Collection device and method of collecting interstitial fluid

Abstract

There is provided a collection device for extracting interstitial fluid that includes an extractor assembly and a collector assembly. The extractor assembly includes a hydrogel based positive electrode, a metallic negative electrode, and a pumping channel adapted to be fluidly connected to a vacuum pump. The collector assembly has a fixed portion and a removable portion, the fixed portion having a fixed support connected to the extractor assembly, a conduit for receiving the interstitial fluid and a needle at the end of the conduit, and the removable portion having a cavity defined by a hydrophobic membrane enclosure in fluid communication with the conduit for receiving the interstitial fluid, a septum adapted to be pierced by the needle, an external enclosure housing the cavity and defining a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for pumping gas out of the gas cavity.

Description

COLLECTION DEVICE AND METHOD OF COLLECTING INTERSTITIAL FLUIDCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is claiming priority from U.S. Provisional Application No. 63 / 513,598 filed July 14, 2023, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of fluid collection devices and methods using same, particularly for the collection of interstitial fluid.BACKGROUND OF THE ART

[0003] Early disease detection and disease monitoring are critical factors in diagnosing the correct physical symptoms and establishing appropriate therapies. This is often done by analyzing body fluids such as plasma, interstitial fluid and cerebral spinal fluid. Moreover, there is a need for constant monitoring of analytes in certain diseases such as diabetes. Diabetic individuals require an accurate and consistent reading of their blood glucose level to avoid complications which can be severe in certain cases. To monitor a body fluid for an extended period of time, different devices have been designed throughout the years, notably wearable devices that are in contact with the skin to monitor interstitial fluid. These devices rely on skin electroporation to collect interstitial fluid through the skin, see for example WO2010094131. However, improvements in the performance of these devices are still desired, in particular for diabetic individuals.SUMMARY

[0004] In one aspect, there is provided a collection device for extracting interstitial fluid comprising: an extractor assembly comprising a positive electrode, at lest one negative electrode, and a pumping channel adapted to be fluidly connected to a vacuum pump; and a collector assembly having a fixed portion and a removable portion, the fixed portion comprising a fixed support connected to the extractor assembly, a conduit for receiving the interstitial fluid and a needle at the end of the conduit, and the removable portion comprising a cavity defined by a hydrophobic membrane enclosure in fluid communication with the conduit for receiving the interstitial fluid, a septum adapted to be pierced by the needle, an external enclosure housing the cavity and defining a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for pumping gas out of the gas cavity.

[0005] In some embodiments, the positive electrode is a current defusing positive electrode, such as a hydrogel based electrode or a dry electrode. In an embodiment, the positive electrode comprises stomic 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 device, such as one or more o-rings, for sealing the connection between the fixed portion and the removable portion. In some embodiments, the collection device further comprises one or more sealing device for sealing the connection between the fixed portion of the collector assembly and the extractor assembly. In some embodiments, the hydrophobic membrane enclosure is a square, a circle or a dome. In some embodiments, the extractor assembly further comprises a doublesided tape adhesive for adhering to skin. In some embodiments, the positive electrode is used as an adhesive at the skin interface. In some embodiments, the positive electrode is a membrane at the skin interface having at least one membrane hole. In some embodiments, the positive electrode is made of a hydrogel assembly comprising a hydrogel layer, a conductive layer and an adhesive layer. In some embodiments, there is a single positive electrode. In an embodiment, more than one positive electrode is used. In some embodiments, the collection device comprises more than one, or 4 or more negative electrodes. In some embodiments, the collection device comprises 8 negative electrodes, or 16 or more negative electrodes. In some embodiments, the collection device comprises 64 or more negative electrodes. In some embodiments, the collection device comprises 64 negative electrodes.

[0006] In a further embodiment, the collection device is connected to a module, wherein said module is connected to the positive and negative electrode and controlling current applied, and the module comprising the vacuum pump for controlling in fluid communication to the vacuum pumping through the vacuum pumping channel. In some further aspect, the module measures variation in resistance between the at least one negative electrode or between the positive electrode and the at least one negative electrode. In another embodiment, the interstitial fluid is collected into a tube or chamber connected to the collector assembly.

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

[0008] In a further aspect, there is provided a method of electroporating the skin of an individual, the method comprising: contacting with the skin a positive electrode and a metallic negative electrode, applying a current between the positive electrode and the metallic negative electrode, stopping the current when pores are formed in the skin, and applying a negative pressure on the skin to maintain the pores in an open state. In some embodiments, the methodfurther comprises the step of determining the electroporation level by measuring the resistance variation between the at least one negative electrode or between the positive electrode and the at least one negative electrode. In some embodiments, the skin is cleaned before contacting the step of contacting.

[0009] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a schematic side view of a collection device according to an embodiment of the present disclosure.

[0011] FIG. 2 is a schematic top view of a collection device according to an embodiment of the present disclosure.

[0012] FIG. 3 is a schematic cross section view of the collector assembly of the collection device according to one embodiment of the present disclosure.

[0013] FIG. 4 is a schematic cross section view of the fixed portion of the collector assembly of the collection device according to one embodiment of the present disclosure.

[0014] FIG. 5 is a schematic cross section view of the removable portion of the collector assembly of the collection device according to one embodiment of the present disclosure.

[0015] FIG. 6A is an exemplary schematic cross section view of square shaped of the hydrophobic membrane enclosure of the collector assembly.

[0016] FIG. 6B is an exemplary schematic cross section view of dome shaped of the hydrophobic membrane enclosure of the collector assembly.

[0017] FIG. 6C is an exemplary schematic top view of circular shaped of the hydrophobic membrane enclosure of the collector assembly.

[0018] FIG. 6D is a cross section view along line A-A of the hydrophobic membrane enclosure of Fig. 6C.

[0019] FIG. 7 is a schematic exploded view of the extractor assembly of the collection device according to one embodiment of the present disclosure.

[0020] FIG. 8A is a schematic top view of the extractor assembly according to one embodiment of the present disclosure.

[0021] FIG. 8B is a bottom side view of the extractor assembly of Fig. 8A.

[0022] FIG. 8C is a bottom elevation view of the extractor assembly of Fig. 8A.

[0023] FIG. 8D is a side view of the extractor assembly of Fig. 8A.

[0024] FIG. 8E is a side top view of the extractor assembly of Fig. 8A.

[0025] FIG. 8F is a cross section view of the extractor assembly of Fig. 8A showing an unexpanded membrane.

[0026] FIG. 8G is a cross section view of the extractor assembly of Fig. 8A showing an expanded membrane.

[0027] FIG. 9A is a schematic top view of the extractor assembly showing exemplary dimensions (in inches at 2:1 scale).

[0028] FIG. 9B is a schematic cross section view of the extractor assembly of Fig. 9A.

[0029] FIG. 9C is a schematic side view of the extractor assembly of Fig. 9A.

[0030] FIG. 9D is a schematic cross section view of Fig. 9C.

[0031] FIG. 10 is a schematic bottom view of an extractor assembly with four membrane holes.

[0032] FIG. 11A is a schematic inside view of an extractor assembly according to an embodiment of the present disclosure.

[0033] FIG. 11B is a schematic cross section of the extractor assembly of Fig. 11A along line E-E.

[0034] FIG. 11C is a schematic cross section of the extractor assembly of Fig. 11 A along line B-B.

[0035] FIG. 12A is a schematic side view of an extractor assembly according to an embodiment of the present disclosure.

[0036] FIG. 12B is a schematic cross section of Fig. 12A.

[0037] FIG. 13A is a top view of an extractor assembly according to one embodiment of the present disclosure assembled with screws.

[0038] FIG. 13B is a schematic cross section of Fig. 13A along line E-E.

[0039] FIG. 13C is a schematic cross section of Fig. 13A along line G-G.

[0040] FIG. 13D is a schematic cross section of Fig. 13A along line H-H.

[0041] FIG. 14A is a schematic top view of the extractor assembly positioned on skin according to one embodiment of the present disclosure.

[0042] FIG. 14B is a cross section of Fig. 14A along line l-l.

[0043] FIG. 14C is a close up of the region labeled “K” in Fig. 14B.

[0044] FIG. 14D is a close up of the region labeled “L” in Fig. 14C.

[0045] FIG. 14E is a close up of the region labeled “M” in Fig. 14C.

[0046] FIG. 14F is a schematic cross section view of the extractor assembly of Fig. 14A showing an unexpanded membrane.

[0047] FIG. 14G is a close up of the region labeled “K” in Fig. 14F.

[0048] FIG. 14H is a close up of the region labeled “M” in Fig. 14G.

[0049] FIG. 141 is a close up of the region labeled “L” in Fig. 14G.

