Sampling device and method for collecting interstitial fluid

Abstract

A sampling device for extracting interstitial fluid is provided, comprising an extraction assembly and a collector assembly. The extraction assembly has a hydrogel-based positive electrode, a metal negative electrode, and a suction channel configured to be fluidly connected to a vacuum pump. The collector assembly has a fixed part and a detachable part, the fixed part having a fixed support connected to the extraction assembly, a conduit for receiving interstitial fluid, and a needle at the end of the conduit, and the detachable part having a cavity defined by a hydrophobic membrane enclosure that is in fluid communication with the conduit for receiving interstitial fluid, a diaphragm adapted to be punctured by the needle, an external enclosure that houses the cavity and defines a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for pumping gas from the gas cavity.

Description

[Technical Field]

[0001] Cross-reference of related applications The present application claims priority from U.S. Provisional Application No. 63 / 513,598, filed on July 14, 2023, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to the field of fluid collection devices and methods using the same, and in particular, to collection of interstitial fluid. [Background Art]

[0003] Early detection of diseases and monitoring of diseases are extremely important factors for diagnosing accurate physical symptoms and establishing appropriate treatment methods. This is often done by analyzing body fluids such as plasma, interstitial fluid and cerebrospinal fluid. Furthermore, for certain diseases such as diabetes, continuous monitoring of analytes is required. People with diabetes need accurate and consistent readings of glucose levels in their blood to avoid complications that can be severe in certain cases. Various devices have been designed over the years for monitoring body fluids over long periods of time, and in particular, wearable devices in contact with the skin have been designed for monitoring interstitial fluid. These devices rely on electroporation through the skin to collect interstitial fluid through the skin (see, for example, WO2010 / 094131). However, improving the performance of these devices remains desirable, particularly for individuals with diabetes. [Summary of the Invention]

[0004] In one respect, a sampling device for extracting interstitial fluid is provided. The sampling device comprises an extraction assembly having a positive electrode, at least one negative electrode, and a suction channel adapted to be fluidly connected to a vacuum pump; and a collector assembly having a fixed part and a detachable part, wherein the fixed part has a fixed support part connected to the extraction assembly, a conduit for receiving the interstitial fluid, and a needle positioned at the end of the conduit; the detachable part has a cavity defined by a hydrophobic membrane enclosure that is in fluid communication with the conduit for receiving the interstitial fluid, a diaphragm adapted to be penetrated by the needle, an external enclosure housing the cavity and defining a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for discharging gas from the gas cavity.

[0005] In some embodiments, the positive electrode is a current-diffusing positive electrode, for example, a hydrogel-based electrode or a dry electrode. In one embodiment, the positive electrode comprises a stoma adhesive hydrocolloid material. In some embodiments, the negative electrode comprises palladium or is made of stainless steel. In some embodiments, the collection device further comprises a sealing gasket or sealing member, such as one or more O-rings, for sealing the connection between the fixed portion and the detachable portion. In some embodiments, the collection device further comprises one or more sealing members for sealing the connection between the fixed portion of the collector assembly and the extraction assembly. In some embodiments, the hydrophobic membrane enclosure is square, circular, or dome-shaped. In some embodiments, the extraction assembly further comprises double-sided adhesive tape for adhesion to the skin. In some embodiments, the positive electrode is used as an electrode having adhesive function at the skin interface. In some embodiments, the positive electrode is a membrane at the skin interface having at least one membrane pore. In some embodiments, the positive electrode comprises a hydrogel assembly having a hydrogel layer, a conductive layer, and an adhesive layer. In some embodiments, a single positive electrode is present. In one embodiment, two or more positive electrodes are used. In some embodiments, the sampling device has more than one or four or more negative electrodes. In some embodiments, the sampling device has eight or sixteen or more negative electrodes. In some embodiments, the sampling device has sixty-four or more negative electrodes. In some embodiments, the sampling device has sixty-four negative electrodes.

[0006] In yet another embodiment, the sampling device is connected to a module, which is connected to the positive and negative electrodes and controls the applied current, and the module has a vacuum pump for controlling vacuum suction through a vacuum suction channel in fluid communication. In yet another aspect, the module measures the variation in resistance between at least one negative electrode and at least one positive electrode. In another embodiment, the interstitial fluid is collected in a tube or chamber connected to a collector assembly.

[0007] In another embodiment, the method further includes the step of measuring the glucose level in the extracted interstitial fluid.

[0008] In another view, a method is provided for electroporating solid skin, which includes bringing a positive electrode and a metallic negative electrode into contact with the skin, applying an electric current between the positive electrode and the metallic negative electrode, stopping the current when a pore is formed in the skin, and applying negative pressure to the skin to keep the pore open. In some embodiments, the method further includes the step of determining the electroporation level by measuring the resistance variation between at least one negative electrode and at least one negative electrode, or between the positive electrode and at least one negative electrode. In some embodiments, the skin is cleaned before the contact step is performed.

[0009] Further features relating to this improvement, and combinations thereof, will become apparent to those skilled in the art after reading this disclosure. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic side view of a sampling apparatus according to an embodiment of the present disclosure. [Figure 2] Figure 2 is a schematic plan view of a sampling apparatus according to one embodiment of the present disclosure. [Figure 3] Figure 3 is a schematic cross-sectional view of a collector assembly of a sampling device according to one embodiment of the present disclosure. [Figure 4] Figure 4 is a schematic cross-sectional view of the fixed portion of the collector assembly of a sampling device according to one embodiment of the present disclosure. [Figure 5] Figure 5 is a schematic cross-sectional view of the attachment / detachment portion of the collector assembly of a sampling device according to one embodiment of the present disclosure. [Figure 6A] Figure 6A is an exemplary schematic cross-sectional view of the square hydrophobic membrane enclosure of the collector assembly. [Figure 6B]Figure 6B is an exemplary schematic cross-sectional view of the dome-shaped hydrophobic membrane enclosure of the collector assembly. [Figure 6C] Figure 6C is an example of a schematic plan view of the circular hydrophobic membrane enclosure of the collector assembly. [Figure 6D] Figure 6D is a cross-sectional view of the hydrophobic membrane enclosure in Figure 6C along line AA. [Figure 7] Figure 7 is a schematic exploded view of the extraction assembly of a sampling device according to one embodiment of the present disclosure. [Figure 8A] Figure 8A is a schematic plan view of an extraction assembly according to one embodiment of the present disclosure. [Figure 8B] Figure 8B is a bottom side view of the extraction assembly shown in Figure 8A. [Figure 8C] Figure 8C is a bottom view of the extraction assembly shown in Figure 8A. [Figure 8D] Figure 8D is a side view of the extraction assembly shown in Figure 8A. [Figure 8E] Figure 8E is a side view of the extraction assembly shown in Figure 8A. [Figure 8F] Figure 8F is a cross-sectional view of the extraction assembly shown in Figure 8A, illustrating the membrane in its unexpanded state. [Figure 8G] Figure 8G is a cross-sectional view of the extraction assembly shown in Figure 8A, illustrating the expanded membrane. [Figure 9A] Figure 9A is a schematic plan view of the extraction assembly, showing exemplary dimensions (in inches, 2:1 scale). [Figure 9B] Figure 9B is a schematic cross-sectional view of the extraction assembly shown in Figure 9A. [Figure 9C] Figure 9C is a schematic side view of the extraction assembly shown in Figure 9A. [Figure 9D] Figure 9D is a schematic cross-sectional view of Figure 9C. [Figure 10] Figure 10 is a schematic bottom view of an extraction assembly having four membrane pores. [Figure 11A] Figure 11A is a schematic diagram of the inside of an extraction assembly according to an embodiment of this disclosure. [Figure 11B]FIG. 11B is a schematic cross-section taken along line E-E of the extraction assembly of FIG. 11A. [Figure 11C] FIG. 11C is a schematic cross-section taken along line B-B of the extraction assembly of FIG. 11A. [Figure 12A] FIG. 12A is a schematic side view of an extraction assembly according to an embodiment of the present disclosure. [Figure 12B] FIG. 12B is a schematic cross-section of FIG. 12A. [Figure 13A] FIG. 13A is a plan view of an extraction assembly assembled with screws according to an embodiment of the present disclosure. [Figure 13B] FIG. 13B is a schematic cross-section taken along line E-E of FIG. 13A. [Figure 13C] FIG. 13C is a schematic cross-section taken along line G-G of FIG. 13A. [Figure 13D] FIG. 13D is a schematic cross-section taken along line H-H of FIG. 13A. [Figure 14A] FIG. 14A is a schematic plan view of an extraction assembly placed on the skin according to an embodiment of the present disclosure. [Figure 14B] FIG. 14B is a cross-section of FIG. 14A taken along line I-I. [Figure 14C] FIG. 14C is an enlarged view of a region labeled "K" in FIG. 14B. [Figure 14D] FIG. 14D is an enlarged view of a region labeled "L" in FIG. 14C. [Figure 14E] FIG. 14E is an enlarged view of a region labeled "M" in FIG. 14C. [Figure 14F] FIG. 14F is a schematic cross-sectional view of the extraction assembly of FIG. 14A, showing the membrane in a non-expanded state. [Figure 14G] FIG. 14G is an enlarged view of a region labeled "K" in FIG. 14F. [Figure 14H] FIG. 14H is an enlarged view of a region labeled "M" in FIG. 14G. [Figure 14I] FIG. 14I is an enlarged view of a region labeled "L" in FIG. 14G. [Figure 15A] Figure 15A is a schematic diagram showing the skin in its resting state before dilation (i.e., undiluted). [Figure 15B] Figure 15B is a schematic diagram showing skin expanded using the sampling device of the present disclosure. [Figure 16A] Figure 16A is a bottom view of an extraction assembly having eight negative electrodes. [Figure 16B] Figure 16B is a bottom view of the extraction assembly shown in Figure 16A, and shows the hydrogel material for the cathode. [Figure 16C] Figure 16C is a side view of the extracted assembly shown in Figure 16A. [Figure 16D] Figure 16D is an exploded view of the extraction assembly shown in Figure 16A. [Figure 16E] Figure 16E is a bottom view of an extraction assembly having 16 negative electrodes. [Figure 16F] Figure 16F is a bottom view of the extraction assembly shown in Figure 16E, and shows the hydrogel material for the cathode. [Figure 16G] Figure 16G is an exploded top view of the extraction assembly shown in Figure 16E. [Figure 16H] Figure 16H is an exploded view of the bottom side of the extraction assembly shown in Figure 16E. [Figure 16I] Figure 16I is a bottom view of an extraction assembly having 64 electrodes. [Figure 16J] Figure 16J is a bottom view of the extracted assembly shown in Figure 16I. [Figure 16K] Figure 16K is a side view of the extracted assembly shown in Figure 16I. [Figure 16L] Figure 16L is an exploded top view of the extraction assembly shown in Figure 16I. [Figure 16M] Figure 16M is a side exploded view of the extraction assembly shown in Figure 16I. [Figure 16N] Figure 16N is a bottom view of the extraction assembly shown in Figure 16I, and shows the hydrogel material for the cathode. [Figure 16O] Figure 16O is a side view of the extraction assembly shown in Figure 16N. [Figure 16P]Figure 16P is a side exploded view of the extraction assembly shown in Figure 16N. [Figure 16Q] Figure 16Q is a bottom-side exploded view of the extraction assembly shown in Figure 16N. [Figure 17A] Figure 17A is a schematic cross-sectional view of a hydrogel assembly according to one embodiment, viewed from above. [Figure 17B] Figure 17B is an exploded view of the hydrogel assembly shown in Figure 17A. [Figure 17C] Figure 17C is an exploded view showing the hydrogel assembly in Figure 17B with its position shifted. [Figure 18A] Figure 18A is a schematic plan view of a hydrogel assembly according to another embodiment. [Figure 18B] Figure 18B is a side elevation view of the hydrogel assembly shown in Figure 18A. [Figure 18C] Figure 18C is an exploded view of the hydrogel assembly shown in Figure 18A. [Figure 19A] Figure 19A is a photograph of double-sided tape placed on electroperforated skin. [Figure 19B] Figure 19B is a photograph of glue adhesive placed on electroporated skin. [Figure 20A] Figure 20A is a photograph of skin electroporated using the apparatus and method described herein. [Figure 20B] Figure 20B is a photograph of a control skin sample that was electroporated using an adhesive-based device. [Figure 21] Figure 21 shows a schematic diagram of a sampling device connected to an electronic module according to an embodiment of the present disclosure. [Modes for carrying out the invention]

