embodiments described in the specification may have other technical effects and can solve other technical problems.