[0050] FIG. 15A is a schematic showing the skin at rest before expansion (i.e., unexpanded).

[0051] FIG. 15B is a schematic showing the skin expanded using the collection device of the present disclosure.

[0052] FIG 16A. is a bottom elevation view of an extractor assembly with eight negative electrodes.

[0053] FIG. 16B is a bottom elevation view of the extractor assembly of Fig. 16A showing the positive electrode hydrogel material.

[0054] FIG. 16C is a side view of the extractor assembly of Fig. 16A.

[0055] FIG. 16D is an exploded view of the extractor assembly of Fig. 16A.

[0056] FIG. 16E is a bottom view of an extractor assembly with sixteen negative electrodes.

[0057] FIG. 16F is a bottom view of the extractor assembly of Fig. 16E showing the positive electrode hydrogel material.

[0058] FIG. 16G is a top-down exploded view of the extractor assembly of Fig. 16E.

[0059] FIG. 16H is a bottom-up exploded view of the extractor assembly of Fig. 16E.

[0060] FIG. 161 is a bottom elevation view of an extractor assembly with sixty-four electrodes.

[0061] FIG. 16J is a bottom view of the extractor assembly of Fig. 161.

[0062] FIG. 16K is a side view of the extractor assembly of Fig. 161.

[0063] FIG. 16L is a top-down exploded view of the extractor assembly of Fig. 161.

[0064] FIG. 16M is a side exploded view of the extractor assembly of Fig. 161.

[0065] FIG. 16N is a bottom view of the extractor assembly of Fig. 16I showing the positive electrode hydrogel material.

[0066] FIG. 160 is a side view of the extractor assembly of Fig. 16N.

[0067] FIG. 16P is a side exploded view of the extractor assembly of Fig. 16N.

[0068] FIG. 16Q is a bottom-up exploded view of the extractor assembly of Fig. 16N.

[0069] FIG. 17A is a schematic top cross section view of a hydrogel assembly according to one embodiment.

[0070] FIG. 17B is an exploded view of the hydrogel assembly of Fig. 17A.

[0071] FIG. 17C is a staggered exploded view of the hydrogel assembly of Fig. 17B.

[0072] FIG. 18A is a schematic top view of a hydrogel assembly according to another embodiment.

[0073] FIG. 18B is a side elevation view of the hydrogel assembly of Fig. 18A.

[0074] FIG. 18C is an exploded view of the hydrogel assembly of Fig. 18A.

[0075] FIG. 19A is a photograph of a double-sided tape set up on electroporated skin.

[0076] FIG. 19B is a photograph of a glue adhesive set up on electroporated skin.

[0077] FIG. 20A is a photograph of skin electroporated using the device and method according to the present disclosure.

[0078] FIG. 20B is a photograph of a comparative skin electroporated with a device that utilises glue.

[0079] FIG. 21 illustrate a schematic view of a collection device according to an embodiment of the present disclosure connecting to an electronic module.DETAILED DESCRIPTION

[0080] There is provided herein a device for obtaining continuously and over long periods of time a sufficient quantity of interstitial fluid from the human body in a non-invasive way. The method and device disclosed herein include the combination of electroporation of human skin and suction of the interstitial fluid through the permeabilized and micro-opened skin. The term “electroporation” as used herein with respect to skin refers to a method of opening permanent pores in the stratum corneum of the skin. Those pores then allow the continuous extraction of interstitial fluid for as long as the extractor is applying a negative pressure on the electroporated area. The pores produced in that manner are said to be permanent for as long as pneumatic and / or mechanical pressure is / are applied on the electroporated skin for the purpose of the extraction of interstitial fluid (IF).

[0081] There is provided herein an improved collection device that has a means for applying mechanical pressure onto the pointed electrode (which is the negative electrode) that is applied on the skin for electroporation. The negative electrode used herein preferably has a small diameter (around 0.5 mm ± 150 pm). The negative electrode can have a working end that is conically shaped with a blunt point which has, for example, a diameter of 0.1 mm ± 25 pm in diameter, to avoid piercing the skin. Suitable materials for the negative electrode include but are not limited to noble metals such as palladium. When the negative electrode is applied on the skin for electroporation, the electrode presses against the skin because of an offset between the seating plane of the complete apparatus on the skin and the electrode’s point. That offset is pushing the electrode into the skin surface. There may also be an additional pressure contributed by the application of the negative pressure during the electroporation.

[0082] The pressure makes a conical deformation on the skin, more specifically in the stratum corneum and epidermis, and possibly further in the dermis. The effects of that deformation are many fold:• thinning out the layers to be electroporated, helping in the electroporation process,• increasing the electrical connectivity through the stratum corneum and epidermis in a very small area of the skin,• concentrating the electrical current application to a small channel, improving on the efficiency of the electrical current application process, and• mechanically improving the opening of the electroporated pore in the stratum corneum and epidermis and possibly in the dermis, by helping the separation of the cells during the electroporation process, to create the electroporated pore.

[0083] However, the mechanical pressure on the skin after the electroporation is completed has the effect of opening up the pores due to the elastic / plastic properties of the skin. This opening is similar to what happens when one is trying to force a fluid through a thin perforation in a septum. At low or very low pressures, no fluid flow will happen. Once the pressure is high enough to make the septum expand / deform, the hole shall then open and a fluid flow will then be possible.

[0084] As to the “permanent state” of the pores electroporated using the present method, the mechanical stress imposed on the electroporated region helps keep the electroporated material apart and distance it, so that after a certain period of time the state of the electroporated region becomes more “permanently electroporated”.

[0085] It was observed that the skin expansion due to the negative pneumatic pressure applied on the skin makes the skin expand as well as countering the capillary pressure that would otherwise tend to keep the IF within the skin. This expansion may bring an important contribution to the fact that the electroporated pore opens for the passage of the extracted IF, but it also may be important for the fact that the electroporated pores are deemed to be “permanent” for as long as the negative pressure, i.e. skin expansion is applied, and so the IF can be extracted for long periods of time, i.e. 21 days continuously.

[0086] Although the present device is generally described as being a collection device designed to extract a body fluid, the device may also be used for introducing a substance to a subject through the electroporated skin. For substance administration, the application ofpressure would be just as important as in the case of extraction. Since the skin expansion helps open the electroporated pores in one direction, the same would happen in the case of the administration of substances through the skin, meaning overcoming the capillary pressure, greater opening of the pore and keeping the pore open.

[0087] Making reference to Fig. 1 , the collection device 1 comprises an extractor assembly 2 which makes contact with skin 28 of an individual to extract a body fluid such as interstitial fluid. The extractor assembly 2 is connected to a collector assembly 3 comprising a fixed portion 3a and a removable portion 3b. A top view of the collection device 1 is shown in Fig. 2. Fig. 3 shows a cross section along line A-A of Fig. 2 of the collector assembly 3. The collector assembly 3 has a conduit 4 which brings interstitial fluid and gas extracted from the skin and flowing from the extractor assembly 2 into a collection cavity 5. The collection cavity 5 is positioned in the removable portion 3b of the collector assembly 3. The removable portion 3b also has a gas cavity 6 in which gas freely circulates around the inner hydrophobic membrane 7 which defines the collection cavity 5. The hydrophobic properties of the membrane 7 allow the gas to pass through it while retaining the aqueous mixture within. The hydrophobic membrane and the septum effectively seal the liquid within (IF) from any external contamination (sterile conditions). A pump conduit 8 is fluidly connected to the gas cavity 6 and leads to a pressure regulated pump system to control the pressure in the cavity 6. The pump conduit 8 allows pumping gas out of the collector assembly 3 in order to pull the interstitial fluid and gas into the collection cavity 5. The hydrophobic inner membrane enclosure 7 is preferably sealed to the solid base 21 of the removable portion 3b. The removable portion 3b is reversibly connected and reversibly sealed to the fixed portion 3a through o-rings 9a. O-rings 9b are also used to connect the fixed portion 3a to the extractor assembly 2. The pumping operation is also sealed by an o-ring.