[0011] This specification provides apparatus for obtaining a sufficient amount of interstitial fluid from the human body continuously and over a long period of time in a non-invasive manner. The methods and apparatus disclosed herein include a combination of electroporation of the skin of the human body and aspiration of interstitial fluid through permeable and micro-opened skin. In this specification, the term “electroporation” as used with respect to skin refers to a method of creating permanent pores in the stratum corneum of the skin. These pores subsequently allow for continuous extraction of interstitial fluid as long as the extractor applies negative pressure to the electroporated area. Pores created in such a manner are considered permanent as long as air pressure and / or mechanical pressure are applied to the electroporated skin for the purpose of extracting interstitial fluid (IF).

[0012] This specification provides a sampling apparatus that improves upon a sampling apparatus by having means for applying mechanical pressure to a pointed electrode (which is the negative electrode) that is in contact with the skin for electroporation. The negative electrode used herein preferably has a small diameter (about 0.5 mm ± 150 μm). To avoid puncturing the skin, the negative electrode may have a cone-shaped working end with a blunt point, which in an example has a diameter of 0.1 mm ± 25 μm. Suitable materials for the negative electrode include, but are not limited to, noble metals such as palladium. When the negative electrode is in contact with the skin for electroporation, the electrode presses against the skin due to the offset between the plane on which the entire apparatus sits on the skin and the point of the electrode. This offset pushes the electrode into the skin surface. Additionally, during electroporation, there may be additional pressure due to the action of negative pressure.

[0013] This pressure causes conical deformation in the skin, more specifically in the stratum corneum and epidermis, and can also occur in the dermis. The effects of this deformation are wide-ranging. • To thin the layer to be electrocuted, to assist in the electrocution process. • To increase electrical connectivity through the stratum corneum and epidermis in a very small area of ​​skin. • By concentrating the application of current to a small channel, the efficiency of the current application process is improved. • Mechanically improves the opening of electroporation holes in the stratum corneum and epidermis, and possibly the dermis, by assisting in the separation of cells during the electroporation process in order to form electroporation holes.

[0014] However, the mechanical pressure on the skin after electroporation is complete has the effect of opening a hole due to the elastic / plastic properties of the skin. This opening is similar to what happens when a person tries to force fluid through a narrow perforation in a diaphragm. At low or very low pressures, no fluid flow occurs. Once the pressure becomes high enough to expand / deform the diaphragm, it then opens a hole, and then fluid flow becomes possible.

[0015] Regarding the "permanent state" of pores electroporated using this method, the mechanical stress applied to the electroporated region helps to maintain the electroporated material in a separated state, creating distance between the materials. As a result, after a certain period, the state of the electroporated region becomes a more "permanent electroporated state."

[0016] It was observed that skin dilation caused by negative air pressure applied to the skin expands the skin and counteracts the capillary pressure that would otherwise tend to retain IF within the skin. This dilation can make a significant contribution to the fact that it opens the electroporation pores for the extracted IF to pass through, and it can also be significant to the fact that the electroporation pores are considered "permanent" as long as negative pressure, i.e., skin dilation, is applied, thus allowing IF to be extracted over a long period, i.e., continuously for 21 days.

[0017] While this device is generally described as a sampling device designed to extract bodily fluids, it can also be used to introduce substances into a subject through electroperforated skin. In substance administration, the effect of pressure will be as important as in extraction. Skin dilation helps to open the electroperforated holes in one direction, and the same thing happens in the case of substance administration through the skin, namely overcoming capillary pressure, making the opening of the holes larger, and maintaining the holes open.

[0018] Referring to Figure 1, the sampling device 1 comprises an extraction assembly 2 that comes into contact with an individual's skin 28 to extract bodily fluids such as interstitial fluid. The extraction assembly 2 is connected to a collector assembly 3 having a fixed section 3a and a detachable section 3b. Figure 2 shows a plan view of the sampling device 1. Figure 3 shows a cross-section of the collector assembly 3 along line AA in Figure 2. The collector assembly 3 has conduits 4 that guide the interstitial fluid and gas extracted from the skin and flowing out of the extraction assembly 2 into a sampling cavity 5. The sampling cavity 5 is located within the detachable section 3b of the collector assembly 3. The detachable section 3b further has a gas cavity 6 around an inner hydrophobic membrane 7 that defines the sampling cavity 5, around which gas circulates freely. The hydrophobic properties of the membrane 7 allow gas to pass through the membrane 7 while retaining an aqueous mixture inside. The hydrophobic membrane and diaphragm effectively seal the internal liquid, i.e., interstitial fluid (IF), from any external contamination (sterile conditions). The pump conduit 8 is in fluid communication with the gas cavity 6 and leads the fluid to a pressure-regulated pump system for controlling the pressure within the cavity 6. The pump conduit 8 allows for pumping of gas out of the collector assembly 3 to draw interstitial fluid and gas into the extraction cavity 5. The hydrophobic inner membrane enclosure 7 is preferably sealed to the solid base 21 of the detachable section 3b. The detachable section 3b is reversibly connected to and reversibly sealed to the fixed section 3a via a plurality of O-rings 9a. The O-rings 9b are also used to connect the fixed section 3a to the extraction assembly 2. The operation of the pump is also sealed by O-rings.