[0088] The fixed portion 3a is shown in Fig. 4 and has a fixed support 11 to hold onto the extraction assembly 2 along with o-rings 9b. The illustrated embodiment containing o-rings should not be interpreted as limitative since there are other means than o-rings to provide sufficient connection between the extraction assembly 2 and the fixed portion 3a. At the end of the conduit 4 there is a needle 12 for piercing the septum of the removable portion 3b of the collector assembly 3. The piercing allows for creating a fluid communication between the fixed portion 3a and the removable portion 3b through the conduit 4. The fixed portion 3a also includes the pump conduit 8 which leads to the regulated pump system. Fig. 5 shows the removable portion 3b separately from the fixed portion 3a. The removable portion 3b can be enclosed in an external enclosure 20 which then may define the gas cavity 6. The solid base 21 comes into contact with the fixed portion 3a when assembled. Additionally, the needle 12pierces the septum 22 forming a hole 22a in the septum 22. The removable portion 3b can also includes an inner enclosure 23 to support the inner hydrophobic membrane enclosure 7. The membrane enclosure 7 may be of different shapes. For example, a square shape membrane is illustrated in Fig. 6A, whereas a dome shaped membrane is illustrated in Fig. 6B. When a dome shape is used the hydrophobic membrane can be a continuous single piece and may not need a support. In another example, Figs. 6C and 6D illustrate a circular hydrophobic membrane 7a which has a solid plastic closure 7c for support which may be welded or glued. The circular hydrophobic membrane can be in the form of a sheet that is rolled and glued.

[0089] Fig. 7 shows an exploded view of the extractor assembly 2 when in operation on the skin 28. The extractor 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 comes into contact with the skin 28. An elastic membrane 100A expands the skin 28 forming an expanded skin region 29. The extractor assembly 2 is also divided into a bottom part 30A and a top part 30B which fits into the bottom part 30A. The negative electrode is positioned inside the top part 30B and makes it way all the way down to make contact with the skin 28 when electroporation is performed.

[0090] It is encompassed that the positive electrode has a large surface to reduce pain when electroporation is ongoing. The positive electrode is a current defusing positive electrode that limits the pain experienced by the patient. In an embodiment, the positive electrode can be a hydrogel electrode having a hydrocolloid material as described herein, but also encompassed is a dry electrode, and not limited to. The electrode can be made of stainless steel and / or comprises palladium for example, and not limited to.

[0091] Figs. 8A-8E show different external views of the extractor assembly 2 which has a bottom surface 40 that comes into contact with the skin 28 and a side surface 41 perpendicular to the bottom surface 40. On the bottom surface 40 there is at least one membrane hole 42 from which interstitial fluid is extracted from the skin and enters the extractor assembly 2. Fig. 8F is a cross section along line A-A of Fig. 8A showing the extractor assembly 2 when the membrane is unexpanded and an expansion space 45 exists at the boundary between the skin 28 and the extractor assembly 2. The cross section also shows the hydrophobic membrane 44 which acts as a positive electrode. Fig. 8G is a cross section along line A-A when the membrane expands 46 in the expansion space 45 driven by a pumping through the pumping channel 43.

[0092] Fig. 9A is a top view of the extractor assembly 2. Fig. 9B shows a cross section along line E-E of the Fig. 9A illustrating the top part 30B and the bottom part 30A, the hydrophobic membrane 44 and a rigid membrane 47. Fig. 9C is a side view of the extractor assembly 2 and Fig. 9D is a cross section along line D-D of Fig. 9C. The cross section shows the top part 30B and the bottom part 30A as well as the pumping channel 43. Embodiments illustrated herein have been shown with a single membrane hole 42 which is preferably centered to avoid slippage. The dimension of the membrane hole 42 can also be optimized to reduce or eliminate any slippage that may happen when dealing with skin and a rigid assembly. However, other embodiments comprising more than one membrane hole 42 are also contemplated herein. For example, Fig. 10 illustrates a four-hole assembly that can advantageously reduce the “dead volume”. The interior of a four-hole embodiment is illustrated in Fig. 11 A and Fig. 11 B shows a cross section along line E on the pump exit section 48 which includes the pumping channel 43. Fig. 11C is a cross section along line B-B which shows exemplary relative dimensions of the extractor assembly 2. Fig. 12A shows yet another side view along with a cross section view in Fig. 12B. In some embodiments, as illustrated in Fig. 13A, a mechanical means such as screws 49 can be used to secure the top part 30B to the bottom part 30A. This is better illustrated in the cross sections shown in Figs. 13B-13D.

[0093] Fig. 14A shows a top view of extractor assembly 2 on skin 28. Figs. 14B-14E are cross sections showing the expansion of the membrane which leaves a small empty space 10 from which the pumping channel 43 can continuous pump out. Figs. 14F-14I show the same device but with the membrane being unexpanded. The extraction conduit sections 32A and 32B are two sections of the pumping channel 43. The elastic membrane 100A and the opening in the elastic membrane 100B are most particularly illustrated in Fig. 14E in the expanded configuration which can be compared with Fig. 14H at rest. The skin expansion is also illustrated in Figs. 15A-15B. As can be seen in Fig. 15B, when the skin expands an opening is created which allows the extraction of body fluids.

[0094] As previously mentioned, the extractor assembly 2 can have a single negative electrode or a plurality of negative electrodes for example 4, 8, 16, 32 or 64 negative electrodes. In preferred embodiments, there is only a single positive electrode which covers the bottom surface of the extractor assembly. Figs. 16A-16D shows an embodiment with a single positive electrode and four negative electrodes. Figs. 16E-16H shows an embodiment with a single positive electrode and eight negative electrodes. Finally, Figs. 16I-16Q shows an embodiment with a single positive electrode and sixty-four negative electrodes.

[0095] The present disclosure contemplates the use of various hydrogel adhesives, for example a configuration as illustrated in Figs. 17A-17C or the configuration illustrated in Figs.18A-18C. The hydrogel assembly 50 comprises a hydrogel 51 , a conductive layer 52, and an adhesive 53 which can be positioned on an extractor head 54 (Figs. 17A-17C). The hydrogel assembly can also be circular (Figs. 18A-18C) with a point of contact 55 extending therefrom. The “extractor head” 55 is the solid piece that is stuck to the skin with an adhesive such as either the hydrogel or a polysaccharide. In some embodiments, the extractor head is the solid material that constitute the vacuum chamber(s) through whose openings the extracted interstitial fluid could be extracted. The polysaccharide is not conductive while the hydrogel is conductive and can have the secondary usage of taking place of the positive electrode in using a hydrogel electrode construction to replace the polysaccharide adhesive. Accordingly, the hydrogel is preferred (i.e. , a hydrocolloid electrode). The connection to the hydrogel positive electrode can be made through a “carbon film” that also can be supported by an additional metal layer / backing. When using a larger current and / or using a large number of electrodes, the hydrocolloid electrode’s surface should be designed proportionally to provide the same electroporation conditions or instead the electric parameters can be adjusted. However, increasing the current too much may lead to undesirable sensations on the skin.

[0096] After a complete electroporation of the skin, pores have been made in the outer part of the skin. Those pores may remain “open” if “unattended” for periods of time over 15 minutes. For continuous extraction of the interstitial fluid and for the pores to remain open, a negative pressure must be applied over the electroporated area. That negative pressure must be sufficient to overcome the capillary effect that would keep the interstitial fluid from flowing through the opened pores. Interstitial fluid extraction has successfully been performed on a continuous basis for periods of up to 21 days. The pressure differential applied tends to draw the skin outwards and into the extraction chamber. The extraction chamber must then be made large enough to avoid the opened pores to touch a solid surface that may imped both the extraction of interstitial fluid and the pore being kept open. Some skin deformation and light bruising or reddening may also occur, which would not be acceptable in some circumstances. That skin deformation is not only making elastic deformation of the skin, but the skin also may be subjected to plastic deformation, which all may greatly differ depending on the subject’s skin characteristics and conditions. Minimizing the skin surface exposed to the negative pressure would also minimize the skin expansion, thus minimizing the “dead volume” to be dealt with in the course of interstitial fluid extraction. Mechanical construction of the extraction chamber, using posts, contributed to limiting skin expansion and minimizing dead volume considerations. Experiments have been performed by using a thin plate of solid material pierced with small holes to be centered on each of the electroporated areas of skin. In that case skin expansion was very much limited, but interstitial fluid extraction also was either very small or even inexistent. One conclusion of that experimentation was that the electroporatedskin was acting like a pharmaceutical vial septum, which can be pierced, but remains “sealed” when the needle is removed. However, when and if such a perforated septum would be subjected to a pressure differential, the septum would be forced to expand and the piercing would then be “expanded” as well, making it into a larger opening, to allow fluids to more easily pass through the opening (piercing). The present collection device was designed with a thin, flexible, elastic membrane with small holes aligned with the electroporated openings in the skin to contribute to minimizing the above problems. Skin deformation and bruising are deemed to be very important for applications like glucose monitoring devices, where those effects on the skin may have detrimental effects on the quality and contents of the extracted interstitial fluid.

[0097] In some embodiments, the extractor assembly can be made from a polymeric material, a combination of a polymeric material or any other material that are electrically conductive or insulating. The collection device may be covered by a material in order to facilitate its use in a swimming pool, during the use of a bath or a shower and / or any situation where a liquid can surround the extraction assembly. The extraction assembly can include a sealed hole so that the collected freshly extracted fluid can be analyzed remotely from the device. Furthermore, in order to increase the fixture of the extraction assembly as described herein, a non allergenic material such as a bracelet or a strap can be used to stabilize the contact between the device and the skin.

[0098] Before the application of the collection device on the skin, the skin can be gently cleaned by any known chemicals used in medicine to clean such skin or simply by the use of a soap and / or water. Preferably, any chemicals that evaporate after cleaning are used such that they do not leave any residue at the surface of the skin. Moreover, the chemicals or the soap should not induce any allergenic reaction of the skin nor modify the structure of the skin. As soon as the skin is gently cleaned, the collection device is attached on the top of the cleaned skin by the help of the non allergenic material such as the adhesive, the strap or bracelet. The preferred part of the skin is any part of the skin covering the arm of a person, but the majority of the body skin can be used with the device and the method described here. Once attached on the skin, the extraction is continuously carried out by the application of a controlled negative pressure that is applied by the pumping system. The controlled negative pressure is maintained by the action of the pump and the level of the negative pressure is preferably continuously monitored and controlled using a pressure sensor.

[0099] The negative pressure is attained by activating the pump in an on / off fashion or in a continuously modulated fashion. The pump and the pressure sensor can be under the control of a microcontroller that shall continuously or discontinuously monitor the actualpressure in the device from reading the actual status of the pressure sensor and accordingly activate or deactivate the operation of the pump in a continuous, discontinuous or modulated fashion in order to achieve the desired negative pressure. A temperature sensor can also be incorporated in order to improve the efficiency of the device and to correct the reading of the biosensor 22 or the plurality of biosensors if needed. The vacuum is generated inside between the skin and the pump in order to improve the adhesion, the continuous extraction of the transdermal fluid, the circulation of the extracted fluid and, consequently, the continuous monitoring of one analyte or a plurality of analytes in the extracted fluid. The vacuum pump provides a vacuum that is sufficient suction to stretch a portion of the skin in the region from which the sample of interstitial fluid is to be extracted. As the suction provided by the vacuum pump is stretching the appropriate portion of the skin, the suction provided by the vacuum pump also causes the stretched portion to become completely filled with interstitial fluid. A vacuum pump that is suitable for the device defined herein can be a peristaltic pump, a diaphragm pump, a piston pump, a rotary vane pump, or any other pump that will perform the required functions set forth previously. Typically, the vacuum pump preferably employs a self- contained permanent magnet DC motor. The vacuum pump is preferably capable of providing a differential pressure down to about -14.7 psi and is more preferably operated at from about -0.3 psi to about -10.0 psi. The vacuum provided by the vacuum pump can be continuous or pulsed. A continuous vacuum is preferred for the reason that it requires fewer components than does related to a pulsed vacuum. It is preferred that the applied vacuum does not cause damage to the skin. It is preferred that the applied vacuum does not produce bruises and discolorations of the skin that persist for several days. It is also preferred that the level of applied vacuum and the duration of application of the vacuum do not be so excessive that it causes the dermis to separate from the epidermis, which results in the formation of a blister filled with fluid. The collection device can also include other electrochemical sensors or optical sensors, a wired or wireless communication device, a sensing electrode, a reference electrode and a counter electrode, and / or a sensing electrode and an accompanying electrode acting both as a reference and a counter electrode.

[0100] The use of a positive displacement pump would not have any effect different to the use of any other type of pump. Since the main parameter in extraction is entertaining a constant differential pressure between the body’s interior and the extraction site itself (assuming, and in fact it is the same, or having a constant relationship, inside the body and the atmosphere), maintaining a differential pressure between the atmosphere and the extraction site. The use of any type of pump, disregarding the differential pressure would put awry the extraction, and would also cause excess differential pressure onto the skin, causingexcessive skin expansion and cause blood pulling up either through the skin, or through the pose, or cause blisters on the skin.

[0101] As described above, the collection device has electrodes with the goal of performing electroporation in the skin. This is an effective electropermeabilization of the stratum corneum non-invasively, with permanent opening(s) as long as the suction is applied, without any pain felling and which can permit a continuous transdermal fluid extraction for a time as long as needed. A continuous fluid extraction is intended herein to refer to a continuous flow of the interstitial liquid through the skin, including the opening(s) in the stratum corneum, into the extractor assembly.

[0102] The present disclosure describes a device and method using same for the non- invasive and continuous extraction of interstitial fluid through the skin. This method as will be seen hereinafter provides an accurate way to obtain openings that last for days or as long as the monitoring of an analyte or a plurality of analytes in the interstitial fluid is carried out. The openings according to the present disclosure are in the order of micrometers and should not be confused with the smaller scale electroporation of individuals cells. Indeed, the electroporation as described in the present disclosure is an electroporation which led to openings of the skin or the stratum corneum. The openings last for days, hence, an accurate and precise monitoring of one or a plurality of analytes in the extracted interstitial fluid can be carried out with and without further electroporation of the same area of the skin.

[0103] The microcontroller can also control the application of the electrical excitation on the skin. If required, the mechanical contact of the electrode with the skin could be controlled by the microcontroller. Once the contact is established between the electrodes with the stratum corneum, the microcontroller then starts the process of the electroporation of the skin. The microcontroller shall be able to control over the time the voltage either the pulsed applied voltage or the non pulsed floor voltage, the current, the repetition rate, the pulse duration or the duty cycle of the pulses, the duration of application of the voltages and / or currents, the measurement of the voltages and the currents and the control of the conditions that may direct the starting and / or stopping of the application of the voltages and the currents.

[0104] An advantage of the present collection device is the use of a hydrogel based positive electrode in contrast to a traditional metal electrode. It was found that the metal positive electrode was not causing sufficient openings in the skin through electroporation. Moreover, the devices with a positive metal electrode were painful to the subject. The large hydrogel surface now used in the present device lowers pain experienced by the subject by dispersing current at the contact site, while causing no significant skin reddening. Thehydrogel electrode is off the shelf tech. The surface of the hydrogel positive electrode is determined based on the number of negative metal electrodes to obtain an efficient electroporation. Exemplary hydrogels include but are not limited to stomic adhesive hydrocolloid material, stomic adhesive hydrocolloid material with polydimethylsiloxane (PDMS) adhesive supplement applied or other similar hydrogel accepted as skin compatible.

[0105] It was sought that unipolar high voltage pulses while using continuous low voltage simultaneously applied between an electrode array including metallic positive electrode installed on the skin were adequate to perform electroporation. The traditional method also includes applying mechanical pressure to apply the electrodes on the skin, to better localize the electroporation points, and using a negative pressure differential between the skin and the atmosphere during the electroporation. More particularly, the metallic positive electrodes used were made of noble metal (Pd in particular), of a small diameter and of a conical shape at the ends in contact with the skin. The present collection device has replaced this electrode with a semi-conductive, large surface hydrogel electrode that may be made of hydrocolloid material.

[0106] After experimenting and looking at the skin opening made with the devices having the traditional metallic electrodes, it was noticed that the skin under the positive electrode(s) was not really “opened” by the electroporation process. Even by reducing the number of positive electrodes relative to the number of negative electrodes no improvement was obtained. Throughout those experimentations there was always pain perceived on the electroporated skin area during the application of the electrical process. There also were concerns for the skin areas under the positive electrodes, which were not making effective openings in the skin but just localized reddening and swelling of the skin.

[0107] In order to reduce the unnecessary skin alterations caused by the positive electrodes and in order to maximize the effectiveness of the opening process due to the negative electrodes, the present collection device utilizes a large-area conductive “positive” electrode to be applied. The concept of the large-area positive electrode is to effectively distribute the current on the positive side over a large area of the skin. That large distribution area is to have the effect of eliminating the useless skin alterations caused by the positive electrode(s) and reducing the skin pain sensations due to the positive electrode(s). This takes advantage of the electrostimulation process which is available and widely used. It makes use of hydrogel-based conductive electrodes that can have areas between 10 and 40 cm2.

[0108] The usage of such hydrogel-based positive electrodes proved to be a major improvement on the electroporation process in that it:• Allowed a more effective and more concentrated positioning of the negative electrodes on the skin, due to the absence of the metallic positive electrode(s).• Reduced the pain sensations felt on the skin due to the electroporation process.• Could be installed at any distance or position from the negative electrode(s).

[0109] The hydrogel electrode also imposes some usage considerations. Since the hydrogel is only installed onto the skin and does not press into the skin, as a negative electrode would, its surface in contact with the skin must be considered in relation with the number of negative electrodes. That is due to the limited conductivity of the hydrogel electrodes and the quantity of total current returned through them, that is due to the number of negative electrodes and their individual currents.