[0019] The fixed portion 3a is shown in Figure 4 and has a fixed support portion 11 that holds the extraction assembly 2 together with an O-ring 9b. The illustrated embodiment with the O-ring should not be interpreted restrictively, as other means other than the O-ring exist to provide sufficient connection between the extraction assembly 2 and the fixed portion 3a. The end of the conduit 4 is provided with a needle 12 for puncturing the diaphragm of the detachable portion 3b of the collector assembly 3. The puncture allows for the formation of fluid communication between the fixed portion 3a and the detachable portion 3b via the conduit 4. The fixed portion 3a also has a pump conduit 8 that leads to a regulated pump system. Figure 5 shows the detachable portion 3b separately from the fixed portion 3a. The detachable portion 3b may be housed in an external enclosure 20, in which case the external enclosure 20 may define a gas cavity 6. The solid base 21 comes into contact with the fixed portion 3a during assembly. Furthermore, the needle 12 punctures the diaphragm 22, forming a hole 22a in the diaphragm 22. The detachable portion 3b may also have an inner enclosure 23 for supporting the inner hydrophobic membrane enclosure 7. The membrane enclosure 7 may have a different shape. As an example, a square membrane is shown in Figure 6A, while a dome-shaped membrane is shown in Figure 6B. When a dome shape is used, the hydrophobic membrane can be a continuous single piece and may not require support. In other examples, Figures 6C and 6D show a circular hydrophobic membrane 7a having a solid plastic closure portion 7c for support that can be welded or bonded. The circular hydrophobic membrane may be in the form of a rolled and bonded sheet.

[0020] Figure 7 shows an exploded view of the extraction assembly 2 in operation on skin 28. The extraction assembly has a negative electrode 14 and a positive electrode having a hydrocolloid material 18A, a conductive material 18B, and an adhesive 18C. The hydrocolloid material 18A is in contact with the skin 28. An elastic membrane 100A expands the skin 28, forming an expanded skin area 29. The extraction assembly 2 is also divided into a lower part 30A and an upper part 30B that fits into the lower part 30A. The negative electrode is located inside the upper part 30B and extends downward to contact the skin 28 when electroporation is performed.

[0021] The disclosure also includes having a large surface area on the positive electrode to reduce pain while electroporation is in progress. The positive electrode is one that diffuses the current and limits the pain experienced by the patient. In embodiments, the positive electrode may be a hydrogel electrode having the hydrocolloid material described herein, but dry electrodes are also included in the disclosure and are not limited thereto. The electrode may be, for example, made of stainless steel and / or may contain palladium, but is not limited thereto.

[0022] Figures 8A–8E show different external views of the extraction assembly 2, which has a bottom surface 40 in contact with the skin 28 and side surfaces 41 perpendicular to the bottom surface 40. The bottom surface 40 has at least one membrane pore 42 through which interstitial fluid is extracted from the skin and enters the extraction assembly 2. Figure 8F is a cross-section along line AA in Figure 8A, showing the extraction assembly 2 when the membrane is unexpanded and an expansion space 45 exists at the boundary between the skin 28 and the extraction assembly 2. The cross-section also shows a hydrophobic membrane 44 that functions as a positive electrode. Figure 8G is a cross-section along line AA when the membrane 46 expands in the expansion space 45, driven by pumping through the suction channel 43.

[0023] Figure 9A is a plan view of the extraction assembly 2. Figure 9B is a cross section along line EE of Figure 9A, showing the upper 30B and lower 30A, the hydrophobic membrane 44 and the rigid membrane 47. Figure 9C is a side view of the extraction assembly 2, and Figure 9D is a cross section along line DD of Figure 9C. The cross section shows the upper 30B and lower 30A as well as the suction channel 43. Embodiments illustrated herein are preferably shown having a single membrane pore 42 centrally located to avoid shear. The dimensions of the membrane pore 42 can also be optimized to reduce or eliminate any slippage that may occur when handling the skin and the rigid assembly. However, other embodiments having more than one membrane pore 42 are also envisioned herein. For example, Figure 10 shows a four-pore assembly which can advantageously reduce dead volume. The interior of the four-pore embodiment is shown in Figure 11A, and a cross section along line E above the pump outlet 48 with the suction channel 43 is shown in Figure 11B. Figure 11C is a cross-section along line BB, showing exemplary relative dimensions of the extraction assembly 2. Figure 12A shows yet another side view, and Figure 12B shows a cross-sectional view. In some embodiments, mechanical means such as screws 49 can be used to fasten the upper part 30B to the lower part 30A, as shown in Figure 13A. This is better illustrated in the cross-sections shown in Figures 13B–13D.

[0024] Figure 14A shows a plan view of the extraction assembly 2 on the skin 28. Figures 14B-14E are cross-sections showing the expansion of the membrane, which leaves a small gap 10 through which the suction channel 43 can continuously pump out fluid. Figures 14F-14I show the same apparatus, but the membrane is not expanded. Sections 32A and 32B of the extraction conduit are two sections of the suction channel 43. The openings in elastic membranes 100A and 100B are most clearly shown in the expanded configuration in Figure 14E, which is comparable to Figure 14H in the static state. Skin expansion is also illustrated in Figures 15A-15B. As shown in Figure 15B, when the skin expands, an opening is formed that allows for the extraction of bodily fluids.

[0025] As described above, the extraction assembly 2 may have a single negative electrode or multiple negative electrodes (4, 8, 16, 32, or 64 negative electrodes as examples). In a preferred embodiment, only a single positive electrode is present, covering the bottom surface of the extraction assembly. Figures 16A–16D show embodiments with a single positive electrode and four negative electrodes. Figures 16E–16H show embodiments with a single positive electrode and eight negative electrodes. Finally, Figures 16I–16Q show embodiments with a single positive electrode and 64 negative electrodes.

[0026] This disclosure assumes the use of various hydrogel adhesives (for example, those in the configurations shown in Figures 17A-17C or Figures 18A-18C). The hydrogel assembly 50 includes a hydrogel 51, a conductive layer 52, and an adhesive 53 that can be placed on an extraction head 54 (Figures 17A-17C). The hydrogel assembly may also be circular (Figures 18A-18C) from which a contact point 55 extends. The "extraction head" 55 is a solid member that is attached to the skin with an adhesive, for example, a hydrogel or a polysaccharide. In some embodiments, the extraction head is a solid material constituting a vacuum chamber through which the extracted interstitial fluid can be extracted. Polysaccharides are not conductive, while hydrogels are and may have a secondary use as a substitute for the positive electrode when using a hydrogel electrode configuration to replace a polysaccharide adhesive. Therefore, hydrogels are preferred (i.e., hydrocolloid electrodes). Connection to the hydrogel cathode may be made via a "carbon film," which may also be supported by an additional metal layer / metal backing. When using a larger current and / or a larger number of electrodes, the surfaces of the hydrocolloid electrodes should be designed proportionally, or the electrical parameters can be adjusted, to provide the same electroporation conditions. However, excessively increasing the current may cause an undesirable sensation on the skin.

[0027] Following complete electroporation of the skin, pores are formed in the outer portion of the skin. These pores may remain "open" if left "until" for a period exceeding 15 minutes. To ensure continuous extraction of interstitial fluid and to maintain the open pores, negative pressure must be applied across the electroporated area. This negative pressure must be sufficient to overcome the capillary effect that would otherwise prevent interstitial fluid flow through the pores. Interstitial fluid extraction has been successfully carried out in a continuous manner for periods of up to 21 days. The applied pressure difference tends to pull the skin outward and into the extraction chamber. The extraction chamber must then be large enough to prevent the pores from coming into contact with solid surfaces that could hinder both the extraction of interstitial fluid and the maintenance of the open pores. Some skin deformation and mild subcutaneous bleeding or redness may occur, which are unacceptable under certain circumstances. This skin deformation can involve not only elastic deformation of the skin but also plastic deformation, all of which can vary greatly depending on the characteristics and conditions of the subject's skin. Minimizing the skin surface exposed to negative pressure also minimizes skin dilation, and therefore minimizes the dead volume to be processed during interstitial fluid extraction. The mechanical structure of the extraction chamber with supports contributed to limiting skin dilation and minimizing considerations regarding dead volume. Experiments were conducted using thin sheets of solid material with small holes perforated to coincide with the center of each electroperforation area of ​​the skin. In this case, skin dilation was very limited, but interstitial fluid extraction was also very small or nonexistent. One conclusion of the experiment was that the electroperforated skin functioned like the diaphragm of a pharmaceutical vial, which is punctureable but remains "sealed" once the needle is removed. However, when such a perforated diaphragm is exposed to a pressure difference, and if exposed to a pressure difference, the diaphragm expands, and the perforations similarly "enlarge," becoming larger openings, allowing fluid to pass through the opening (perforation) more easily. To help minimize the above-mentioned problems, this sampling device is designed to include a thin, flexible elastic membrane with small holes aligned with electroperforated openings in the skin.Skin deformation and subcutaneous hemorrhage are considered very serious problems in applications such as glucose monitoring devices, where these effects on the skin can have detrimental effects on the quality and content of the extracted interstitial fluid.