[0110] In an exemplary experimental condition tested, it was found that using as the positive electrode one hydrogel electrode with a surface of 2 to 3 square centimeters is sufficient to conduct successful electroporation with four negative electrodes, each using a nominal current of 130 micro-amperes. The manufacturing process for that type of electrodes is of the low-technology type, and they could be manufactured in any shape and size desirable to fit better with our devices and processes.

[0111] The usage of the hydrogel positive electrode is limited to the electroporation phase of the process and is not needed anymore after the electroporation is completed. The installation, connection and placement of the positive electrode is an additional operation, as well as is the removal of the positive electrode and its connection after the electroporation is completed. It is also contemplated that the adhesive properties of the hydrogel material could also be used as the adhesive / sealant required for the successful usage of the interstitial fluid extractor system thereby achieving a dual function. This construction would eliminate the installation / removal of the positive electrode and its connection would improve the useability of the interstitial fluid electroporator / extractor device, while improving its reliability and looks. This simpler and more compact construction would improve the reliability of the whole device. In general terms, the adhesive used for mechanical holding of the extractor assembly on the skin, as well as for maintaining a reliable vacuum tightness should be long-term compatible with skin. In preferred embodiments, a double-sided tape adhesive is used for more clear and defined electroporation holes.

[0112] One challenge previous collection devices and methods have struggled with is how to effectively determine when the electroporation has been completed and is opened pores ofthe desired size and that they are stable and permanent. It was presently found that measuring the resistance of the negative electrodes to monitor the electroporation is a suitable means of determining the electroporation level. The basic principle of impedance monitoring is that the impedance between two pores reduces as opening attains an efficient and more permanent state. That change can be measured in the reduction in the impedance when not electroporating as well as during electroporation. The measure of the rate of closure after electroporation stops also is provided as a method of evaluating the opening efficiency. In particular, the method comprises measuring “kilohms” of resistance or complex impedance using voltage changes, rate of rise and fall of voltage in the electrode arrangement. Resistance or impedance is basically defined as the ratio between voltage over current. In some embodiments, a target value or range of value of resistance can be indicative of sufficient electroporation. This target value or range can be calibrated generally based on the device while accounting for skin variations or can be calibrated on an individual basis since there can be a lot of variation in skin properties between individuals. Even without a target value or range, the rate of change and “holding patterns” of the impedance (or resistance values) can be used as the indicator of the opening state of the pore(s). The actual resistance can depend on the electrode(s) size(s), electrode pressure on the skin, skin’s own characteristics, electrode separation, electrode size etc.

[0113] Resistance measurement and variation can be related to opening made in the skin as being “holes” that let leak out the IF. If the hole is sufficiently “opened” in terms of “correctly electroporated skin” it will tend to be opened longer. That implies that if the holes are opened on the long term, some additional pressure has to be applied to extract the IF. A hole or opening will tend to revert to a “closed” condition at a lower rate than an “under-electroporated” one or even an “over-electroporated” one. In over-electroporated situations, the resistance tended to re-increase when in “over-electroporated” conditions. The measured resistance values can be variable, in absolute terms (as measured in ohms or kilohms) depending on the measurement method, the electrode separation, skin thickness etc.

[0114] But a good opened “hole” in the skin will tend to “keep” its post-electroporated conditions for a longer time than a lesser opened “hole”. So, the resistance measurement method relies on the comparative measurements. The basic measurements are taken e.g between two. negative electrodes in a set but can also be taken between the positive electrode and one negative electrode, but with possibly more uncontrolled variables than in the case of “negative electrode to negative electrode” measurements. During an electroporation the resistance values tend to decrease in time from the unelectroporated to the electroporated skin measurements, from over 100 kohms down to a few kohms.

[0115] For taking the resistance measurements, the electroporation is stopped and measurements are taken (for example) every second for 8 to 15 seconds, using a very low voltage so that the skin condition would not change due to the measuring currents / voltages. Studying the series of measurements always shows a re-increase of the resistance values between the first and the last measurement of a series. If the rate of increase is large the electroporation can be deemed not to be sufficient. For example, when the increase between the first and the tenth measurement represents an increase of 10% to 15%, this has resulted in efficient electroporation conditions. It should be noted that when the electroporation is pushed too far, the percentage increase and gets larger.

[0116] Resistance measurements are used to evaluate the durability of an electroporated opening. The evaluation of such condition relies on the lowest increase of resistance between the first and last resistance values taken in a series of, for example 8 to 15 measurements taken every second, periodically, for example every minute to 5 minutes.

[0117] The application of high-voltage electrical pulses along with a low-level continuous voltage makes durable (permanent) perforations across the stratum corneum. Those perforations will let interstitial fluid flow across the stratum corneum for as long as a negative pressure is applied on the skin, relative to atmosphere, which is deemed to be in equilibrium with the inside of the body. The application of negative pressure has two aims: (a) to counter the capillary pressure that would keep the fluids within the body, and (b) to entertain a flow of liquid between the body and the external device, called the extractor. The application of negative pressure has a mechanical effect, proportional to the value of the negative pressure maintained. The mechanical effect manifests itself by an expansion of the skin towards the side where the pressure is lowest, thus the outside of the body. That expansion of the skin causes a dilation of the electroporated pore and maintains it open. For different reasons, the actual extraction devices use a completely open cavity where the negative pressure is set and maintained by an active pumping system. The skin being both elastic and to a certain point plastic, in expanding it tends to flow into the pumping cavity. Depending on the value of the negative pressure used, the skin’s thickness, and the duration of the application of the negative pressure, the skin may: deform and expand, develop some wounding along the sides of the extraction cavity, show blasted capillaries in the pumping area, and / or make blisters (under specific conditions, though). In order to reduce the skin expansion and ancillary defects, a preferred embodiment a design with a symmetrical shape, with 4 to 6 active electrodes (pores), around a center protruding post. The role of that post is to prevent the skin from expanding too much, thus minimizing what is called the “dead” or unused volume necessary to maintain the pores away from any surfaces to prevent the blocking of the liquid flow fromthe skin. In order to further reduce that effect, extractor assemblies with a single hole per opening were made. Those were made of rigid polycarbonate plastic. If the holes were too small, they could often block the electroporated holes, because of adhesive slippage. If the holes were too small, they would prevent the holes from expanding, thus preventing liquid flow through the stratum corneum. If the holes were made larger, they would require a larger total volume and area to be used. Therefore, an elastic membrane was used to limit skin expansion, deformation, limit damage and wounding, limit or prevent capillary breaking and limit blister production.

[0118] As described above, the electroporation process is a method of opening permanent pores in the stratum corneum. Those pores will then allow the continuous extraction of interstitial fluid for as long as the extractor applying a negative pressure on the electroporated area shall be active. The pores to be opened in the skin have to be opened in an optimal way. Skin is a very variable assembly of living and dead cells that show widely differing behaviours in terms of thickness, elasticity, dryness, fat contents etc. Those highly variable factors will cause very differing behaviours in response to the application of the electroporation process that uses both current-limited voltage pulses and continuous voltage between positive and negative electrodes installed on the skin. After a certain time, the high-voltage pulses will have disrupted the first layers of the stratum corneum. That process should be continued further on in order to make certain the pores will remain open. If the electroporation process is not applied for a sufficiently long time, the pores will not remain open, and blisters will appear when the extraction process is applied on said under-electroporated skin. If the electroporation process is continued over too long a period of time, there will be over-electroporation, that will show in over-reddening of the electroporated skin, and, more importantly in the appearance of red cells of the blood in the extracted fluid.

[0119] Plotting of the voltages / currents on the electrodes shows that after just a few minutes, the resistance measured between any two negative electrodes will show very low values. Resistance between the electrodes on non-electroporated skin shows values above 100 thousand ohms (100 kQ). After a few minutes of electroporation, the resistance falls into the 5 kQ range. From that point on, the resistance will continue getting into lower values, but in a very much slower fashion. The determination of an absolute value of resistance to be attained for the “optimal electroporation” is extremely vague because of the large variability in skin properties. Inasmuch as the resistance evolution remains in a very flat curve, preventing any easy determination of a “stopping point”.