[0028] In some embodiments, the extraction assembly may be manufactured from a polymer material, a combination of polymer materials, or any other material, which may be conductive or insulating. The sampling device may be covered with a material to facilitate its use in a swimming pool, while using a bathtub or shower, and / or in any situation where liquid may surround the extraction assembly. The extraction assembly may include sealed holes so that the collected, freshly extracted extract can be analyzed away from the device. Furthermore, to enhance the fixation of the extraction assembly described herein, a non-allergenic material such as a bracelet or strap may be used to stabilize contact between the device and the skin.

[0029] Before the sampling device is brought into contact with the skin, the skin may be gently cleansed using any known chemicals used in medical cleansing of such skin, or simply with soap and / or water. Preferably, any chemicals that evaporate after cleansing are used so as not to leave any residue on the skin surface. Furthermore, the chemicals or soaps must not induce any allergic reaction of the skin, nor alter the structure of the skin. As soon as the skin is gently cleansed, the sampling device is attached to the cleansed skin with the help of a non-allergenic material such as an adhesive, strap, or bracelet. The preferred portion of the skin is any portion of the skin covering a person's arm, but most of the skin of the human body can be used with the device and method described herein. Once attached to the skin, continuous extraction is carried out by the action of controlled negative pressure applied by a pumping system. The controlled negative pressure is maintained by the action of a pump, and the level of negative pressure is preferably continuously monitored and controlled using a pressure sensor.

[0030] Negative pressure is achieved by operating the pump in an on / off manner or in a continuously modulated manner. The pump and pressure sensor may be under the control of a microcontroller that monitors the actual pressure within the device in a continuous or discontinuous manner by reading the actual state of the pressure sensor, and accordingly starts or stops the pump operation in a continuous, discontinuous, or modulated manner to achieve the desired negative pressure. A temperature sensor may also be incorporated to improve the efficiency of the device and, if necessary, to compensate for the readings of the biosensor 22 or multiple biosensors. Vacuum is generated internally between the skin and the pump to improve contact, continuous extraction of transdermal fluid, and circulation of the extract, and as a result, one or more analytes in the extract can be monitored in a continuous manner. The vacuum pump provides a vacuum sufficient to stretch a portion of the skin in the area from which the interstitial fluid sample is extracted. While the suction provided by the vacuum pump stretches the appropriate portion of the skin, the suction provided by the vacuum pump also ensures that the stretched portion is completely filled with interstitial fluid. Vacuum pumps suitable for the apparatus defined herein may be peristaltic pumps, diaphragm pumps, piston pumps, rotary vane pumps, or other pumps capable of performing the required functions described herein. Typically, the vacuum pump preferably employs a self-contained, permanent magnet DC motor. The vacuum pump is preferably capable of providing a pressure difference up to about -14.7 psi, and more preferably operates from about -0.3 psi to about -10.0 psi. The vacuum provided by the vacuum pump may be continuous or pulsed. Continuous vacuum is preferred because it requires fewer parts than those required by pulsed vacuum. It is preferable that the applied vacuum does not damage the skin. It is preferable that the applied vacuum does not cause skin bruising and discoloration that lasts for several days. It is also preferable that the level of vacuum applied and the duration of its action are not excessive enough to cause the dermis to peel away from the epidermis, resulting in the formation of fluid-filled blisters.The sampling device may also include other electrochemical or optical sensors, wired or wireless communication devices, a detection electrode, a reference electrode and a counter electrode, and / or an accompanying electrode that acts as both the detection electrode and the reference electrode and counter electrode.

[0031] The use of a pump with positive displacement has no different effect than the use of any other type of pump. The main parameter in extraction is to maintain a constant pressure difference between the inside of the human body and the extraction site itself (assuming that the inside of the human body and the atmosphere are the same or have a constant relationship, and in fact this is the case), and therefore maintain the pressure difference between the atmosphere and the extraction site. The use of any type of pump that ignores the pressure difference will disrupt the extraction, create an excessive pressure difference in the skin, cause excessive skin dilation, and further cause blood to be drawn through the skin or pores, or cause blisters on the skin.

[0032] As described above, the sampling device has electrodes for the purpose of performing electroporation in the skin. This is non-invasive, provides effective electrical permeability to the stratum corneum, has a permanent opening as long as suction is applied, and can allow continuous transcutaneous fluid extraction for as long as required without any sensation of pain. In this specification, continuous fluid extraction refers to the continuous flow of interstitial fluid into the extraction assembly through the skin having an opening in the stratum corneum.

[0033] This disclosure describes an apparatus and method for non-invasive, continuous extraction of interstitial fluid through the skin. The method, detailed below, provides a precise method for obtaining openings that persist over several days or over a period of time during which one or more analytes are being monitored in the interstitial fluid. The openings relating to this disclosure are on the order of micrometers and should not be confused with electroporation at a smaller scale for individual cells. In fact, the electroporation described in this disclosure is electroporation that results in openings in the skin or stratum corneum. Because multiple openings persist over several days, one or more analytes in the extracted interstitial fluid can be accurately and precisely monitored, whether or not further electroporation is performed on the same area of ​​skin.

[0034] The microcontroller can also control the application of electrical excitation to the skin. If necessary, mechanical contact between the electrode and the skin can be controlled by the microcontroller. Once contact between the electrode and the stratum corneum is established, the microcontroller then initiates the process of electroporation of the skin. The microcontroller can control, over time, the voltage (either pulsed applied voltage or non-pulsed floor voltage), current, repetition rate, pulse duration or pulse duty cycle, duration of voltage and / or current application, control the measurement of voltage and current, and further control conditions that can instruct the start and / or stop of voltage and current application.

[0035] One of the advantages of this sampling device is the use of a hydrogel-based positive electrode, in contrast to conventional metal electrodes. It has been found that metal positive electrodes did not produce sufficient openings in the skin through electroporation. Furthermore, devices with metal positive electrodes were painful for subjects. The large hydrogel surface currently used in this device reduces the pain experienced by subjects and does not cause significant skin redness by dispersing the current at the contact site. Hydrogel electrodes are a commercially available technology. The surface of the hydrogel positive electrode is determined based on the number of negative metal electrodes to obtain efficient electroporation. Exemplary hydrogels include, but are not limited to, stoma adhesive hydrocolloid materials, stoma adhesive hydrocolloid materials coated with polydimethylsiloxane (PDMS) adhesive auxiliaries, or other similar hydrogels accepted as skin-compatible.

[0036] It has been found that a unipolar high-voltage pulse, applied simultaneously with a continuous low voltage between electrodes of an electrode array having a metallic positive electrode placed on the skin, is sufficient to perform electroporation. Conventional methods also include applying mechanical pressure to bring the electrodes into contact with the skin and to better localize the electroporation point, as well as using a negative pressure difference between the skin and the atmosphere during electroporation. More specifically, the metallic positive electrodes used were made of a noble metal (particularly Pd), were small in diameter, and had a conical end in contact with the skin. This sampling device replaces these electrodes with a large-surface semiconducting hydrogel electrode, which may be made from a hydrocolloid material.

[0037] Experiments conducted and observations of skin openings formed with conventional devices using metal electrodes revealed that the electroporation process did not actually open the skin beneath the positive electrode. Reducing the number of positive electrodes relative to the number of negative electrodes did not improve the situation. Throughout these experiments, pain was consistently perceived in the electroporated skin area during the application of the electrical process. Furthermore, there were concerns that the positive electrode did not form an effective opening in the skin beneath the positive electrode, but merely caused localized redness and swelling of the skin.

[0038] To reduce unwanted skin changes caused by the positive electrode and to maximize the effectiveness of the opening process by the negative electrode, the sampling device uses a large-area conductive positive electrode in contact with the skin. The concept of a large-area positive electrode is to effectively distribute the positive-side current over a large area of ​​skin. This large distribution area has the effect of eliminating unwanted skin changes caused by the positive electrode and reducing skin pain caused by the positive electrode. This utilizes an available and widely used electrical stimulation process. It is 10-40 cm 2 A hydrogel-based conductive electrode that may have a region is used.

[0039] The use of such hydrogel-based cathodes has been demonstrated to be a significant improvement over the electroporation process. Because there is no metal positive electrode, it is possible to position multiple negative electrodes more effectively and more centrally on the skin. • Reduced the sensation of pain felt in the skin due to the electroporation process. It can be installed at any distance or position from the negative electrode.

[0040] Hydrogel electrodes also impose several considerations for use. Since the hydrogel is placed only on the skin and not pressed into the skin, as is the case with the negative electrode, the surface area of ​​the hydrogel in contact with the skin must be considered in relation to the number of negative electrodes. This is due to the limited conductivity of the hydrogel electrodes and the amount of total current returned through them, the amount of which depends on the number of negative electrodes and the individual currents of the negative electrodes.

[0041] Under exemplary experimental conditions tested, it was found that using one hydrogel electrode with a surface area of ​​2-3 square centimeters as the positive electrode was sufficient to perform successful electroporation using four negative electrodes, each negative electrode with a nominal current of 130 microamperes. The manufacturing process for this type of electrode belongs to the low-tech category, and they can be manufactured in any shape and size, which is preferable to our equipment and processes.

[0042] The use of a hydrogel cathode is limited to the electroporation stage of the process and is no longer required after electroporation is complete. Installation, connection, and positioning of the cathode are additional operations, as are removal and connection of the cathode after electroporation is complete. Furthermore, the adhesive properties of the hydrogel material are assumed to allow its use as an adhesive / sealant required for the successful operation of the interstitial fluid extractor system, thereby achieving a dual function. This structure eliminates the installation, removal, and connection of the cathode, improving the usability of the interstitial fluid electroporation / extractor apparatus, as well as improving the reliability and appearance of the apparatus. This simpler and more compact configuration can improve the overall reliability of the apparatus. Generally, the adhesive used for mechanical retention of the extraction assembly on the skin, and for maintaining reliable vacuum airtightness, should have long-term compatibility with the skin. In preferred embodiments, double-sided tape adhesive is used to obtain clearer and more defined electroporation holes.

[0043] One of the challenges that conventional sampling devices and methods have struggled with is how to effectively determine when electroporation is complete, whether the openings are of the desired size, and whether they are stable and permanent. Currently, measuring the resistance of the negative electrode to monitor electroporation has been found to be a suitable means of determining the level of electroporation. The basic principle of monitoring impedance is that as the openings reach an efficient and more permanent state, the impedance between the two holes decreases. This change can be measured as a decrease in impedance when electroporation is not being performed and during electroporation. A method for measuring the rate of closure after electroporation has been stopped is also provided as a way to evaluate opening efficiency. In particular, this method involves measuring resistance or complex impedance in "kilohms" using the change in voltage in the electrode arrangement and the rate of increase and decrease in voltage. Resistance or impedance is basically defined as the ratio of voltage to current. In some embodiments, a target value of resistance or a range of resistance values ​​can serve as an indicator of sufficient electroporation. This target value or range can generally be calibrated based on the device, taking into account skin variations, and can also be calibrated individually, as there can be considerable variation in skin characteristics between individuals. Even without a target value or range, the rate of change in impedance (or resistance) and the "holding pattern" can be used as indicators of the pore opening state. Actual resistance may depend on the electrode size, the pressure of the electrodes on the skin, the characteristics of the skin itself, the distance between electrodes, the size of the electrodes, etc.

[0044] Resistance measurements and variations can be associated with openings formed in the skin, which are "holes" that allow IF to leak out. If the holes are "open" to a sufficient degree in the term "properly electroporated skin," they tend to remain open for longer periods. This means that if the holes are open for a long period, some additional pressure may be needed to extract the IF. Holes or openings tend to return to a "closed" state at a lower rate than those that are "under-electroporated" or even "over-electroporated." In over-electroporated situations, resistance tends to increase again when under "over-electroporation" conditions. Measured resistance values, in absolute terms (when measured in ohms or kiloohms), may be variable depending on the measurement method, electrode distance, skin thickness, etc.

[0045] However, well-drilled holes in the skin tend to maintain their post-electroporation conditions for longer periods than poorly drilled holes. Therefore, resistance measurement methods rely on comparative measurements. Basic measurements are performed, for example, between two negative electrodes in a set, but can also be performed between a positive electrode and one negative electrode. However, in that case, there may be more uncontrolled variables than in the case of "negative electrode to negative electrode" measurements. During electroporation, resistance values ​​tend to decrease over time from measurements in unelectroporated skin to measurements in electroporated skin, decreasing from over 100 kilohms to a few kilohms.

[0046] To perform resistance measurement, electroporation is stopped, and a very low voltage is used to avoid altering the skin condition due to the measurement current / voltage. As an example, resistance is measured every second for 8 to 15 seconds. When examining a series of measurements, a re-increase in resistance is always observed between the first and last measurements in the series. If the rate of increase is large, electroporation may be considered insufficient. For example, if the increase between the first and tenth measurements represents a 10% to 15% increase, this is a result of efficient electroporation conditions. Note that if electroporation is pushed too far, the percentage increase will be even larger.

[0047] Resistance measurement is used to evaluate the durability of electro-drilled openings. The evaluation of this condition relies on the smallest resistance increase between the first and last resistance values ​​obtained in a series of measurements, consisting of 8 to 15 measurements, performed every second, as in the example, every 1 to 5 minutes.

[0048] Applying high-voltage electrical pulses along with a low-level continuous voltage creates durable (permanent) perforations across the stratum corneum. These perforations allow interstitial fluid to flow across the stratum corneum, as long as negative pressure is applied to the skin relative to the atmosphere (which is considered to be in equilibrium with the inside of the human body). The negative pressure serves two purposes: (a) to counteract the capillary pressure that keeps fluid within the human body, and (b) to allow the flow of fluid between the human body and an external device called an extractor. The negative pressure has a mechanical effect proportional to the value of the negative pressure, if maintained. The mechanical effect manifests as expansion of the skin toward the side with the lowest pressure, i.e., toward the outside of the human body. This skin expansion causes expansion of the electroporation holes, keeping them open. For various reasons, actual extraction devices use a fully open cavity where negative pressure is set and maintained by an active pumping system. The skin is elastic and, to a certain point, plastic, and when it expands, tends to create flow into the pump cavity. Depending on the value of the negative pressure used, the thickness of the skin, and the duration of the negative pressure's action, the skin may deform and expand, develop some damage along the sides of the extraction cavity, show ruptured capillaries in the negative pressure area, and / or develop blisters (under certain conditions). To reduce skin expansion and associated defects, a preferred embodiment is a symmetrical design with 4 to 6 active electrodes (holes) around a centrally projecting post. The role of the post is to prevent the skin from expanding excessively, thereby minimizing the so-called "dead" or unused volume necessary to keep the holes away from any surface to prevent obstruction of the fluid flow from the skin. To further reduce this effect, extractor assemblies with one hole per opening were fabricated. They were made of rigid polycarbonate resin. If the pores are too small, they can often block the electroporated pores due to adhesive displacement. If the pores are excessively small, they prevent them from expanding and thus prevent the flow of fluid through the stratum corneum. If the pores are made larger, they require a larger total volume and total surface area.Therefore, elastic membranes have been used to limit skin dilation, deformation, damage, and wounding, to limit or prevent capillary rupture, and to limit blister formation.

[0049] As described above, the electroporation process is a method for creating permanent pores in the stratum corneum. These pores then allow for continuous extraction of interstitial fluid as long as the extractor, which applies negative pressure to the electroporated area, is in operation. The pores to be created in the skin must be created in an optimal manner. Skin is a highly variable aggregate composed of living and dead cells, exhibiting greatly different behaviors in terms of thickness, elasticity, dryness, and fat content. These highly variable factors cause very different behaviors in response to the application of the electroporation process, which uses both current-limited voltage pulses and continuous voltage between the positive and negative electrodes placed on the skin. After a certain period of time, the high-voltage pulse will have disturbed the outermost layer of the stratum corneum. This process should be continued to ensure that the pores remain open. If the electroporation process is not applied for a sufficiently long time, the pores cannot be maintained open, and blisters will appear when the extraction process is applied to skin that has not been electroporated sufficiently. If the electroporation process is continued for too long a period of time, excessive electroporation occurs, which manifests as excessive redness of the electroporated skin and, more importantly, as the appearance of red blood cells in the extract.

[0050] A plot of voltage / current on the electrodes shows that, after just a few minutes, the resistance between any two negative electrodes is extremely low. The resistance between electrodes on unelectroporated skin is above 100,000 ohms (100 kΩ). A few minutes after electroporation, the resistance drops to the 5 kΩ range. Beyond that point, the resistance continues to decrease to lower values, but at a much slower pace. Determining the absolute value of the resistance to be reached for "optimal electroporation" is extremely ambiguous due to the large variation in skin characteristics. The resistance transition remains on a very flat curve, preventing an easy determination of the "point where the resistance transition stops."

[0051] The negative electrodes used in the electroporation process are small in diameter, (nominal 0.5 mm ± 0.1 mm). Their ends can be shaped conically and applied with sufficient pressure to localize the point of action of electroporation. The ends of the cone are not perfectly pointed to avoid mechanically puncturing the skin. The high-voltage pulse will, after a certain period of time, destroy the outermost layer of the stratum corneum. This process should be continued to maintain the pores open under the slight negative pressure necessary to extract interstitial fluid through the skin. If the electroporation process is not applied for a sufficiently long time, the pores will not remain open, and blistering may occur if the extraction process is applied to insufficiently electroporated skin. If the electroporation process is continued for an excessively long period, hyper-electroporation occurs, which is indicated by excessive redness of the electroporated skin, and more importantly, by the presence of red blood cells in the extract. Measurements of voltage and / or current at electrodes show that, after just a few minutes, the resistance (or impedance) measured between any two negative electrodes changes considerably from the initial state of the electroporation process. Resistance measurements between electrodes in contact with unelectroporated skin show values ​​exceeding 100,000 ohms (100 kΩ). After a few minutes of electroporation, the resistance falls into the 5 kΩ range. Beyond that point, the measured resistance continues to decrease at a much slower rate. As initially stated, skin properties are not only dependent on the subject's age and location on the body, but are also highly variable. Furthermore, there is the often overlooked factor of skin anisotropy, which can add considerable variability to the progression of electroporation. Therefore, the absolute value of resistance to be achieved for determining "optimal electroporation" is extremely ambiguous. Also, the resistance changes in a very flat curve after a few minutes, preventing any easy determination of a "point to stop electroporation." Therefore, this method for determining the optimal electroporation parameters focuses on "durability" as the primary determining factor.