[0120] The negative electrodes used in the electroporation process are of a small diameter, (nominally 0.5 mm ±0.1 mm) in diameter. Their end can be conically shaped andapplied with a sufficient pressure to localize the point of action of the electroporation. The cone’s end is not perfectly pointed to avoid mechanically piercing the skin. The high-voltage pulses will after a certain time have disrupted the first layers of the stratum corneum. That process should be continued further on to make certain the pores will remain open afterwards under the slight negative pressure needed to extract the interstitial fluid through the skin. If the electroporation process is not applied for a sufficiently long time, the pores will not remain open and blisters may appear when the extraction process is applied on the said underelectroporated skin. If the electroporation process is continued over too long a period of time, there will be over-electroporation, that will show by an over-reddening of the electroporated skin and, more importantly in the presence of red blood cells in the extracted fluid. Measurements of the voltages and / or currents on the electrodes system shows that after just a few minutes, the resistance (or impedance) measured between any two negative electrodes will show considerable changes from the situation at the start of the electroporation process. Resistance measurements between the electrodes applied on non-electroporated skin show values above 100 thousand ohms (100 kQ). After a few minutes of electroporation, the resistance falls into the 5 kQ range. From that point on, the measured resistance will continue getting into lower values, but in a very much slower fashion. As originally said, the skin characteristics are very variable as well as depending on the age of the subject or the location on the subject’s body etc. There also is the often disregarded factor of the skin’s anisotropic structure, which may add considerable variability to the progress of an electroporation. An absolute value of resistance to be attained for the determination of an “optimal electroporation” is thus extremely vague. Also, as the resistance evolution progresses in a very flat curve after a few minutes, it prevents any easy determination of an “electroporation stopping point”. Thus, the present method of determining the optimal electroporation parameters focuses on the “durability” as the primary determinant.

[0121] Accordingly, a feedback method can be used to better define the completion of a successful electroporation. This would allow automatic (or manual) control over the electroporation parameters: time, current, pulse voltage, continuous voltage, pulse repetition rate, pulse duty cycle, and total time of electroporation. In the past and up to now, the optimal electroporation times were determined by past experimentation, while using a set of operational electrical parameters that also had been experimentally determined and verified. The way it was done was to stop the electroporation, remove the electroporation head and look at the skin where the negative electrodes were applied. Correlating what was seen with past observations was how the “correct electroporation” was determined. The standard continuous electroporation sequence was a continuous electroporation of 12 minutes, stopping for 30 seconds and restarting the electroporation for 3 minutes more.

[0122] When performing resistance measurements, it was noticed that a surprising event can occur: when the electroporation would have been completed, because of the negative pressure that had to be applied during the electroporation, small drops of interstitial fluid appeared in at least two of the negative electrode sites (the electrodes still being in place). Those drops of interstitial fluid, if they became large enough, could “short-circuit” the space between those two electrodes (or more) and then it was causing resistance readings that were suddenly lower than the “through-the-skin” resistance that was seen slowly decreasing. For example, the resistance could pass from 2 kQ to 300Q (0.3kQ). In some cases, this event can be used as an indicator for a completed electroporation sequence, although this is generally an off-chance event or may not appear at all the electrodes (if at all). While a continuous measurement of the resistance(s) with adequate analysis could present a more solid determination means for the effective completion of an electroporation process, the aim of all that is to take into account the variability in skin thickness, need for changing the electrical electroporation parameters, etc. The aim of that measurement is to provide a feedback method to modulate the electroporation process for minimizing pain, while keeping the electroporation time to an effective minimum. If the opening remains open in a stable fashion during extraction, it means that the conditions make it stay “stable”. The farther the opening is from the optimal, the faster it would revert to a higher resistance value.

[0123] An exemplary method is as follows:• Electroporation is stopped,• Resistance measurements are performed at pre-determined intervals, and• Determining the slope of the resistance measurements over time to determine if the pore opening is sufficiently stable, measurements could be performed, for example, over 15 seconds, at 1 -second intervals, then(a) Immediately terminating the electroporation process if the pore openings are stable, or(b) Pursuing electroporation for an additional period, such as one minute to give a “topping off” or “lackering over”.

[0124] The present collection device and method are particularly relevant in the domain of glucose monitoring because of the following: glucose monitoring does not require high flowrates, since they may not be desirable, the membrane greatly limits skin deformation and wounding, due to the expected and desired long-term usage of the glucose monitoringdevices, the membrane possibly limits the capillary breakage, and the membrane limits the formation of blisters. To maximize its usefulness, such a membrane should be sticking to the skin to: reduce as much as possible the skin slippage to keep the hole (pore) centered on the hole in the membrane, help keep the “dead volume”, or the extraction chamber’s dimensions to the strict minimum.

[0125] One potential concern for glucose readings is that density and speed of extraction may impact the accuracy of the readings. The dynamic control of pressure is key to avoid this. By decreasing the pressure for low sample volume testing, it improves the integrity of the sample in the process. “Decrease” in this case means using the lowest possible pressure. The range of 5-10 kilopascals is about a normal range so preferably the pressure is below 5 kPa, preferably in the range of from 0.1 to 1 kPa.

[0126] The present disclosure should not be limited to glucose and diabetes as any analytes found in the IF can be monitored for example lactate, cortisol, triglyceride, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), C-reactive protein, interleukin-1 , 3 or 18, and cystatin A or C as well as many others. The collection device is sealed, extractable, and requires minimal manipulation, which lowers the risk of spillage or contamination. The collection device can itself separate aqueous fluid the collected sample, so that only ISF is obtained. The collection device is configurable so that various sizes and shapes are contemplated herein.

[0127] Also encompassed herein is the reading and the analysis of the signal obtained from the biosensor of the collection device in order to transform the information and send the information to an alarm or a cell phone, for example, for further diffusion of the obtained information. The sent information can have many meanings such as the pure concentration of the analyte or the plurality of the analytes, a decision that the concerned person should take or simply a warning. In one example, when the biosensor for the continuous monitoring of glucose is based on the electrochemical methods, it can comprise three or two electrodes. In the case of three electrodes configuration, the first electrode is a common Ag / AgCI reference electrode; an inert counter-electrode constitutes the second electrode; and the third electrode is the sensing electrode. The sensing electrode comprises an inert electronic layer surrounded by a sensing layer. The sensing layer is obtained by the deposition of a homogenous mixture of a glucose oxidoreductase enzyme such as glucose oxidase, a mediator, a nano / micro powder of an electronic conductor, a cross-linking agent, a hydrophilic material, a hydrophilic additive and an adhesion promoter on the electronic layer-based sensing electrode.

[0128] In summary, the design of the present collection device includes many advantages such as but not limited to:- The design makes the device a sealed container,- The design makes the device a removable sealed container,- The design makes the device a minimum-manipulation collection unit,- The design allows the efficient collection of liquid (ISF) very close to the extraction point,- The design makes the device a spill-avoidance container and assembly,- The design makes the device an aqueous fluid separator to collect only the IF,- The design makes the device an easily reconfigurable collection device for any volume, large or small,- The design may be amenable to a sterilizable version,- Creates permanent pores,- Uses low power, no electroporation required after first electroporation,- Simple and continuous extraction,- High flow rate extraction possible,- Continuous extraction of IF,- Continuous substance administration is possible, and- Non-invasive out-of-body operation.EXAMPLE I

[0129] It was observed that in a 4 electrode pattern, there was different rates of electroporation amongst the electrodes. The difference may result from a difference in the limiting current. The installation of the extraction device may stretch the skin in more than one direction, thus affecting the actual mechanical static pressure effected by the negative electrode(s) onto the skin.

[0130] It was shown otherwise in an experiment performing electroporation in two different configurations on the same person. Both electroporations have been performed placing the negative electrodes (set of 4 electrodes) on the subject’s forearm. In both the electroporations exactly the same electrical and timing parameters have been used. The first electroporation used the positive electrode placed on the forearm and very close to the extractor chamber (bearing the four negative electrodes) position. The second electroporation placed the positive electrode on the subject’s calf on the same side of the body as the forearm bearing the extraction chamber with the negative electrodes. In both cases the electroporation was successful and the markings on the skin were similar. It was then deduced that the position of the positive electrode is immaterial. This was also demonstrated that an array of 64 negative electrode is possible. The advantage of increasing the number of electrodes is to increase the speed at which the sample is obtained.

[0131] The preferred electroporation parameters to be used on the inside forearm of a human adult were determined. However, even while using those parameters, the electroporation result may widely differ due to the skin’s conditions: dryness, stratum corneum thickness, lipid layer thickness, age, etc.• Pulse voltage (high voltage): 75 V• Pulse repetition rate: 4000 / s (4 kiloHertz) (period of 250 microseconds)• Pulse duty cycle: 2,375%• Continuous voltage (low voltage): 7V• Negative electrode current (limitation): 130 microAmperes• Time duration: One period of 720 seconds (T1), followed by a stoppage of 30 seconds (T2) and a last electroporation period of 180 seconds (T3).• Negative pressure: typical 10kPa.