[0052] Therefore, feedback methods can be used to more clearly define the completion of successful electroporation. This allows for automatic (or manual) control of electroporation parameters, namely time, current, pulse voltage, continuous voltage, pulse repetition frequency, pulse duty cycle, and total electroporation time. From the past to the present, the optimal electroporation time has been determined by past experiments using a set of operating electrical parameters that were similarly determined and validated by experiment. The method used was to stop electroporation, remove the electroporation head, and observe the skin to which multiple negative electrodes had been applied. Correlating what was observed with past observations was the method used to determine the "correct electroporation". The standard continuous electroporation sequence was 12 minutes of continuous electroporation, followed by a 30-second pause in electroporation and then a 3-minute resumption of electroporation.

[0053] During resistance measurements, a surprising phenomenon was observed: small droplets of interstitial fluid appeared at at least two locations on the negative electrode (the electrode was still in place) due to the negative pressure that had to act during electroporation, even though electroporation should have been complete. These interstitial fluid droplets, when large enough, could "short-circuit" the space between two (or more) electrodes, resulting in a sudden decrease in resistance readings compared to the previously observed "through-skin" resistance. For example, resistance could drop from 2 kΩ to 300 Ω (0.3 kΩ). In some cases, this phenomenon can be used as an indicator that the electroporation sequence is complete. However, this is generally an uncommon phenomenon, or (if it does occur) it may not occur on all electrodes. While continuous resistance measurements with appropriate analysis can provide a more reliable means of determining the effective completion of the electroporation process, the overall purpose is to take into account variations in skin thickness and the need to change electrical electroporation parameters. The purpose of the measurement is to provide a feedback method for adjusting the electroporation process to minimize pain and to maintain the electroporation time at an effective minimum. During extraction, if the opening is maintained in a stable manner, it means that the conditions keep the opening in a "stable" state. The further the opening is from the optimal value, the faster it will return to a higher resistance value.

[0054] An example method is as follows: • Electroporation has been stopped. • Resistance measurements are performed at predetermined intervals. • To determine whether the opening of the hole is sufficiently stable, and to determine the slope of the resistance measurement with respect to time, the measurement may, as an example, be performed over 15 seconds at 1-second intervals, then, (a) If the hole opening is stable, immediately terminate the electroporation process, or (b) Continue electroporation for an additional period, such as one minute, to give a “topping off” or “lacquering over.”

[0055] This sampling device and method are particularly relevant in the field of glucose monitoring for the following reasons: glucose monitoring does not require high flow rates (high flow rates can be undesirable); the membrane significantly reduces skin deformation and skin wounding; long-term use of the glucose monitoring device is desirable; the membrane may suppress capillary rupture; and the membrane suppresses blister formation. To maximize its usefulness, such a membrane should adhere closely to the skin, minimizing skin slippage as much as possible to keep the skin pores centered in the membrane pores, and helping to maintain a strict minimum "dead volume," i.e., the dimensions of the extraction chamber.

[0056] One potential concern regarding glucose measurements is that density and extraction rate can affect the accuracy of the measurement. Dynamic pressure control is key to avoiding this. Reducing the pressure in low-sample-volume tests improves the integrity of the sample in the process. In this case, "reduce" means using the lowest possible pressure. Since the range of 5 to 10 kilopascals is roughly the normal range, the pressure is preferably less than 5 kPa, and preferably in the range of 0.1 to 1 kPa.

[0057] This disclosure should not be limited to glucose and diabetes, but can monitor any analytes found in IF (for example, lactate, cortisol, triglycerides, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), C-reactive proteins, interleukin-1, -3 or -18, and cystatin A or C, and many others). The sampling device is sealed, allows for extraction of contents, and requires minimal handling, thus reducing the risk of leakage or contamination. The sampling device can itself separate the aqueous fluid from the collected sample, thus obtaining only ISF. The sampling device can be configured as various sizes and shapes are assumed herein.

[0058] Furthermore, this specification also includes reading and analyzing signals obtained from a biosensor of a sampling device for transforming the acquired information and transmitting it, as an example, to an alarm or mobile phone, for the purpose of further disseminating the acquired information. The transmitted information may have many meanings, such as the net concentration of the analyte, measurements for multiple analytes, decisions to be made by the parties involved, or simply a warning. In one embodiment, if a continuous biosensor monitoring glucose is based on an electrochemical method, the biosensor may include three or two electrodes. In the case of a three-electrode configuration, the first electrode is a general Ag / AgCl reference electrode, an inert counter electrode constitutes the second electrode, and the third electrode is the detection electrode. The detection electrode has an inert electron layer surrounded by a detection layer. The detection layer is obtained by depositing a homogeneous mixture of glucose oxidoreductase (e.g., glucose oxidase), mediators, nano / micro powders of conductive materials, crosslinkers, hydrophilic materials, hydrophilic additives, and adhesion promoters onto the electron layer-based detection electrode.

[0059] In summary, the design of this sampling device has many advantages, not limited to the following: -This design uses a sealed container for the device. - This design uses a removable sealed container for the device. - This design aims to create a sampling unit that can be used with minimal operation. - This design enables efficient collection of liquid (ISF) very close to the extraction point. - This design makes the device a spill-proof container and assembly. -This design uses an aqueous fluid separator that collects only IF (infrared filtration). - This design makes the device an easily reconfigurable sampling device for any volume (large or small), - This design allows for a sterilizable configuration. - Forms permanent pores, - Using low power, and not requiring electroporation after the first electroporation, - Simple continuous extraction, - High-flow extraction is possible. - Sequential extraction of IF statements, - Continuous administration of substances is possible, - Operates non-invasively, while placed outside the body. [Examples]

[0060] Example I In the 4-electrode pattern, it was observed that the rate of electroporation differed between electrodes. This difference may result from a difference in the limiting current. The attachment of the extraction device may stretch the skin in multiple directions, which may affect the actual mechanical and static pressure applied by the negative electrode to the skin.

[0061] Experiments involving electroporation of the same individual using two different configurations yielded results contrary to conventional assumptions. Both electroporations were performed with the negative electrode (a set of four electrodes) placed on the subject's forearm. Identical electrical and timing parameters were used in both electroporations. The first electroporation used a positive electrode positioned on the forearm, very close to the extraction chamber (equipped with four negative electrodes). The second electroporation used a positive electrode on the calf of the subject, on the same side of the body as the forearm where the extraction chamber with negative electrodes was attached. In both cases, the electroporation was successful, and the markings on the skin were similar. It was then inferred that the position of the positive electrode was not critical. Furthermore, it was demonstrated that an array of 64 negative electrodes is possible. The advantage of increasing the number of electrodes is an increase in the rate at which the sample is obtained.

[0062] Preferred electroporation parameters for use on the medial forearm of adult humans were determined. However, even when using those parameters, the results of electroporation can vary greatly depending on skin conditions (dryness, stratum corneum thickness, lipid layer thickness, age, etc.). • Pulse voltage (high voltage): 75V • Pulse repetition frequency: 4000 / s (4 kHz) (period 250 microseconds) • Pulse duty cycle: 2.375% • Continuous voltage (low voltage): 7V • Negative electrode current (limit): 130 microamperes • Duration: One period of 720 seconds (T1), followed by a 30-second pause (T2) and a final electroporation period of 180 seconds (T3). • Negative pressure: Typically 10 kPa.

[0063] The following devices were used to perform electroporation of the skin: • An extraction chamber that is airtightly bonded to the skin surface using an adhesive layer. • A pumping system to maintain negative pressure in the extraction chamber during electroporation. • An electroporation apparatus for generating and controlling electrical and other conditions necessary for electroporation. • Electrode holder for positioning the electrode within the opening of the extraction chamber and sealing the extraction chamber during electroporation. A positive electrode, specifically referred to as a "positive electrode," provides a positive electrical return path via (for example) a hydrocolloidal material.