[0132] The equipment used for performing the skin electroporation according were:• An extraction chamber to be hermetically affixed onto the skin surface, using an adhesive layer.• A pumping system for maintaining negative pressure in the extraction chamber during electroporation.• An electroporator to generate and control the conditions, electrical and other, to perform the electroporation.• An electrode holder, to position the electrodes in the opening of the extraction chamber and seal up the extraction chamber during electroporation.• A specifically called “positive electrode” to provide the positive electrical return path through a hydrocolloid (for example) material.

[0133] The skin should be minimally selected and prepared in the following way. If the skin seems to be greasy, wash with light soap and thoroughly dry or wipe with a light alcohol solution. One may wait for 20 minutes before the next steps. If there is hair on the skin’s surface, it is recommended to remove the hair by shaving. Avoid scraping the surface of the skin. It is preferable to wait a few hours before the next steps. If the skin is too dry, a sticky tape can be used to remove the outer flakes from the skin, to ensure good adhesion of the adhesive / dealing surface(s) to the skin. The extractor assembly is installed onto the skin by making sure the skin remains flat to avoid wrinkles that may disrupt the vacuum. A belt, strap or adhesive tape is used to further secure the extractor head on the skin. The pumping unit is then connected to the vacuum connector on the extractor head. The electrode holder assembly is added (electroporator head) on the extractor head, taking care to insert it perfectly vertically. The electrodes length should be so that some mechanical pressure is exerted on the skin so that the electroporation points be well defined and localized. A vacuum test is produced to verify for possible leaks. The leaks are corrected, if any. The pump / vacuum control active is maintained at pressure P1. The hydrocolloid positive electrode is installed on the skin and connected to the electroporation head or to the electroporator. Then, the electroporator is connected to the electroporation head. A software is used to check / adjust the electroporation parameters. The electroporation begins for the T 1 time period. Preferably, resistance measurements are performed to identify whether the electroporation is complete. If the resistance measurement is used, the automatic measurement can be set to interrupt the electroporation every N1 seconds for N2 seconds, during which time the resistance measurements are made every M1 milliseconds and successively recorded. The percentage of change between the first and the last measurement should be used for determination of a successful electroporation or for adjusting the electrical electroporation parameters. Waiting step for a period of time T2 to restart the electroporation (if needed or desired) and optionally for a T3 time period if needed. Phase M- could also be performed for electroporation completion verification. At this point the electroporation should have successfully completed the opening(s) in the skin. If using the resistance measurement method, the electroporation could have been terminated before and / or after the typical sequence of T1 , T2 and T3. Oncethe electroporation is deemed to be completed, the pumping unit is stopped. Then the electroporator head is disconnected from the electroporator itself and from the hydrogel positive electrode (if so used). The electroporator head is removed. Each of the negative electrode locations the skin should show a slightly darker tint, indicating a successful electroporation. Because of the negative pressure applied, and in the case of a successful electroporation, there may be the presence of drops of interstitial fluid at the electroporation points, indicating a successful electroporation.

[0134] As soon as possible the extractor head should be re-closed to again with the extractor head top set up and maintain the required negative pressure to extract the interstitial fluid from the skin through the already made pores. The following steps were performed: Reconnecting the vacuum connection to the pumping unit. Starting the pumping unit for it to maintain the P1 pressure. Verifying that the vacuum is stable and constant, that there are no leaks either in the adhesive-to-skin interface nor in the extractor head top set up nor in the tubing connecting the extractor head to the pumping unit. Correcting if necessary. The pumping unit may be operating from time to time, to equalize / control the pressure at the P1 point. The pumping also may be operating because of the outcoming fluid. There may also be some gas along with the liquid: dissolved gas from the interstitial fluid. After a few tens of minutes, fluid should be seen trying to fill the extraction chamber internal space. After the extraction chamber is filled, the fluid will be seen slowly exiting the extraction chamber through the tubing connecting it to the pumping unit. For collection purposes, the extracted fluid is collected at the exit of the extraction chamber.

[0135] The mechanism that makes the electroporated zone to revert rapidly to the nonelectroporated state is an indication of the progress of the electroporation leading to “permanent pores”. Electroporated “holes” or “pores” rapidly tend to revert to their nonelectroporated state. The conditions under which the process operates makes the opened pores “permanent”, but after a certain amount of time. It has been noted that for a successful electroporation to occur, one must apply the conditions must comprise a certain number of factors:- Pulse repetition rate- Pulse duty cycle- Pulse voltage- Continuous (base) voltage- Current- Time of application

[0136] Those factors also have to be adjusted depending on the characteristics of the skin to be electroporated. One of them being the “time of application”. A successful electroporation is currently being determined in a very relative way, using the following observations:- Visual observation of the skin under the electrode.- Apparition of liquid around the electrode position (if negative pressure is used during the electroporation.

[0137] That method being very approximate and requiring the opening of the electroporation set-up applied on the skin, it makes the determination of a successful electroporation quite approximative and may yield either under or over-electroporation, which in turn give additional problems, like blistering, blood in the IF etc.

[0138] The collection device of the present disclosure was tested with measurements on a benchtop skin model using inter-electrode resistance measurements. The electroporation was be stopped for approximately 10 to 15 seconds. The measurements were made every 1 or 1 second and the resistance increase rate was evaluated. If over a period of 10 to 15 seconds the resistance values increased rapidly, it would indicate incomplete electroporation. If to the contrary, the resistance values were to keep very close to the initial values, it would indicate a successful electroporation. This is because if a pore re-closed the resistance would increase, else if the pore were to be in a “permanently open” state its resistance would exhibit low values. This method is thus deemed to yield an accurate view of the state of electroporation in the electroporation process. Different skin conditions may then indicate an acceptable pattern of resistance measurements after varying times, but always yielding a much more accurate indication of the end of electroporation than estimation and looking into the extraction / electroporation chamber.

[0139] In an embodiment, resistance or impedance can be measured during the process of electroporation to insure e.g., optimal contact to the skin by measuring changes in resistance. Adjustment to current level can be also made following measurement of resistance or impedance if needed. By measuring the resistance or impedance, it is encompassed that the user could then detect if the electrodes are in good contact with the skin, thus prevent failed poration due to poor contact. In addition, it will allow efficient detection when poration has occurred so that the clinician can stop poration immediately, reduce the size of the defectand speed up the procedure. It is encompassed adjusting poration parameters to improve efficacy of poration depending on data such that if someone is trending that the pores are opening slower, then the intensity of poration could be increased to attempt to speed them up.Table 1. Measurements over 10 seconds after 4 minutes of electroporationTable 2. Measurements over 10 seconds after 9 minutes of electroporationTable 3. Measurements over 10 seconds after 14 minutes of electroporation

[0140] As can be seen in Tables 1-3, by 14 minutes the resistance values measured tend to remain quite stable. It is an indication that the electroporation may have reached its final stages. This avoids the previous practice of opening the electroporation chamber to try and monitor the state of the pores because there were no accurate ways to determine when to stop the electroporation.

[0141] The collection device of the present disclosure was installed on a skin used a double-sided tape adhesive in a first condition and with a glue adhesive in a second condition.It was found that the glue adhesive set up affects the pores and the results and should be avoided (Figs. 19A-19B). It was found that the diameter of the pores was consistent between the electrodes when the double-sided adhesive tape is used (Fig. 20A) but an inconsistent diameter was found when glue was used (Fig. 20B).

[0142] The electroporation progress was assessed using resistance measurements. The apparatus used to measure the resistance between two electrodes was a completely isolated device meaning that it does not impose either resistance nor capacitance on the electroporator electrodes and device, thus not affecting the electroporation by itself.

[0143] The measurement procedure was performed as follows:• At periodic intervals, programmable from 30 seconds to 999 seconds, the electroporation was stopped.• The electroporation device was functionally disconnected from the electrodes.• A low-voltage apparatus measured the resistance across each pair of electrodes.• The measurement was made by measuring the resistance by applying a low voltage between the electrodes and measuring the current flowing through, thus yielding the resistance value.• The measurements were reversed to eliminate any “ionic” or “battery effect”.• The measurements were made over a very short period of time to eliminate or minimize “ionic” or “battery effects”.• The process was repeated each second for a programmable number of times, from 1 to 15 times.