[0064] The skin should be prepared with minimal selection in the following manner: If the skin appears oily, wash the skin with mild soap and dry it thoroughly, or wipe it with a mild alcohol solution. You may wait 20 minutes before the next step. If there is body hair on the surface of the skin, it is recommended to remove the hair by shaving. Avoid rubbing the surface of the skin. It is preferable to wait several hours before the next step. If the skin is too dry, adhesive tape can be used to remove the outer flakes from the skin to ensure that the adhesive / sealing surface adheres well to the skin. The extraction assembly is mounted on the skin by ensuring that the skin remains flat to avoid wrinkles that may disrupt the vacuum. Belts, straps, or adhesive tape are used to further secure the extraction head on the skin. The pump unit is then connected to the vacuum connector on the extraction head. The electrode holder assembly (electroporation head) is added to the extraction head, taking care to insert the electrode holder assembly perfectly vertically. The electrode length should be determined so that the electroporation point is clearly defined and localized, allowing a certain amount of mechanical pressure to be applied to the skin. A vacuum test is performed to verify the possibility of leakage. If leakage is present, it is corrected. The pump / vacuum control is maintained at a working pressure P1. The hydrocolloid positive electrode is placed on the skin and connected to the electroporation head or electroporation device. The electroporation device is then connected to the electroporation head. Software is used to verify or adjust the electroporation parameters. Electroporation is started, and its duration is T1. Preferably, resistance measurement is performed to determine whether electroporation is complete. When using resistance measurement, automatic measurement can be set to interrupt electroporation for N2 seconds every N1 seconds, during which resistance measurement is taken every M1 milliseconds and recorded sequentially. The percentage change between the first and last measurement should be used to determine successful electroporation or to adjust the electroporation parameters. If necessary or desired, wait for a period T2 to resume electroporation, and optionally wait for a period T3 if further necessary.Phase M- can also be performed to verify the completion of electroporation. At this point, electroporation should have successfully completed openings within the skin. When using resistance measurement methods, electroporation could be terminated before and / or after the typical sequence of T1, T2, and T3. Once electroporation is deemed complete, the pump unit is stopped. The electroporation head is then detached from the electroporation device itself and from the hydrogel cathode (if such a hydrogel cathode is used). The electroporation head is removed. At each negative electrode position, the skin should show a slightly darker tone, indicating successful electroporation. Due to the applied negative pressure, and in the case of successful electroporation, droplets of interstitial fluid may be present at the electroporation points, indicating successful electroporation.

[0065] As quickly as possible, the extraction head should be closed again with the top of the extraction head attached, maintaining the required negative pressure to extract interstitial fluid from the skin through the already formed hole. The following steps were performed: The vacuum connection was reconnected to the pump unit. The pump unit was started to maintain the pressure at P1. It was verified that the vacuum was a stable and constant vacuum, and that there were no leaks at the skin interface with the adhesive, the top of the extraction head set, and the tubing connecting the extraction head to the pump unit. Corrections were made if necessary. The pump unit may operate occasionally to equalize / control the pressure at point P1. Its pumping may also be caused by the outflowing fluid. Along with the liquid, some gas may also be present, i.e., dissolved gases from the interstitial fluid. After several tens of minutes, it should be observed that the fluid is attempting to fill the internal space of the extraction chamber. After the extraction chamber is filled, it is observed that the fluid is slowly flowing out of the extraction chamber through the tubing connecting the extraction chamber to the pump unit. For sampling purposes, the extract is collected at the outlet of the extraction chamber.

[0066] The mechanism that allows electroporated areas to rapidly revert to a non-electroporated state is an indicator of the progression of electroporation to "permanent pores." Electroporated "pores" or "micropores" tend to rapidly revert to their non-electroporated state. The conditions under which the process operates make the pores "permanent" after a certain period of time. It has been noted that for successful electroporation to occur, the conditions that must be applied must include a certain number of factors. - Pulse repetition frequency - Pulse duty cycle - Pulse voltage - Continuous (base) voltage -Current - Application time

[0067] These factors also need to be adjusted according to the characteristics of the skin being electroporated. One of them is the "application time." Successful electroporation is currently determined in a very relative way using the following observations. - Visual inspection of the skin beneath the electrodes. - Appearance of liquid around the electrode position (when negative pressure is used during electroporation).

[0068] The method is very imprecise and requires opening of the electropermeabilization set in contact with the skin, making the determination of successful electropermeabilization quite rough and potentially resulting in either under- or over-permeabilization, which can further lead to additional problems such as blister formation and bleeding during IF.

[0069] The sampling apparatus of this disclosure was tested by measurements on a benchtop skin model using resistance measurement between electrodes. Electroporation was paused for approximately 10–15 seconds. Measurements were taken every 0.5 or 1 second, and the rate of increase in resistance was evaluated. A rapid increase in resistance over a 10–15 second period would indicate incomplete electroporation. Conversely, if the resistance remains very close to the initial value, it indicates successful electroporation. This is because an increase in resistance occurs when the pore closes again, while a low resistance indicates that the pore remains "permanently open." Thus, this method is considered to provide an accurate understanding of the state of electroporation in the electroporation process. Different skin conditions may then show acceptable patterns of resistance measurements after various time periods, but this always provides a much more accurate indicator of the completion of electroporation than estimation and looking inside the extraction / electroporation chamber.

[0070] In embodiments, during the electroporation process, resistance or impedance can be measured, for example, by measuring changes in resistance, to confirm optimal contact with the skin. If necessary, the current level can also be adjusted after measuring resistance or impedance. Measuring resistance or impedance allows the user to subsequently detect whether the electrodes are in good contact with the skin, thus preventing electroporation failure due to poor contact. Furthermore, it enables efficient detection when perforation occurs, allowing the clinician to immediately stop the perforation, reduce the size of the defect, and increase the speed of the procedure. It also includes adjusting perforation parameters according to the data to improve the effectiveness of the perforation; if the data indicates a tendency for slow hole opening, the perforation intensity may be increased to attempt to increase the speed of hole opening. Measurement taken over 10 seconds, 4 minutes after electroporation. [Table 1] Measurement taken over 10 seconds, 9 minutes after electroporation. [Table 2] Measurement over 10 seconds at 14 minutes after the start of electroporation. [Table 3]

[0071] As shown in Tables 1-3, at 14 minutes, the measured resistance values ​​tend to remain fairly stable. This is an indicator that electroporation may have reached its final stage. This avoids the conventional practice of opening the electroporation chamber to monitor the condition of the hole, which was done because there was no precise way to determine when to stop electroporation.

[0072] The sampling device of this disclosure was attached to the skin using double-sided adhesive tape under a first condition and using glue adhesive under a second condition. The fixation configuration using glue adhesive was found to affect the holes and results, and adhesive setting should be avoided (Figures 19A-19B). When using double-sided adhesive tape (Figure 20A), the hole diameters were found to be consistent between electrodes, but when using adhesive (Figure 20B), the hole diameters were found to be inconsistent between electrodes.

[0073] The progress of electroporation was evaluated using resistance measurement. The apparatus used to measure the resistance between the two electrodes was a completely insulated apparatus, meaning it did not add any resistance or capacitance to the electrodes of the electroporation apparatus or the electroporation apparatus itself, and therefore did not affect the electroporation.

[0074] The measurement procedure was carried out as follows: Electroporation was stopped at programmable periodic intervals ranging from 30 to 999 seconds. The electroporation device was functionally separated from the electrodes. A low-voltage device measured the resistance between each pair of electrodes. The measurement was performed by applying a low voltage between electrodes and measuring the current flowing between them to determine the resistance, thereby obtaining the value of the resistance. • The measurements were performed with the polarity reversed to eliminate any "ionic effects" or "battery effects." Measurements were performed over very short periods to eliminate or minimize ionic or battery effects. The process was repeated a programmable number of times, ranging from 1 to 15 times per second.

[0075] The reasoning behind this measurement method is as follows: • The electroporation process is not instantaneous, but progresses over time. Instantaneous measurements of resistance or impedance may not provide a good assessment of the "persistence" or "durability" of the electroporation results. The ease with which electric current passes through the electroporated hole depends on the progress of the electroporation process. If electroperforated skin is allowed to rest, the holes will close after a certain period of time. The time it takes for the closure to become more complete depends on the "quality" or "progress" of the electroporation. While we do not wish to be bound by theory, there is an assumption that the skin rests for a period of time, and then the electrical resistance of the electroporation holes is evaluated. There is no precise value or absolute value of resistance that can be used to determine the "integrity" of electroporation using the methods described herein. Rather than simply waiting and then measuring the resistance, it is decided to perform continuous measurements over a period of time to observe the "rate of change" of the resistance and determine the "point" at which one can reasonably be confident that it is a "good" electroporation. The absolute value of the resistance can be helpful, but it may not always be the determining factor.

[0076] To illustrate examples of resistance measurements when electroporation is performed on different parts of the human body, a series of measurements were performed on the same human subject. The following parameters were used: duration 250 μs, duty cycle 2.375%, duration 960 seconds, pulse voltage 75 V, base voltage 7.5 V, resistance measurement interval every 120 seconds, number of measurements per interval 10 (1 per second), and duration of electroporation between 960 and 1600 seconds.

[0077] The first measurement was performed on the inner side of the forearm of a human subject. The electroporation duration was 960 seconds. When electroporation was completed, fluid was already present at the electrode sites, but the fluids at each electrode site were not interconnected. There may have been some over-electroporation, but the extracted fluid did not contain blood. Resistance measurement [Table 4] JPEG2026531708000006.jpg196166 JPEG2026531708000007.jpg195166 JPEG2026531708000008.jpg196166

[0078] The point to note from the table above is the rate of increase in resistance over time. The slower the increase in resistance, the better the electroporation.

[0079] The second set of measurements was performed on the lower part of the upper thigh of a human subject. This part of the human body has somewhat thick skin and may have more fat beneath the epidermis, and as an example, it has properties similar to pig skin. Electroporation was performed for a full period of 960 seconds, and upon completion, electroporation was restarted for another 960-second period (the latter not fully completed). At the end of this final electroporation, fluid was found to be present at the electrode locations, but they were not interconnected. However, since the electrode locations were not "blackened", it was certainly not excessive electroporation. Second resistance measurement [Table 5] JPEG2026531708000010.jpg187166 JPEG2026531708000011.jpg196166 JPEG2026531708000012.jpg185166 JPEG2026531708000013.jpg203166 JPEG2026531708000014.jpg196166 JPEG2026531708000015.jpg195166 JPEG2026531708000016.jpg107166

[0080] By comparing the final measurement described above with the final measurement of the forearm, it can be observed that the resistance ratio between measurement 2 and measurement 10 is greater during this total electroporation time, and therefore, it can be concluded that electroporation should be performed for a further period of time. Visual observation supported the electrical "blind measurement."

[0081] However, it is interesting that in those limited cases, when good electroporation is achieved, there appears to be an "absolute value" of resistance. This is likely to differ depending on whether the subject is human or animal, or even on the part of the human body.

[0082] The evaluation of the "rate of change" was better performed between measurement 2 and measurement 10, as the first measurement may be affected by misplaced setup values. Furthermore, what may be more suggestive is not the value of the ratio itself, but the stability of that ratio.

[0083] The measurements performed so far support the importance of using this method to understand the progress of electroporation. This is because this method has the ability to help determine when to introduce changes in electroporation parameters or the duration of electroporation for refinement of the approach or for dynamic adjustment or tuning during electroporation. Example II

[0084] Extraction was performed on two volunteers (males under 60 years of age). Following 30 minutes of electroporation using the sampling apparatus configuration shown in Figure 21, 400-500 μL of interstitial fluid was obtained over a 5-hour extraction period.

[0085] In a further configuration, as shown in Figure 21, a sampling device 60 is included, comprising an extraction assembly 62 connected to an electronic module 64. As incorporated herein, the extraction assembly comprises a negative electrode 66 and a positive electrode 68 having a hydrocolloidal material. In this configuration, the negative electrode 66 is deposited on the positive electrode, which is on top of a vacuum plug 72, or secured with a clip, via an electrode dock 70 on the positive electrode. In operation, once a fluid is collected, it moves through a fluid path or tube 74 to a sampling container 76 (which may, in some embodiments, be a tube or a chamber, but is not limited thereto). The fluid path 74 can be connected to the sampling container 76 via a connector 78. As defined herein, a continuous vacuum is preferred, which is applied to the extraction assembly 62 via a vacuum conduit 82 using the vacuum plug 72, via a connection 80, by the electronic module 64. To ensure the sterility of the extraction assembly 62 system, a sterile filter 84 can be integrated into the system. The negative electrode 66 is connected to the electronic module 64 via a hole 86, and the positive electrode 68 is connected to the electronic module 64 via a hole 88, so that the electronic module 64 controls the current and vacuum applied to the extraction assembly 62.

[0086] Using the sampling device incorporated herein, extraction was carried out in a two-step process for accessing and collecting interstitial fluid from the human body. The first step involved creating micro-openings in the epidermis (stratum corneum), and the second step involved extracting the interstitial fluid through these micro-openings. Step 1 - Electroporation

[0087] The electroporation process has three components: an extraction head (for example, a square block placed on the surface of the skin via an adhesive patch, a circuit board on which an electroporation probe is mounted that forms pulsed, mild stimuli, which in turn form micro-openings, and an electroporation module which is a driver and Bluetooth link to a computer program that delivers stimuli to the extraction site and monitors the stimuli).

[0088] The extraction head contains multiple probe electroporation heads and multiple chambers. Each chamber has space to accommodate multiple electroporation probes, which are mounted on a circuit board and operate as a single device.

[0089] The extraction site on the skin was pre-treated with an alcohol swab, and an extraction head with adhesive was placed on the extraction site. A wire harness connected a circuit board to the electroporation module, and the circuit board was secured to the extraction head assembly. A Bluetooth connection was established between the computer program and the electroporation module, and the duration (920 seconds in this embodiment) and appropriate voltage settings were configured. After completion, the electroporation assembly (electroporation head, multiple positive electrodes, and electroporation module) was removed from the extraction head and site. Step 2 - Extraction of ISF

[0090] In the extraction process, the pump module described herein, attached to the extraction head, was used. The extraction head was fitted with a sealed transparent cover, sealing the extraction site within the vacuum chamber. In this embodiment, tubes were connected from the extraction head to a sampling tube, and from the sampling tube to the pump module, generating the negative pressure necessary to extract ISF from the interstitial tissue. Once the pump was started, it maintained a predetermined pressure level within the extraction chamber and over the micro-opening. The pump was kept on until the desired amount of fluid was extracted.

[0091] The amount of fluid collected from the first subject was approximately 450 μL to 500 μL, and the amount of fluid collected from the second subject was approximately 500 μL to 550 μL.

[0092] Although this disclosure has been described with particular reference to the illustrated embodiments, those skilled in the art will understand that numerous modifications thereto will be conceivable. Accordingly, the above description and accompanying drawings should be construed as illustrative and not as limiting. One positive electrode was attached to the skin for each probe cluster.

[0093] While this specification has described connections to specific embodiments, it will be understood that further modifications are possible. This application is intended to encompass any variations, uses, or applications, including deviations from this disclosure that fall under prior art knowledge or common practice and are applicable to the essential features described above, and that fall within the scope of the appended claims.

Claims

1. An extraction assembly having a positive electrode, at least one negative electrode, and a suction channel adapted to be fluidly connected to a vacuum pump, A collector assembly having a fixed part and a detachable part, Equipped with, A sampling device for extracting interstitial fluid, wherein the fixed part has a fixed support part connected to the extraction assembly, a conduit for receiving interstitial fluid, and a needle positioned at the end of the conduit, and the detachable part has a cavity defined by a hydrophobic membrane enclosure that is in fluid communication with the conduit for receiving interstitial fluid, a diaphragm adapted to be penetrated by the needle, an external enclosure that houses the cavity and defines a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for discharging gas from the gas cavity.

2. The sampling device according to claim 1, wherein the positive electrode is a positive electrode that diffuses electric current.

3. The sampling apparatus according to claim 1, wherein the positive electrode is a hydrogel-based electrode or a dry electrode.

4. The sampling apparatus according to any one of claims 1 to 3, wherein the negative electrode contains palladium or is made of stainless steel.

5. The sampling device according to any one of claims 1 to 4, further comprising one or more sealing members for sealing the connection between the fixed portion and the detachable portion.

6. The sampling device according to any one of claims 1 to 5, further comprising one or more sealing members for sealing the connection between the fixed portion of the collector assembly and the extraction assembly.

7. The sampling apparatus according to any one of claims 1 to 6, wherein the hydrophobic membrane enclosure is square, circular, or dome-shaped.

8. The sampling device according to any one of claims 1 to 7, wherein the extraction assembly further comprises a double-sided adhesive tape for adhesion to the skin.

9. The sampling device according to any one of claims 1 to 8, wherein the positive electrode is used as an electrode having an adhesive function at the skin interface.

10. The sampling device according to any one of claims 1 to 9, wherein the positive electrode is a membrane at the skin interface having at least one membrane pore.

11. The sampling apparatus according to any one of claims 1 to 10, wherein the positive electrode comprises a hydrogel assembly having a hydrogel layer, a conductive layer, and an adhesive layer.

12. The sampling device according to any one of claims 1 to 11, wherein the sampling device has four or more negative electrodes.

13. The sampling device is the sampling device according to any one of claims 1 to 12, wherein the sampling device has eight negative electrodes.

14. The sampling device according to any one of claims 1 to 13, wherein the sampling device is connected to a module, the module is connected to the positive electrode and the negative electrode and controls the applied current, and the module has a vacuum pump for controlling vacuum suction through a vacuum suction channel in fluid communication.

15. The sampling device according to claim 14, wherein the module measures the variation in resistance between at least one negative electrode or between a positive electrode and at least one negative electrode.

16. The sampling device according to any one of claims 1 to 15, wherein the interstitial fluid is collected in a tube or chamber connected to a collector assembly.

17. The sampling apparatus according to any one of claims 1 to 16, further comprising the step of measuring the glucose level in the extracted interstitial fluid.

18. The positive electrode and at least one metallic negative electrode are brought into contact with the skin. Applying current between the positive electrode and the at least one metal negative electrode, The current is stopped when a hole is formed in the skin. Applying negative pressure to the skin in order to keep the pore open, A method for electroporating the skin of an individual, including [specific component].

19. The method according to claim 18, further comprising the step of determining the electroporation level by measuring the resistance variation between at least one negative electrode and at least one negative electrode, or between a positive electrode and at least one negative electrode.

20. The method according to claim 18 or 19, wherein the skin is washed before the contact step.

21. The method according to any one of claims 18 to 20, comprising attaching the sampling device according to any one of claims 1 to 15 to solid skin and applying an electric current.

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