[0144] The reasons behind that method of measurement are the following:• the electroporation process is not immediate and progresses over time,• instantaneous measurements of resistance or impedance may not give a good evaluation of the “permanence” or “durability” of the result of the electroporation,• the ease of current passage through the electroporated pores depends on the advancement of the electroporation process,• if the electroporated skin is allowed to rest the pore(s) will close after a period of time,• the period of time the closure becomes more complete depends on the “quality” or “advancement” of the electroporation,• without wishing to be bound by theory, there is an assumption that the skin rests for some time and evaluate the electrical resistance of the electroporated pores,• there is no exact or absolute value of resistance that could be used to determine the “completeness” of an electroporation using the method described herein,• instead of just waiting and then measuring the resistance, it was decided to make successive measurements over a period of time and observe the “rate-of- change” of the resistance, to determine the point from which we could be reasonably confident of a “good” electroporation, and• the absolute value of resistance may also help but may not be determinant.

[0145] A series of measurements was performed on the same human subject to show examples of those resistance measurements as electroporations were performed on different parts of the body. The following parameters were used: Period 250 ps, Duty 2,375%, Duration 960 seconds, Pulse voltage 75V, Base voltage 7.5V, Resistance measurement interval every 120 seconds, Number of measurements per interval 10 (one per second), and Duration of the electroporation: between 960 and 1600 seconds.

[0146] The first measurements were made on the inner forearm of a human subject. The duration of the electroporation was 960 seconds. Once the electroporation was ended, liquid was already present at the electrodes locations, but were not inter-connected. There may have been some over-electroporation, but no blood came with the extracted liquid.Table 4. Resistance measurement

[0147] What should be noted from the table above is the rate of increase of the resistance values over time. The slower the resistance increases, the better the electroporation is.

[0148] The second set of measurements was made on the lower topside thigh of the human subject. That part of the body has somewhat thicker skin and may have more fat underneath the epidermis, like pig skin, for example. A complete electroporation period of 960 seconds was performed and once it was completed it was re-started for another 960-second period (that was not fully completed). At the end of this last electroporation liquid was found to be present at the electrodes locations but were not inter-connected. However, it certainly was not over-electroporation, because the electrode locations were not "blackened”.Table 5. Second round of resistance measurement

[0149] By comparing the above last measurements with the last measurements of the forearm, it can be noticed that the ratio of resistance between measurements 2 and 10 is larger for this total electroporation time, and therefore it can be concluded to electroporate for a further period of time. The visual observations corroborated the electrical “blind measurements”.

[0150] It however is interesting to see that in those limited cases, there seems to be an “absolute value” of resistance when good electroporation is achieved. This likely varies based on subjects, human or animal, or based on the body part as well.

[0151] The “rate-of-change” evaluations were better made between measurements 2 and 10, because the first measurement may be affected by stray set-up values. Moreover, it might be the stability of that ratio rather than the value of that ratio that is more indicative.

[0152] The presently made measurements support the importance of making measurements using this method to assert the state of advancement of an electroporation because of its ability to help determining when either electroporation parameters or electroporation duration changes would have to be brought in, either for refining the approaches or for dynamic adjustments or tuning during electroporation.EXAMPLE II

[0153] An extraction was conducted on two volunteers (males under 60). 400-500 ul of interstitial fluid was obtained over a 5-hour extraction, following a 30-minute electroporation using the collection device configuration as illustrated in Fig. 21.

[0154] In a further configuration, as seen in Fig. 21 , it is encompassed a collection device 60 comprising an extractor assembly 62 connected to an electronic module 64. As encompassed herewith, the extractor assemble has a negative electrode 66 and a positive electrode 68 having a hydrocolloid material. In this configuration, the negative electrode 66 will be deposited or clipped through an electrode dock 70 on the positive electrode on top of a vacuum plug 72. In operation, once the fluid collected will travel through a fluid path or tube 74 to a collection vessel 76 which can be a tube or chamber, for example, and not limited to. The fluid path 74 can be connected to the collection vessel 76 through a connector 78. As provided herewith, a continuous vacuum is preferred and is applied by the electronic module 64, though a connection 80, to the extractor assembly 62 using the vacuum plug 72 and through a vacuum conduit 82. A sterile filter 84 can be integrated to ensure sterility of the extractor assembly 62 system. The negative electrode 66 is connected to the electronic module 64 through pore 86 and the positive electrode 68 connected to the electronic module 64 through pore 88 such that the electronic module 64 controls the current and vacuum applied on the extractor assembly 62.

[0155] Using a collection device as encompassed herein, the extraction was conducted in a two-step process for accessing and harvesting interstitial fluid from the body. The first step involves the creation of the micro-openings in the epidermis (stratum corneum) and the second step is the extraction of interstitial fluid through these micro-openings.Step 1- Electroporation

[0156] The electroporation process involves three components. The extraction head (e.g., a square block that sits on top of the skin via an adhesive patch, a circuit board mounted with the electroporation probes that create the pulsed mild stimulation which creates the micro-openings and an electroporation module, which is the driver and Bluetooth link to the computer program monitoring and delivering the stimulation to the extraction site.

[0157] In the extraction head there are multiple probe electroporation heads and chambers. Each chamber has room for multiple electroporation probes, which are attached to a circuit board and operated as a single device.

[0158] The extraction site on the skin was prepped with an alcohol swab, and the extraction head with the adhesive was placed on the extraction site. A wire harness connected the circuit board to the electroporation module and circuit board was affixed to the extraction head assembly. The blue tooth connection was made between the computer program and the electroporation module, and the settings were established for the duration (in this example 920 seconds), along with the appropriate voltage settings. Once completed, the electroporation assembly (electroporation head, positive electrodes and electroporation module) were removed from the extraction head and site.Step 2 - Extraction of ISF

[0159] The Extraction process involved a pump module as described herein attached to the extraction head. The extraction head was fitted with a sealed clear cover, sealing the extraction sites in a vacuum chamber. In this embodiment, tubing was connected from the extraction head to the collection tube and from the collection tube to the pump module, creating the necessary negative pressure to extract the ISF from the interstitium. Once the pump was turned on, it maintained a predetermined level of pressure in the extraction chamber and over the micro-openings. The pump was left on until the desired amount of fluid was extracted.

[0160] The amount of fluid collected from the first subject was about approximately of 450ul to 500ul and for the second subject, approximately 500ul to 550ul of fluid.

[0161] While the disclosure has been described with particular reference to the illustrated embodiment, it will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative and not in a limiting sense. A positive electrode was affixed to the skin, one for each of the probe clusters.

[0162] While the description has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may beapplied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A collection device for extracting interstitial fluid comprising: an extractor assembly comprising a positive electrode, at least one negative electrode, and a pumping channel adapted to be fluidly connected to a vacuum pump; a collector assembly having a fixed portion and a removable portion, the fixed portion comprising a fixed support connected to the extractor assembly, a conduit for receiving the interstitial fluid and a needle at the end of the conduit, and the removable portion comprising a cavity defined by a hydrophobic membrane enclosure in fluid communication with the conduit for receiving the interstitial fluid, a septum adapted to be pierced by the needle, an external enclosure housing the cavity and defining a gas cavity around the hydrophobic membrane enclosure, and a pump conduit for pumping gas out of the gas cavity.

2. The collection device of claim 1 , wherein the positive is current defusing positive electrode.

3. The collection device of claim 1 , wherein the positive is a hydrogel based electrode or a dry electrode.

4. The collection device of any one of claims 1 to 3, wherein the negative electrode comprises palladium or is made of stainless steel.

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

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

7. The collection device of any one of claims 1 to 6, wherein the hydrophobic membrane enclosure is a square, a circle or a dome.

8. The collection device of any one of claims 1 to 7, wherein the extractor assembly further comprises a double-sided tape adhesive for adhering to skin.

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

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

11. The collection device of any one of claims 1 to 10, wherein the positive electrode is made of a hydrogel assembly comprising a hydrogel layer, a conductive layer and an adhesive layer.

12. The collection device of any one of claims 1 to 11 , the collection device comprises 4 or more negative electrodes.

13. The collection device of any one of claims 1 to 12, wherein the collection device comprises 8 negative electrodes.

14. The collection device of any one of claims 1 to 13, wherein the collection device is connected to a module, wherein said module is connected to the positive and negative electrode and controlling current applied, and the module comprising the vacuum pump for controlling in fluid communication to the vacuum pumping through the vacuum pumping channel.

15. The collection device of claim 14, wherein the module measures variation in resistance between the at least one negative electrode or between the positive electrode and the at least one negative electrode.

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

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

18. A method of electroporating the skin of an individual, the method comprising: contacting with the skin a positive electrode and at least one metallic negative electrode,applying a current between the positive electrode and the at least one metallic negative electrode, stopping the current when pores are formed in the skin, and applying a negative pressure on the skin to maintain the pores in an open state.

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

20. The method of claim 18 or 19, wherein the skin is cleaned before contacting the step of contacting.

21. The method of any one of claims 18-20, comprising installing the collection device as defined in any one of claims 1 to 15 on the skin of the individual and applying a current.

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